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• Published: 09 June 2023

Disentangling the cultural evolution of ancient China: a digital humanities perspective

• Siyu Duan 1 , 2 ,
• Jun Wang 1 , 2 , 3 ,
• Hao Yang 2 , 3 &
• Qi Su 2 , 3 , 4  

Humanities and Social Sciences Communications volume  10 , Article number:  310 ( 2023 ) Cite this article

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• Anthropology
• Cultural and media studies
• Language and linguistics

Being recognized among the cradles of human civilization, ancient China nurtured the longest continuous academic traditions and humanistic spirits, which continue to impact today’s society. With an unprecedented large-scale corpus spanning 3000 years, this paper presents a quantitative analysis of cultural evolution in ancient China. Millions of intertextual associations are identified and modelled with a hierarchical framework via deep neural network and graph computation, thus allowing us to answer three progressive questions quantitatively: (1) What is the interaction between individual scholars and philosophical schools? (2) What are the vicissitudes of schools in ancient Chinese history? (3) How did ancient China develop a cross-cultural exchange with an externally introduced religion such as Buddhism? The results suggest that the proposed hierarchical framework for intertextuality modelling can provide sound suggestions for large-scale quantitative studies of ancient literature. An online platform is developed for custom data analysis within this corpus, which encourages researchers and enthusiasts to gain insight into this work. This interdisciplinary study inspires the re-understanding of ancient Chinese culture from a digital humanities perspective and prompts the collaboration between humanities and computer science.

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Introduction.

Although still in its infancy, digital humanities research supported by big data and deep learning has become a hot topic in recent years. Researchers began to use digital methods to study cultural issues quantitatively, such as examining cultural evolution (Lewens 2015 ) through the diachronic changes of n-gram frequency (Michel et al. 2011 ; Lansdall-Welfare et al. 2017 ; Alshaabi et al. 2021 ; Newberry and Plotkin 2022 ) and word-level semantics (Newberry et al. 2017 ; Garg et al. 2018 ; Kozlowski et al. 2019 ; Giulianelli et al. 2020 ). This trend also spread to the study of ancient civilizations. Scholars from different cultural backgrounds have investigated the culture of ancient Rome (Dexter et al. 2017 ), ancient Greece (Assael et al. 2022 ), and Natufian (Resler et al. 2021 ) with the assistance of computer technology. It is acknowledged that ancient China was one of the longest-standing civilizations in human history, with a culture that evolved over the past thousands of years. Various ancient literature has been handed down over time, providing extensive textual records of Chinese culture. With the digitized versions of these classics, we can gain a glimpse into the cultural evolution in ancient China.

Ancient Chinese classics are highly intertextual texts. Since the doctrine “A transmitter and not a maker, believing in and loving the ancients” proposed in Analects (Legge 1861 . VII.I), quoting previous texts became a convention of literary creation in ancient China. Chinese scholars have long studied this cultural phenomenon from different perspectives. For example, Pan-ma i-t’ung (published around AD 1200) demonstrated the character differences between two history books, Records of the Grand Historian (published around 91 BC) and Book of Han (published around AD 82). Since Qing Dynasty, scholars began to enumerate parallel intertextual associations between ancient classics (Chen 1989 ; He et al. 2004 ). However, intertextuality (Kristeva 1980 ) is not only the connections of words and phrases but also manifests at higher levels hierarchically (Riffaterre 1994 ; Alfaro 1996 ), such as document, author, and community. The traditional form of high-level intertextuality studies was the overall literary criticism by scholars. For example, Ming dynasty scholar Ling Zhilong compiled previous scholars’ literary criticism of the above two history books. Literary criticism was themed on the style, skill, and viewpoints of literature, which was seen as a formidable endeavour due to the complexity of Chinese culture. Both parallel enumeration and literary criticism are limited by the reading and memory of scholars, which restricts the discussion on the large-scale corpus. Assisted by computer technology and digital literature, scholars recently began to study intertextuality within large-scale data.

Various natural language processing (NLP) methods have been applied to the intertextuality modelling of ancient literature. The previous automatic detection methods of text-level intertextuality aimed to discover similar phrases or sequences by lexical matching approach (Lee 2007 ; Coffee et al. 2012a ; Coffee et al. 2012b ; Ganascia et al. 2014 ; Forstall et al. 2015 ), which are insufficient and rigid in semantic modelling. The non-literal feature like synonym (Büchler et al. 2014 ; Moritz et al. 2016 ) and rhythm (Neidorf et al. 2019 ) also implies intertextuality, yet it requires language-specific design. Topic modelling lends a hand to passage-level modelling (Scheirer et al. 2016 ), while its dependence on expert annotation limits its generalization on diverse corpora. Simple statistics on text-level results contribute to document-level modelling (Hartberg and Wilson 2017 ). However, it ignores their overall connections. Besides, graph structure seems to be an appropriate way for the community-level modelling of intertextuality (Romanello 2016 ; Rockmore et al. 2018 ). Intertextuality modelling on classical literature widely supports cultural studies, such as quantitative literary criticism and stylometry (Forstall et al. 2011 ; Burns et al. 2021 ). Existing related studies on Chinese literature were limited to the detection methods (Liang et al. 2021 ; Li et al. 2022 ; Yu et al. 2022 ) and shallow studies of intertextual texts on small corpora (Sturgeon 2018a ; Sturgeon 2018b ; Huang et al. 2021 ; Deng et al. 2022 ), short of macroanalysis (Jockers 2013 ) on Chinese culture.

In this paper, we conducted a macroanalysis of ancient Chinese culture on an unprecedented large-scale corpus spanning nearly 3000 years. Figure 1a presents a schematic of this corpus. This corpus consists of 30,880 articles from 201 ancient Chinese books (or anthologies). It covers various topics, such as philosophy, religion, and politics, including the famous works of major cultural groups (e.g., Analects of Confucianism; Tao Te Ching of Taoism). The history books (e.g., Book of Han) and comprehensive anthologies (e.g., Collected Works of Han) of each era are also involved.

a The dataset of ancient Chinese literature with an instance in each era. The names of the dynasties and the approximate AD years are marked on the timeline. For each period, it gives one instance book and indicates its subject. b Hierarchical framework with three modules for multilevel intertextuality modelling.

In this work, we modelled ancient Chinese literature with a hierarchical framework. The cultural thought of civilization is composed of multiple levels, such as doctrines, individuals, and communities. Moreover, cultural evolution manifests hierarchically with microevolution and macroevolution (Mesoudi 2017 ; Gray and Watts 2017 ). A comprehensive discussion of cultural evolution requires multilevel perspectives. Therefore, this framework models intertextual associations from the text level to the community level with three modules. A schematic of the framework is shown in Fig. 1b . The text-level detection module tracks intertextual sentences with deep-learning models. The book-level aggregation module gathers text-level clues and abstracts various books into an association graph. The community-level inference module applies topological propagation to explore intertextual associations in the cultural community. After the modelling, millions of intertextual sentence pairs and a book-level intertextual association graph are ready for cultural analysis.

In the experiment, we detected 2.6 million pairs of intertextual sentences and then built them into an association graph. For a specific text collection, its intertextual distribution refers to its quantitative intertextual associations with other texts. Based on the modelling results, we can study ancient Chinese culture through the intertextual distribution among ancient literature.

In the cultural analysis, we considered cultural evolution from the perspective of cultural groups and religions. Schools of thought and religions were part and parcel of ancient Chinese culture (Schwartz 1985 ). The Hundred Schools of Thought that originated in the axial age were the prototype of ancient Chinese philosophy (Graham 1989 ). They rose and fell over the millennia that followed. The introduction of foreign cultures, like Buddhism (Chen 1964 ), also influenced the evolution of native culture. In this paper, we disentangled the cultural evolution of ancient China on three levels: (1) The interaction between individual scholars and philosophical schools; (2) The rise and fall of schools in Chinese history and culture; (3) The cross-culture communication with Buddhism.

Specifically, we validated several acknowledged cultural phenomena: the evolutionary paths of Confucianism and Taoism, and the booms and declines of the Hundred Schools of Thought. We also provided quantitative suggestions for cultural problems that are yet to be definitely resolved, such as the school attribution of Lüshi Chunqiu , the authorship attribution of Collected Works of Tao Yuanming , and the influence of Confucianism and Taoism across different cultural domains. Furthermore, we quantitatively discussed the interaction between Buddhism and native culture, revealing how cultural integration has evolved over time.

In addition, we have developed an online platform to display this corpus, along with millions of intertextual associations detected in this work. The platform supports custom data analysis, which encourages researchers and enthusiasts to gain insight into this work.

Two datasets were built respectively, the classic dataset and the era-text dataset. We considered several factors when building the dataset: era balance, representativeness, and official-folk balance. Two datasets consist of 30,880 articles from 201 books (or anthologies).

Classic dataset

The classic dataset is composed of the most prominent and influential books that represent the core culture of ancient China. Before the Tang Dynasty (618–907), literature was copied manually. Due to the long history and the limitations of publishing technology, only time-tested classics have been handed down to this day. Therefore, we added all the collected pre-Tang literature to the classic dataset. In the Tang Dynasty, the invention of block printing led to the rapid development of the publishing industry, resulting in explosive growth in the amount of literature. Until the mid-18th century, China printed more books than the rest of the world combined (Gernet 1996 ). Considering that this study focuses on the evolution of early thought in ancient China, we selected several most famous classics after Tang Dynasty. The well-known digital library of ancient Chinese classics, CTEXT ( https://ctext.org/ ), also adopted similar rules to build a collection of core classics. We considered the literature samples of CTEXT and built the classic dataset.

Our research focuses on ideological evolution, so books in the classic dataset should reflect cultural thought with good data quality. Therefore, we further screened the classic dataset to filter out inappropriate books, including commentary books, mathematics books, dictionaries, excavated literature (e.g., Mawangdui Silk Texts), and lengthy novels.

Finally, the dataset of ancient Chinese classics contains 133 books, including 8984 articles. Table 1 shows the time-period statistics of this corpus. It covers various aspects of culture, such as philosophy, mythology, politics, and religion. In this dataset, the earliest book was created around 1000 BC (e.g., Book of Documents), while the latest book was published around AD 1750 (e.g., The Scholars).

Era-text dataset

We aim for the era-text dataset to reflect the contemporary culture of each period, encompassing both official and folk traditions. To achieve this, we set our sights on history books and anthologies. As ancient China had a tradition of producing history books for each dynasty, history books typically reflected official attitudes. We added the official history (Twenty-Four Histories), large-scale chronicle history books ( Zizhi Tongjian and Continued Zizhi Tongjian Changbian ), and 15 other influential history books to the era dataset. In addition, we included Quan shang gu san dai Qin Han San guo Liu chao wen , a series of large-scale anthologies organized by era. It collected a wide variety of works from numerous authors, including prose, essays, religious scriptures, inscriptions, etc. These anthologies comprehensively record the contemporary culture of ancient China. To further enrich the era-text dataset, we added 13 well-proofread anthologies.

We categorized these history books and anthologies by era. For history books (e.g., Zizhi Tongjian ) that cover multiple eras, we divided them into corresponding eras. Finally, we got 55 history books and 13 anthologies, containing 21,896 articles. Table 2 shows the time-period statistics of this corpus. These works chronicle Chinese history and culture from the legendary period (e.g., Bamboo Annals , from 2600 BC) to the Ming Dynasty (e.g., History of Ming , ending in AD 1644).

Data processing

Ancient Chinese characters may have multiple written forms, we use the open-source toolkit OpenCC ( https://github.com/BYVoid/OpenCC ) to map them to a unique root character before encoding them using deep learning models. The maximum sentence length was set to 50 characters. Sentences exceeding this length were divided into two sentences. This setting can cover more than 99% of sentences.

Intertextuality detection usually aims to discover meaningful textual connections. It is important to note that texts without actual meaning cannot indicate the ideological connection between texts. Therefore, we use additional computational rules to avoid inappropriate text pairs. First, we filtered out sentences (clauses) with less than three remaining characters after removing the stopwords (such as prepositions and pronouns). Then, with predefined rules, we filtered out meaningless sentences, such as tone, dates, lengths, quantities, and formats. After filtering, there are about 436,000 sentences with 840,000 clauses in the classic’s dataset, and 2,113,000 sentences with 4,526,000 clauses in the era-text dataset.

Challenge and limitation

The collection and processing of ancient Chinese literature present challenges and limitations. Although we used punctuated text in this work, the original ancient Chinese literature has no punctuation. When it comes to no-punctuation data, an automatic punctuation model should be applied beforehand. Moreover, ancient literature could have multiple versions. In our dataset, we opted to include only one widely circulated version of each book. It may restrict the applicability of the dataset for researchers interested in different versions.

Additionally, the selection of appropriate literature collections for cultural analysis from a vast pool of ancient literature requires expert knowledge. In our study, humanities scholars specializing in Chinese history and philosophy were consulted.

Modelling framework

Considering that intertextuality and cultural evolution can manifest at multiple levels, we developed a hierarchical framework to analyze ancient literature. This framework captures intertextuality at three levels, ranging from micro to macro. At the text level, similar sentence pairs shared between books are detected by deep neural networks. At the book level, books are abstracted into an intertextual association graph based on the text-level results. At the community level, information propagates through the topological structure of the book-level graph, thus exploring intertextuality in the cultural community. This hierarchical approach provides both micro-evidence and macro-quantification for intertextual associations and cultural evolution.

Text-level detection

The study of cultural evolution is concerned with the connections of thoughts. Each sentence often expresses a distinct thought, making it a suitable quantitative unit. Therefore, we traced the intertextuality at the sentence level. We considered that the more similar sentences the two books share, the more closely connected they are.

The dissemination of text is not static but mutates. The micro-evolution of texts has multiple patterns (Tamariz 2019 ), such as replication, expansion, and succession. Therefore, this module traced similar sentence pairs shared between books with three patterns: overall similarity, partial similarity, and paraphrased similarity. A sketch is given in Fig. 2a .

a Three patterns of similarity between sentences. Darker colour indicates more similar text. b The explicit intertextuality and implicit intertextuality between the three books.

Overall similarity

Two sentences explain the similar meaning with close language expressions.

Partial similarity

Two sentences share similar parts.

Paraphrased similarity

The similar meaning is explained by different language expressions. The text may be disrupted and reorganized.

Deep neural networks (Vaswani et al. 2017 ) and pre-train methods (Devlin et al. 2019 ) have shown excellent performance in text feature extraction. Contrastive learning (Chen et al. 2020 ) can help to obtain personal-defined text similarity models without supervision, which is suitable for text-level intertextuality detection. To get sentence representation for these three patterns, we introduced the RoBERTa base (Liu et al. 2019 ), a pre-trained language model that can be further fine-tuned for our task using different training strategies.

For the overall similarity pattern, it can be treated as the overall semantic similarity between sentences. To train the model 1 , we used SimCSE (Gao et al. 2021 ), a contrastive learning method for extracting sentence embeddings.

For the paraphrased similarity pattern, the sentence structure could be reconstructed. We trained another model 2 for this pattern, with its loss being a weighted sum of loss 1 and loss 2 . The loss 1 was calculated in the same way as for model 1 .

For loss 2 , we randomly dropped and shuffled the clauses and n-grams in the original sentence to obtain a new sentence. It serves as another positive sample of contrastive learning. Negative samples are other sentences in the batch. The final loss for the model 2 is:

r is a hyperparameter that modulates the emphasis between sentence structure and semantics.

For the partial similarity pattern, sentences are considered similar if they share similar clauses. We detected similarities at the clause level using both model 1 and model 2 .

In large-scale information retrieval, brute-force search is often impractical due to the time and resources required. Therefore, it usually follows a multi-step process for the balance of precision and efficiency.

The first step is to recall potential candidates. In our work, we identified K members that were most similar to each sentence embedding. Then, we selected appropriate candidates and calculated a threshold to further filter out similar candidates.

For each pattern, we used the following steps to detect:

1. Extract embeddings of all sentences using the RoBERTa model.

2. De-duplicate embeddings. For each embedding, find its Top K similar embeddings. Denote all embedding pairs obtained as P .

3. Calculate the Euclidean distance of embedding in P and find the t th percentile as the similarity threshold d thr .

4. Filter out sentence pairs whose embedding distance is closer than d thr .

We detected similar pairs with these three strategies separately and gathered their results. The detected similar sentence pairs give concrete evidence of text-level intertextuality.

Book-level aggregation

Text-level results can support textual research on microevolution. However, to analyze at the macro level, text-level results must be gathered and aggregated. In this module, we aggregated text-level intertextuality results and synthesized them into a book-level intertextual association graph g . In this graph, each node B i represents a book, and there are N books in total. The edges indicate the intertextual associations between books. Suppose there are two books B i and B j , they contain n i and n j distinct sentences, respectively, and s ij distinct similar sentence pairs were detected between them. The edge weight α ij between B i and B j is calculated as follows:

For node B i , it has a one-hot node feature \(x_i = [x_{i1},x_{i2} \ldots x_{iN}]\) , where \({x_{ii}} = {1}\) .

Community-level inference

Text-level intertextuality can be observed explicitly. However, some intertextual connections can be implicit, with no direct textual association. In this study, we treated these classics as a cultural community and explored the implicit intertextuality at the community level. A schematic is shown in Fig. 2b .

Explicit intertextuality

If two books share similar sentences, they are explicitly intertextual.

Implicit intertextuality

If Book 1 and Book 2 are explicitly intertextual, and Book 2 and Book 3 are explicitly intertextual, then it can be inferred that Book 1 and Book 3 are implicitly intertextual.

This module performs inference by propagating and aggregating information through the topology of the intertextual association graph:

The first operation gathers explicit intertextuality I ex to the node feature. The second operation infers and integrates the implicit intertextuality I im . r ′ is a custom weight that adjusts the emphasis of implicit intertextuality. After twice graph computations, the node feature of B i is \(y_i = [y_{i1},y_{i2} \ldots y_{iN}]\) , where y ij indicates the united intertextual score I ij between B i and B j .

The node feature reflects the distribution of intertextuality for each book within the community. Excessive aggregation of information on the graph can lead to over-smoothing, which is detrimental to node features. Therefore, we set the number of graph computations to twice. Sparsity is an issue that often plagues text-based cultural analysis. With this method, the sparsity of intertextuality detection results can be alleviated.

Settings and modelling results

In text-level detection, we trained the model on an Nvidia 1080ti GPU. The optimizer is Adam (Kingma and Ba 2015 ). We took the pre-trained ancient Chinese RoBERTa base model as a basis. For both model 1 and model 2 , we fine-tuned the base model 10 epochs at a learning rate of 1e-6. The batch size was 32. The r for the loss of model 2 was set to 0.2. For similarity detection, we set K to 100 and t to 1 based on our data scale and observations. The large-scale vector searching tool Faiss (Johnson et al. 2019 ) was applied to speed up vector retrieval.

In book-level aggregation, we found that diverse genres have variant punctuation styles, disturbing the total number of sentences. After observation, we found that in this dataset, the number of sentences with at least two clauses is relatively stable. Therefore, we set the number of sentences n i of the book B i to the number of sentences with at least two clauses.

In community-level inference, r ′ was set to a value that makes implicit intertextuality one-fifth of explicit intertextuality. \(x_i^\prime\) and I im were clipped with a ten-fold mean. In the modelling after adding era-text, the information propagation between era-text nodes was blocked to evaluate each era independently.

As a result, the detection module identified over 411,000 pairs of similar sentences between classics and 2,209,000 pairs between classics and era-text. An intertextual association graph was built from these pairs.

Manual evaluation of text-level detection

Note that in this corpus, each sentence has millions of intertextual candidates from books on diverse topics. As a result, the likelihood of any two sentences being intertextual is extremely low. Building a hand-labelled test set to evaluate the recall rate is nearly impossible. Therefore, we manually evaluated the accuracy rate with the same number of recalled sentences.

We invited three people with graduate degrees and research experience in Chinese classical literature to conduct the manual evaluation. The evaluators were asked to assess the intertextuality of each detected sentence pair. If the two sentences share a similar meaning, topic, or structural style, give 1 point. Otherwise, give 0 points. We took the single-pattern methods as baselines. We used the SIMCSE (Gao et al. 2021 ) model to detect the same number of pairs at the sentence and clause levels, respectively. One pair is randomly sampled from each book in the dataset of classics. There are three groups with 133 pairs each.

The results are shown in Table 3 . The average accuracy of our proposed multi-pattern detection model is 82.22% ( Pearson ’ s r  = 0.74), while the single-pattern baseline is 73.70% and 45.92%. It suggests that the multi-pattern design can improve intertextuality detection performance.

Ablation of community-level inference

We performed an ablation study on a specific book to validate the designed inference module. Figure 3 shows the intertextual connection between Analects and other classics. To compare the modules fairly, we adjusted the weights r ′ so that explicit and implicit intertextuality have equal status in united intertextuality.

They are mean-normalized, and their standard deviations are given respectively. a Number of similar sentence pairs s . b Explicit intertextual scores I ex . c Implicit intertextual scores I im . d United intertextual scores I .

The number of similar pairs varies widely due to the varying length of books. After aggregation, normalized explicit intertextual scores are obtained. However, some books do not share similar sentences, resulting in vacancies. Implicit intertextual scores are positively correlated with explicit intertextuality. It fills the gap of explicit intertextuality and alleviates sparsity. In addition, the introduction of implicit intertextuality brings smoothness, leading to more robust united intertextual scores ( std  = 0.81) than explicit intertextual scores ( std  = 1.07).

Indicator robustness

A metric that is susceptible to data variance is not ideal. Therefore, we examined these two concerns regarding the intertextual score I :

• Q 1 : Is the intertextual score affected evidently by data size?

• Q 2 : Does the intertextual score decrease noticeably due to language discrepancy in different eras?

For Q 1 , we calculated the correlation between data size and intertextuality with the classic dataset. The two variables used in the correlation calculation are as follows:

Data Size: the number of sentences involved in intertextuality detection for each book.

Intertextual Score: The average intertextual score of each book with all other books.

Our results show that there was no significant correlation between data size and intertextual score ( \(r = - 0.1427,\,P = 0.1025,\,n = 133\) ). Therefore, we considered that the decrease in the H index is not due to data size.

For Q 2 , let us examine some cases. Jin Si Lu of the Song Dynasty (published around 1175) and Chuan Xi Lu of the Ming Dynasty (published around 1472–1529) are two famous works of Neo-Confucianism, which emerged as a continuation of Confucianism thousands of years after its birth. Compared with previous books, is the intertextuality between these two books and Confucianism prominent?

To answer this question, we ranked the intertextual scores between all books and keystone works of Confucianism and observed where these two books are placed. We found that these two books rank highly (1/131, 2/131), even surpassing Confucian books that are more recent to the Axial period. Therefore, we consider that language differences across different eras do not have an obvious impact on the intertextual score.

Through these two examinations, we can conclude that the indicator, intertextual score I , is robust to data variance.

Study 1. Interaction between scholars and schools

At the first level, we discussed the interaction between scholars and schools. Schools can be remoulded by later generations of scholars during their thousands of years of evolution. Confucianism and Taoism were the most influential philosophical schools in ancient China. We examined their evolutionary paths by assessing the preference of their followers through intertextual distributions of literary works. Besides, some literature is controversial or ambiguous in the mists of antiquity. To clarify the true path of cultural evolution, we provided quantitative suggestions for the school attribution of Lüshi Chunqiu and the authorship attribution of the Collected Works of Tao Yuanming .

In the axial age, religion and philosophy transformed drastically in various civilizations. The Hundred Schools of Thought, which arose in the Eastern Zhou Dynasty (500 BC), were the prototype of Chinese philosophy. The enduring and pervasive influence of schools such as Confucianism, Taoism, Mohism, Legalism, and Military make them essential to any discussion of ancient Chinese culture (Sima 1959 ; Ban 1962 ).

Scholars and schools are symbiotic. Scholars were inevitably exposed to mainstream schools of their periods, while the doctrines of schools needed to be passed down to subsequent scholars. In this section, we investigated the interaction between scholars and schools through the intertextual associations of their literature. We calculated the Tendency Index T between 125 ancient Chinese classics and the five schools mentioned above. This index shows the ideological tendency of a particular collection of texts toward each school. The schematic diagram of this index is shown in Fig. 4a , and the details of its design are as follows.

a Calculation of Tendency Index T . b Calculation of Historical Status Index H .

Based on the consensus of Chinese philosophy (Feng and Bodde 1948 ), we selected the keystone works as the benchmarks for each school. We first calculated the average intertextual score between a book and the keystone works of each school. The Tendency Index is defined as the ratio of the average intertextual score with a specific school to its means across all schools. Suppose there are books \(B = \{ B_1,B_2 \ldots B_m\}\) and schools \(S = \{ S_1,S_2 \ldots S_v\}\) . The intertextual score between any two books B i and B j is I ij , which can be obtained from the node features of the association graph. For the book B i and the school S k , the school S k has l keystone works, T ik is calculated as follows:

T ik reflects the tendency of book B i for school S k compared to other schools. When T ik  > 1, Book B i has an above-average preference for school S k .

We also examined the significance of text-level intertextuality. Specifically, we investigated whether sentences from a specific book have a significantly greater probability of being detected in the keystone works of a school than the average of other schools. Considering that these books typically contain a large number of sentences \(({\text{Median}} = 2739)\) , we employed a one-tailed Z -test statistic. This statistic was constructed from the similar sentence pairs detected. Suppose there are books B i and B j containing n i and n j sentences after data processing. There are s ij distinct similar sentence pairs detected between them. For book B i and school S k , the calculation of test statistic Z is as follows:

We set the significance level α to 0.05. With the Tendency Index and P-value , we developed quantitative discussions on the scholar-school linkages.

Evolutionary path of philosophical schools

The schools in ancient China were constantly evolving as scholars reshaped previous theories. As acknowledged in the history of Chinese philosophy (Feng and Bodde 1948 ), the original Taoist philosophy inspired the Taoist religion and Wei Jin metaphysics, while Neo-Confucianism inherited the theories of Confucianism. This section validates these evolutionary paths of Taoism and Confucianism quantitatively.

Taoism was a philosophical school that mainly advocated conformity to nature. Taoist religion evolved from Taoist philosophy, developing into the most prominent native religion until now (Raz 2012 ). The representatives of Taoist philosophy, Laozi and Zhuangzi, were revered as the founder and patriarch of the Taoist religion respectively. Figure 5a shows the Tendency Index of two Taoist religious classics, Cantongqi and Wen Shi Zhen Jing . They were significantly inclined towards Taoist philosophy ( Cantongqi , \(T = 2.48\) , \(P = 0.0142\) , \(n = 529\) ; Wen Shi Zhen Jing , \(T = 2.62\) , \(P = 0.0019\) , \(n = 879\) , for Taoism). It demonstrates the consistency between Taoist religion and Taoist philosophy in their evolution.

The dynasty of publication and the corresponding AD years of each book are shown below. The keystone works of each school are listed on the right, including the time of the publication. a Tendency Index of two Taoist classics, Cantongqi and Wen Shi Zhen Jing . And the Tendency Index of the collected works of two scholars, Ruan Ji and Ji Kang. b Tendency Index of two Neo-Confucianism classics, Jin Si Lu and Chuan Xi Lu . c Tendency Index of Lüshi Chunqiu . d Tendency Index of the Collected Works of Tao Yuanming . And the Tendency Index of its widely accepted and controversial parts.

Apart from the religious re-creation, Taoism inspired a new school of philosophy. Wei Jin metaphysics, a variant of Taoist philosophy, arose during the Three Kingdoms period (220–280) and flourished in the Jin Dynasty (266–420). Ruan Ji and Ji Kang were two representative scholars. Figure 5a shows the Tendency Index of their collected works. Compared with the other four schools, scholars of Wei Jin metaphysics were closer to the theories of Taoism ( Collected Works of Ruan Ji , \(n = 1590\) ; Collected Works of Ji Kang , \(P = 3.57e - 06\) , \(n = 2209\) , for Taoism).

This kind of transformation also occurred in Confucianism. Confucianism, which originated in 500 BC (Yao 2000 ), had an extensive impact on ancient Chinese culture and spread throughout East Asia. Over millennia, the philosophy evolved, and Neo-Confucianism became the new representative of Confucianism in the Song Dynasty (960–1279) and Ming Dynasty (1368–1644) (Bol 2008 ). Jin Si Lu and Chuan Xi Lu , written by Zhu Xi, Lv Zuqian, and Wang Yanming, were two of the most famous classics. Their Tendency Index is shown in Fig. 5b . The significant intertextual connection between the two works and Confucianism confirms their inheritance ( Jin Si Lu , \(T = 2.84\) , \(P = 1.01e - 13\) , \(n = 2914\) ; Chuan Xi Lu , \(T = 3.36\) , \(P = 2.33e - 15\) , \(n = 2495\) , for Confucianism).

Controversial literature attribution

Because of its antiquity, the information of some classics has become vague over thousands of years of circulation. Attributing ancient literature to appropriate schools and original authors has been a long-discussed issue in Chinese cultural studies, and in recent times, scholars have embarked upon quantitative investigations in this regard (Zhu et al. 2021 ; Zhou et al. 2023 ). In this section, we provide quantitative suggestions for controversial literature based on its intertextual distributions among schools.

Appropriate school attribution could contribute to the study of the influence and evolution of cultural thought. For example, Lüshi Chunqiu , an encyclopedic classic from the Warring States Period, was compiled in 239 BC with the support of the politician Lü Buwei. It brought together doctrines from various schools. However, there is no conclusion about its predilection among them (e.g., Syncretism theory, Taoism theory, and Confucianism theory (Chen 2001 )).

In Fig. 5c , our quantitative modelling result shows that Lüshi Chunqiu is a syncretic work ( \(T = 0.78\sim 1.43\) ) led by Taoism ( \(T = 1.43\) , \(P = 0.0004\) , \(n = 6118\) , for Taoism). It indicates that the editors have done a syncretic compilation of the theories in that period, with a slight inclination toward Taoism.

The variation of intertextual distributions can also be applied to controversial authorship attribution. Some ancient books were published in the name of famous scholars, but the real authors maybe someone else. However, the creations by different people have their own styles. The thought divergence between the real celebrity and their impostor could be implied in the intertextual variation of their works.

For example, Tao Yuanming is widely recognized as a representative of Chinese individual liberalism (Swartz 2008 ). He refused to serve the government and pursued a pastoral life. His yearning for a free life was depicted in his poems, which is highly consistent with the claim of Taoism. He is considered to have a strong predilection for Taoism and was slightly affected by Confucianism. Therefore, it is puzzling to find that the Tendency Index shown in Fig. 5d indicates a significant predilection for Confucianism in the Collected Works of Tao Yuanming ( \(T = 2.41\) , \(P = 0.0007\) , \(n = 2119\) , for Confucianism).

Further investigation revealed that the authorship of some parts of the Collected Works of Tao Yuanming is controversial. The version compiled by Xiao Tong (501–531) did not contain Five Sets of Filial Piety Biographies and Book of Ministers , while the version of Yang Xiuzhi (509–582) added them. Yang Xiuzhi mentioned in the preface that Xiao Tong’s version was missing these two parts, so he added them to prevent them from being lost in future generations.

However, later scholars gradually became suspicious of these two parts. The most famous one is the assertion in Siku Quanshu (Ji 1997 ). For its “self-contradictory” and “meaningless”, Siku Quanshu declared that Five Sets of Filial Piety Biographies and Book of Ministers were counterfeit. This view remains popular today, owing to the authority of Siku Quanshu .

To find clues to this dispute, we compared the intertextual distributions of the widely accepted and controversial parts. We divided the Collected Works of Tao Yuanming into two parts: collection 1 included Five Sets of Filial Piety Biographies and Book of Ministers , while collection 2 contains the remaining works. The Tendency Index for the two collections is shown in Fig. 5d . The “Tao Yuanming” of collection 1 exhibited a significant preference for Confucianism ( \(T = 3.02\) , \(P = 0.0002\) , \(n = 436\) , for Confucianism), while the “Tao Yuanming” of collection 2 inclined towards Taoism ( \(T = 2.45\) , \(P = 0.0658\) , \(n=1683\) , for Taoism) and has an above-average preference for Confucianism ( \(T = 1.51\) , \(P = 0.1985\) , \(n = 1683\) , for Confucianism). The modelling result of collection 2 is consistent with the actual behaviours and mainstream cognition of Tao Yuanming.

The Tendency Index shows an antithesis between the controversial sections and other parts in terms of their intertextual connections to Confucianism and Taoism. Considering the life experience of Tao Yuanming, our finding lends further support to the speculation: Five Sets of Filial Piety Biographies and Book of Ministers were forged by others in the name of Tao Yuanming.

Nevertheless, it is also worth considering that these two books were intended as textbooks for family education. If we treat them as the original works of Tao Yuanming, the intertextual discrepancy in the results reveals the divergence between the personal pursuits of Tao Yuanming (Taoism) and his aim to educate future generations (Confucianism).

Study 2. Vicissitudes of schools

At the second level, we studied the rise and fall of schools in different eras and domains. Scholars have employed character co-occurrence (Yang and Song 2022 ), syntactic patterns (Lee et al. 2018 ), and topic analysis (Nichols et al. 2018 ) to quantitatively measure the grammatical and ideological connections in ancient Chinese literature, thus supporting research into cultural differences and thought evolution. In this section, we studied cultural phenomena through diachronic and field-specific intertextual distributions. We investigated quantitative evidence for the connections between historical events and school status. Besides, schools’ claims have their own focus, making them favoured by different aspects of culture. We quantitatively discussed the status of Confucianism and Taoism in various cultural domains.

To achieve this, we divided ancient China into 12 eras and built an era-text corpus from history books and anthologies. The era-text corpus is a comprehensive collection of literature from official and folk sources, allowing them to be taken as indicators of the prevailing thought of that time. The era-text was then classified into 12 eras and added to the intertextuality modelling. For a specific collection of text, its intertextual association with the era-text implies its popularity in that era. The Historical Status Index H was designed to measure the school status in each era. The schematic diagram of this index is shown in Fig. 4b , and the details of the calculation are as follows.

We first calculated the average intertextual score between the keystone works of each school and the era-text in each era. For each school, its index H is defined as the ratio of the average intertextual score in a specific era to its mean across all eras and schools. Let \(S = \{ S_1,S_2 \ldots S_v\}\) denote the set of schools, and \(E = \{ E_1,E_2 \ldots E_f\}\) denote the set of eras. For a given school S k and era E e , where school S k has l keystone works and era E e has c books in era-text, the Historical Status Index H ke is calculated as follows:

H ke reflects the status of school S k in the era E e . If its mean value \(\bar H\) in era E e exceeds 1.00, it suggests that the school had an above-average influence in era E e . The Historical Status Index of five schools in 12 eras is shown in Fig. 6 .

The timeline gives the name of each era, with the approximate AD years of its beginning and end. The histogram shows the H of each school, while the line chart indicates its mean value in each era.

School transformation in history

As society changed, schools of thought experienced booms and declines in Chinese history. Historical events like wars, policies and regime changes have impacted the school’s evolution. In this section, we investigated the quantitative textual evidence of these connections through the diachronic changes in their intertextual distributions.

The results show that the keystone classics of these five schools were highly intertextual with era-text within about a thousand years ( \(\bar H > 1\) ) and then gradually decreased ( \(\bar H < 1\) ). Although the original texts created during the axial period were still classic, they gradually became unsuitable for the new era (Feng and Bodde 1948 ). This could be the reason for the decrease in the \(\bar H\) index. Throughout the millennium of prosperity, we can observe the connections between school transformation and historical events.

The popularity of the school of Military was affected by the division of the country in the Three Kingdoms period (220–280), when China was divided into three comparable kingdoms. The country was in turmoil, and wars often broke out between these three kingdoms. Against this background, the school of Military, which was themed on the philosophy of war, reached its heyday ( \(H = 3.15\) , for Military in the Three Kingdoms period).

The linkage between political events and the prosperity and decline of the school manifested in the quantitative results. Confucianism was a school of humanism (Juergensmeyer 2005 ), while Legalism was a school that advocated legal institutions. Some scholars believe that ancient China was influenced by both Confucianism and Legalism (Zhou 2011 ; Zhao 2015 ). In Qin (221 BC - 207 BC) and Han (202 BC - 220) Dynasties, favour from the government made two schools stand out rapidly. The Shang Yang Reform (356 BC & 350 BC) and the advocation from the emperor Qin Shi Huang brought Legalism to a peak in the Qin Dynasty ( \(H = 3.67\) , for Legalism in the Qin Dynasty). However, this brief prosperity ended with the demise of the Qin Dynasty. The policy implemented in the Han Dynasty, which banned other philosophical schools and venerated Confucianism, caused the drop of \(\bar H\) and made Confucianism ( \(H = 1.73\) , for Confucianism in the Han Dynasty) exceed others ( \(H = 0.94\sim 1.23\) , other schools in the Han Dynasty). This advantage continued since then, and Confucianism had long been the dominant philosophical school in ancient China.

School influence in various domains

Confucianism and Taoism were representative schools of collectivism and individualism in ancient China (Munro 1985 ). As the two most prominent native philosophical schools, Confucianism and Taoism have often been compared. In this section, we studied the influence of Confucianism and Taoism through their intertextual distributions among various cultural domains.

Confucianism placed greater emphasis on family and social relations, whereas Taoism focused more on nature and the spirit. For most of the time since the Han Dynasty (202 BC - 220), Confucianism was far superior to other schools of thought. Nevertheless, there was an anomaly in history. As shown in Fig. 6 , Taoism experienced a revival from the Three Kingdoms period (220–280) to the Jin Dynasty (266–420). In the Jin Dynasty, the status of Confucianism ( \(H = 2.14\) , for Confucianism in the Jin Dynasty) and Taoism ( \(H = 2.10\) , for Taoism in the Jin Dynasty) was very close. It stemmed from the collapse of the Han Dynasty, which advocated Confucianism. During this period, people sought to find a successor from the theories of other schools (Feng and Bodde, 1948 ). In the background, Wei Jin metaphysics developed from Taoism theory. However, this prosperity did not last long. After the brief revival, Taoism decayed while Confucianism remained the mainstream.

In addition to the diachronic investigation, we discussed the status of Confucianism and Taoism in different cultural domains according to their intertextuality with texts on related topics. History books in ancient China tended to record political events. Therefore, we took the intertextual associations with history books to indicate the political status of a school. The average Tendency Index of history books is shown in Fig. 7a . We also test whether the Tendency Index of Confucianism exceeds Taoism significantly with a one-tailed paired samples t-test. The distribution of their difference value is shown in Fig. 7b , which corresponds to normal distribution. The significance level α is set to 0.05. Confucianism exceeded Taoism significantly in the political domain ( \(P = 2.22e - 15\) , \(n = 55\) ).

a The average Tendency Index of history books. b Difference value distribution of Tendency Index between Confucianism and Taoism among 55 history books. c The average Tendency Index of 125 classics from various cultural domains. d Difference value distribution of Tendency Index between Confucianism and Taoism among 125 classics. e Tendency Index of 125 classics towards Confucianism and Taoism, sorted by their difference value.

Although Taoism did not replace Confucianism in the political domain, it is comparable to Confucianism in broader cultural communities. We calculated the average Tendency Index of 125 classics from various cultural domains, and the result is shown in Fig. 7c . We test whether their Tendency Index is variant with a two-tailed paired samples t -test. The distribution of their difference value is shown in Fig. 7d , which corresponds to normal distribution. The significance level α is set to 0.05. There is no significant difference between Confucianism and Taoism among these classics ( \(P = 0.8014\) , \(n = 125\) , not rejecting the null hypothesis). Specifically, Fig. 7e shows the Tendency Index of 125 classics towards two schools. Among these classics, Confucianism and Taoism had respective advocacy groups. Books on politics and regulations are highly intertextual with Confucianism, while books on mythology, occultism, and medicine are close to Taoism.

These indicators show that Confucianism has advantages in the political field, while Taoism attempted to surpass Confucianism yet failed. However, Taoism was on par with Confucianism in other fields of ancient Chinese culture. Thus, it is suggested that in ancient China, the political domain was the territory of collectivism, while individualism flourished in the diverse cultural fields.

Study 3. Communication with foreign culture

At the third level, we investigated the communication between ancient China and foreign cultures, with a focus on Buddhism, one of the most influential foreign religions in ancient China. The preaching of Buddhism experienced imitation and integration (Zürcher 2007 ). We started by identifying the native schools that are most intertextual with Buddhism and then discussed the different stages of the infiltration between Buddhism and native Chinese culture.

Although the dissemination of information in ancient times was much slower than it is now, ancient China had extensive communication with foreign cultures. As a result, the cultural evolution of ancient China was not isolated. Buddhism, a religion that originated in ancient India, spread to ancient China during Han Dynasty. Buddhist scriptures were translated into Chinese versions, which were widely disseminated over the next millennia.

In this section, we investigated the preaching of Buddhism in ancient China through the intertextual association between Buddhist scriptures and native classics. We selected the four most influential Buddhist scriptures in ancient China ( Diamond Sutra , Lotus Sutra , Shurangama Sutra , and Avatamsaka Sutra ) as the keystone work of Buddhism and added them to the modelling. The diachronic changes of intertextual distributions reveal the evolution of cultural integration in different stages. The topics of intertextual associations show the commonalities between Buddhism and native culture.

Analogue in native cultural groups

As a newly introduced religion, Buddhism inevitably interacted with the existing native cultural groups in its preaching. Taoist religion, which developed from Taoist philosophy, was the dominant indigenous religion in China. Scholars generally believe that Buddhism and Taoism imitated each other in many ways (Mollier 2008 ), including textual scriptures, image symbols, and organization. In this study, we concentrate on textual scriptures. Figure 8a shows the Tendency Index of Buddhist scriptures towards the five native schools. Taoism is the closest native school as expected ( \(T = 1.83\) , \(P = 0.0131\) , \(n = 62693\) , for Taoism).

Besides, we found that Buddhist scriptures borrowed language expressions from existing Chinese terms in the translations of Buddhist concepts. Figure 8b shows two cases from the detected intertextual sentences. The term “Amrita” (meaning “immortality drink”) was borrowed from the word “甘露“ (gan lu, meaning “sweet dew”) when translated into Chinese. This word refers to “rain” in the native Taoist classic Tao Te Ching . Similarly, the Chinese translation of “Sattva” (meaning “sentient beings”) employed the term “众生“ (zhong sheng, meaning “all living beings”), as found in the Taoist classic Zhuangzi .

Evolution of cultural integration

Apart from the philosophical schools, intercultural communication manifested in various aspects of society. Therefore, we expanded the horizons to broader cultural domains. In this section, we compared the intertextual associations between Buddhism and native literature before and after its introduction.

During the Jin Dynasty (266–420), these four Buddhist scriptures were translated into Chinese, paving the way for Buddhism to flourish in ancient China. After separating the texts before and after AD 420, we ranked native classics based on their intertextual scores with Buddhist scriptures. The top 10 classics are shown in Fig. 8c and e . We also juxtaposed Buddhism with five native schools and calculated the Tendency Index of these classics.

Before the introduction of Buddhism, its similar native classics often focused on myth and religion, implying that the Chinese version of Buddhist scriptures retained the original theme. Besides, it may attribute to their assimilation of the corresponding native literature in the Chinese translation of Buddhist scriptures. Specifically, three similar cases from the top three classics are shown in Fig. 8d . The Chinese version of Buddhist scriptures shares similar phrases with native myths in their discussions of mysteries, including the control of ghosts and gods, and the description of the mysterious phenomenon of “burning day and night”. It also mimicked the language expression of native religious discourses. For example, the description of the choice between justice and evil is highly consistent between Cantongqi and Avatamsaka Sutra .

After the introduction of Buddhism, Buddhist doctrines diffused into various domains of native culture. Compared to the previous period, there was an overall increase in the Tendency Index of Buddhism among the top 10 classics. It indicates the promotion of Buddhism’s influence on Chinese culture. One notable change is the emergence of three native Buddhist works. It symbolizes that Buddhism built its advocacy group in ancient China. These works remoulded Buddhism in a new cultural environment with localized doctrines. In addition to expanding its own religious territory, Buddhism integrated into other native religions (Zürcher 1980 ). For example, the top 1 work shown in Fig. 8e is the native religious classic named Xuan Zhu Lu , which deeply absorbed Buddhist theories. In terms of missionary targets, the preaching of Buddhism was not limited to ordinary people and even reached the supreme ruler, such as the Emperor Wu of Liang (464–549), which ranks third in Fig. 8e . With the advocacy of the emperor, the Liang Dynasty was the heyday of Buddhism in the Southern Dynasty (Strange 2011 ). For details, Fig. 8f shows three similar cases from the top three native classics after the introduction of Buddhism. Religious concepts from Buddhism were mixed into Chinese as new words (e.g., ten directions, immeasurable and Buddha). India’s “Ganges River” flowed into ancient China along with Buddhist scriptures.

Online platform

In this paper, we focused on the theme of cultural evolution. However, there are many other meaningful findings in our modelling results, which await further explanation by relevant scholars. Therefore, we have developed an online platform ( http://evolution.pkudh.xyz/ ) featuring an interactive visualization system that displays the corpus and intertextual sentences. This platform shows millions of intertextual cases detected in this work and provides support for further data analysis. Even researchers without programming backgrounds can gain valuable insights into our work and develop further studies using this convenient tool. We gave several screenshots of the platform in Fig. 9 .

a Intertextual sentence browsing from corpus. b Intertextual sentence statistics and visualization within a custom collection. c Visualization of intertextual sentences distribution among different chapters of a book within a custom collection.

With the leap forward of big data and AI technology, computer-assisted cultural studies have expanded in both scale and depth. Intricate cultural problems can be discussed quantitatively with the support of large-scale data. In this paper, we used digital methods to quantify the cultural evolution of China over the past thousands of years within a large-scale corpus of ancient literature.

We gave validated results for several acknowledged cultural phenomena. The two evolutionary paths of Taoism and Confucianism, inspiring new branches of school and migrating to religious fields, were confirmed by intertextual associations. Besides, we provided quantitative evidence for the connections between the schools’ status and several historical events. It shows the intertwining of philosophical schools and politics in ancient China.

Through our analysis, we gained quantitative insights into some long-debated cultural problems. For literature with controversial school attribution, our findings suggest that Lüshi Chunqiu is a syncretic work headed by the theory of Taoism. As for literature with controversial authorship attribution, we revealed that Five Sets of Filial Piety Biographies and Book of Ministers are divergent from other works of Tao Yuanming in ideological preference. In the comparison between Confucianism and Taoism, we propose that collectivism represented by Confucianism was mainstream in the political domain, while individualism represented by Taoism was active in extensive fields of ancient Chinese culture.

Furthermore, we investigated intercultural communication between Buddhism and Chinese native culture. The results suggest that the influence of this foreign culture evolved at different stages, from imitation to integration. In the early days, Buddhism imitated similar aspects of native culture to ease resistance (Kohn 1995 ). After the initial prosperity of Buddhism in China, it was remoulded through localized Buddhist works. As time went by, Buddhism became a part of the local culture. It was evident in various cultural domains of ancient China.

Our study demonstrates that hierarchical intertextuality modelling is a promising tool for cultural analysis within the large-scale corpus. However, there are still limitations in quantitative intertextuality research on Chinese literature. The evolution of language over time presents challenges in detecting intertextuality between ancient Chinese and modern Chinese is challenging. Besides, intercultural communication from different languages requires cross-lingual detection, which is still an area that remains underexplored.

This research represents an innovative attempt to study the evolution of Chinese culture from a digital perspective. It provides new insights into the interpretation of ancient Chinese culture and raises important questions for further exploration: How did ancient Chinese culture evolve into its modern form? What was the impact of global culture on this process of evolution? To conduct more comprehensive research, interdisciplinary and intercultural collaboration is necessary.

Data availability

The open-sourced code, data catalogue and online platform can be found here: https://github.com/CissyDuan/Evol . The textual data can be downloaded from open websites: http://www.xueheng.net and http://www.daizhige.org/ . The pre-trained model is accessed from an open-sourced model: https://github.com/ethan-yt/guwenbert .

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This research is supported by the NSFC project ‘the Construction of the Knowledge Graph for the History of Chinese Confucianism’ (Grant No. 72010107003).

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Evolution of Indian cuisine: a socio-historical review

• Vishu Antani 1 , 2 &
• Santosh Mahapatra   ORCID: orcid.org/0000-0002-0077-2882 3  

Journal of Ethnic Foods volume  9 , Article number:  15 ( 2022 ) Cite this article

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This paper aims to present a socio-historical review of Indian cuisine and its evolution. Based on a thorough analysis of a wide range of documents, the investigation focuses on four eras, each having a significant and lasting effect on Indian cuisine. Parallels have been drawn to modern Indian cuisine wherever possible. This study follows the advances in Indian cuisine through the prehistoric era, into the Vedic era, where religious scriptures, especially the Hindu scriptures, affected what was cooked during the time, followed by the invasion of the subcontinent by the Mughals. Finally, the effects of European colonization on Indian cuisine are critically analysed and an attempt is made to examine threads, if any, connecting culinary preferences of people across these phases. The results of the analysis indicate the intriguing impact of intricate connections among several sociocultural factors on the evolution of Indian cuisine. At the end of the paper, a few areas are identified for future exploration.

Introduction

This paper tries to trace the history of Indian cuisine, a topic that remains surprisingly underexplored in the gastronomy literature. Unique in its standing and mysterious in many ways, as claimed by the famous novelist E. M. Forster [ 20 ], India remains a curious case of interest. Among other things, its food culture has a vibrant history filled with tasty twists and turns, which is in agreement with the claim that Indian culture can be explored through its foods. In fact, across many cultures, one can historically trace sociocultural reasons behind culinary choices [ 1 ]. Tracing the history of food in India is pivotal to understanding Indian cuisine. For a very long time, each of India’s conglomeration of kingdoms had a royal court tended by chefs and innovations in food took place in these kitchens; new dishes were created, and old dishes were recreated. In the kitchens of commoners, cooks or housewives would try to recreate those dishes using ingredients at their disposal. Modern Indian cuisine is the result of the flavourful adventure India has been through, and in this adventure, one can socio-historically trace the interaction between personal and social choices, the divine and the mundane obligations [ 8 ], and the karma and its repercussions. The beginnings of India’s food history, especially during Vedic times, were driven by the attributive property of Guṇa , a Hindu philosophical concept referring to quality or peculiarity. Sattva , rajas , and tamas , the three Guṇas , were believed to have manifestations in the form of ‘vegetarian, spicy and carnivorous’ [ 6 , 43 ]. Even the philosophy of ahimsa shaped the food choices of people in ancient Hindu civilizations [ 42 ]. This philosophical tradition underwent a series of changes, challenged by the geopolitical and sociocultural changes that came to India with the Mughal invasion and European colonization. In this paper, we have presented a brief historical review of Indian cuisine that covers a period starting with the prehistoric era and ending with European colonization and have demonstrated how the evolution of food in India is deeply enmeshed in various factors like culture, religion, ethnicity, financial background, social class, and availability of and access to raw materials.

Methodology

We collected data for the study through thorough document analysis. We surveyed ancient religious scriptures from archives, newspapers, memoirs, travelogues, research articles, poems, history books and books written on food cultures, and made comprehensive notes. All the notes were verified through information collected from more than one source. Then, we searched for patterns in the data and organized the details under the four most frequently found patterns in food culture: Prehistoric, Vedic, Mughal, and European colonization. For each era, we arranged the information in a systematic manner which mainly focused on discussions on the evolution of food through a close interplay between sociocultural factors and the availability of raw materials required for the preparation of food.

Results and discussion

Prehistoric era.

Prehistoric food habits in India still remain partly shrouded in uncertainties [ 33 ]. Like literature, cuisines from a different era have proved difficult to understand. Written recipes may be one of the sources of determining the type of food eaten by the people then, but language still remains a barrier [ 11 , 16 , 32 ], which is especially true in the case of prehistoric India. What we know about people’s food habits is an episodic story of survival and a transition to becoming an agrarian community [ 33 ]. The information is obtained through anthropological evidence and cave paintings found in various parts of Indian states such as Rajasthan, Gujrat and Punjab [ 1 ].

Bones of cattle, goats, sheep, buffalo, and pigs have been found at Harappan excavation sites. While cattle, goats, and sheep may have been domesticated, it is also possible that they were hunted or raised for their flesh. Cattle farming was mainly restricted to the Zebu cattle, native to the Indus Valley region (see Fig.  1 ) [ 21 ]. Bones of wild animals like boar, deer, and gharial have also been found. Along with these, the remains of fish and fowl were also discovered. Grilling was the preferred way to cook meat. The animal would be roasted whole on an open fire or cut into bite-size chunks [ 33 , 35 , 36 ]. These chunks were pierced onto a skewer and roasted, like modern-day tikkas . The animal was lodged onto a stick, which could be rotated, and placed over the fire. Pottery from prehistoric times has revealed the presence of bowls with wide mouths, which indicates the use of boiling as another method of preparation [ 24 ].

Source : Wikimedia Commons https://commons.wikimedia.org/wiki/File:Indus_Valley_Civilization,_Mature_Phase_(2600-1900_BCE).png

A map of the Indus Valley Civilization dating back to 2600–1900 BCE. It was a Bronze Age civilization and one of the most widespread civilizations in Asia, spanning modern-day north and north-western India, most of Pakistan and Afghanistan.

Pulses and grains were consumed by the people of the Indus Valley [ 23 ]. Various pulses like chickpeas, red lentil, and green peas were introduced from Western Asia to the Indus Valley in around 4000 B.C. Barley and wheat were also found along with the pulses at archaeological sites. Their use carried over into the Vedic era, when barley was one of the primary grains. Rice was consumed much later.

Several stone tools of varying sizes have been discovered at excavation sites. Large saddle querns have been found, probably used for grinding grains to make flour. Two types of stones may have been used with the querns: a small one for rolling, like a rolling pin, and a larger, heavier one to pound on materials. Curry stones would have been used to grind spices. However, it remains unclear whether the spices were grown within the region or obtained through trade or barter.

Many important texts on medicine, yoga, literature, religion, etc., from the Vedic era, such as Patanjali’s Yoga Sutras, Caraka’s Caraka-Saṃhitā, and  the Upanishads, have discussed foods and their impact on the human body. The Vedas have emphasized the connection between spirituality and the choice of food without relinquishing the need for taste and health [ 27 ]. Recent research throws light on linguistic similarities of food items consumed across cultures making it slightly easier to connect the finer dots of Indian cuisine from the Vedic era [ 1 ]. The decline of the Indus Valley Civilization was followed by the Vedic period. During this period, the north-western region of the Indian subcontinent was occupied by Indo-Aryans. Most of the Aryans then were pastoralists. Milk provided for their food and other products like butter and curd.

The Aryans considered food to be a gift from God and a source of strength. In the four Vedas , Rigveda , Samaveda , Yajurveda , and Atharvaveda, there are various mentions of the grains used during those times [ 38 ]. Initially, barley was a staple food of most Aryans . Cultivation of other crops such as wheat, sugarcane, and millets followed. Lentils, mainly red, green, and black, were also grown. Later, the Aryans added rice and other cereals to their diet [ 13 ].

Sweetcakes called apupa have been mentioned multiple times in the Vedas . Apupa or Malpua (see Fig.  2 ), as it is called today, was made with barley flour. The batter would be flattened into cakes and fried in ghee. Before serving, the fried cake was dipped in honey. Many variations have developed over time, and malpua is now paired with various condiments. Today, the batter is made with refined wheat flour or semolina flour and milk. Like the Vedic version, the batter is spread into flat cakes and fried in ghee. Instead of honey, the cakes are soaked in sugar syrup flavoured with saffron or jaggery (cane sugar) syrup. They are topped with chopped almonds and pistachios and paired with rabri , a sweet made by boiling milk at low heat with sugar and spices.

Malpua and Rabri: Malpua is an Indian sweet fried pancake, made from a batter of wheat or barley flour. It is fried in ghee and then placed in sugar syrup. Souce : Shutterstock https://www.shutterstock.com/image-photo/malpua-traditional-indian-sweet-pancake-coated-1035620161

Fried barley called dhana was not just eaten as food but was also offered to the gods. Pulverized barley seeds, called saktu , have been mentioned in the Vedas. Sattu , a flour made from ground pulses, is used in many kitchens today, and it may have roots in saktu . A porridge called odana was made by boiling barley in milk. Yet another preparation called karambha had yogurt mixed with barley, and it is still popular in parts of Gujarat.

Animal meat was presumably used by Hindus in what is now Punjab. Cows, goats, buffalo, and bulls were killed for their meat, and slaughterhouses have been mentioned in the texts. The practice of eating horse flesh was introduced by the Aryans, but its consumption did not seem to settle well with the natives. However, there have been accounts of slaughter of cows and bulls for their meat [ 24 ]. Meats were mostly roasted in clay ovens or on spits over charcoal. The consumption of meat as a survival strategy can be contrasted with the current debates about meat eating and its consequences [ 46 ].

Sesame seeds were important as both food and in rituals. As food, they would be cooked with vegetables, added to bread, or crushed to extract oil. Turmeric, pepper, and mustard seeds were used as flavourings. Various fruits and vegetables are mentioned in the Vedas including the bael fruit (Aegle marmelos), mangoes, dates, lotus stalks and roots, gourds, jujubes, and water chestnuts. These are not very different from the ingredients used in Indian kitchens today.

Alcoholic beverages existed in Vedic times, as suggested by the mention of sura in the Rigveda but disapproved for consumption. The drink has been mentioned only in a few places, and at times, in conjunction with soma . Soma was considered the drink of Gods and an elixir. The plant from which the drink was made has remained a mystery, but historians have speculated candidate plants [ 34 ]. It could be a type of millet, called ragi . Ragi is still used to make marua , an intoxicating drink in the Eastern Himalayas. Cannabis could also be the plant referred to in the texts which was used for preparing the soma drink [ 4 ]. Yet another suggestion is the fly agaric mushroom, because of its intoxicating effect when consumed [ 51 ]. Soma was prepared and offered to gods by Brahmans [ 17 ]. It is unclear as to whether or when fermentation took place [ 19 ]. There is debate surrounding the effects of soma [ 7 ]. While one branch of research views the description of the drink in the Vedas as intoxicating, other researchers argue that it was simply exhilarating, not intoxicating [ 44 ].

An important aspect of the social system in India that determined food culture and consumption among Indo-Aryans was the caste system [ 40 ] which can be traced back to around 1000 BC. The structural embodiments of the caste system, a legitimised practice of inequity believed to be inherited through one’s parentage and occupation, engendered a sense of purity in the culinary and consumption habits of people. Brahmins, the upper caste priests, often adhered to vegetarian food habits led by the philosophy of sattva . In contrast, Shudras, the lowest group in the caste ladder, engaged in eating animal meat and other kinds of tamasic habits. The possibility of upper caste politics to portray certain kinds of food habits as superior to others could be indeed the case. It is possible that the caste system was strengthened with the help of systemic food demonisation. Unfortunately, the caste system continues to shape food habits even in present times [ 39 ].

The descriptions about the culinary culture around 500 BC are not separately dealt with in any treatises as such. Rather, one can find these details in the memoirs of foreign writers and philosophers who visited India during those times. Megasthenes, an ambassador of Alexander and Chinese visitors such as Fa-Hsien and I-Ching, and Xuan Zang, a learned monk from China, have vividly described sociocultural practices including food and drinks [ 1 ].

Muslim rulers who were based in Sind (currently in Pakistan) started invading India after 700 AD with a desire to have access to the material riches the country possessed. In the consecutive millennia, continual attempts were made by several warlords such as Mahmud of Ghazni, Muhammad of Ghor, Bakhtiyar Khalji, and so on. However, no Muslim empire could flourish and sustain till Mughals came to India in the sixteenth century [ 22 ]. With Mughals came a flood of culinary inspirations and innovations that remain highly influential in the culinary practices of India [ 29 ]. Indian food culture, much like architecture, art, music, and language, would be unthinkable without considering the Mughal influence [ 10 ]. The predecessors of the Mughals, the Afghans, had brought with them a lot of culinary wealth. Afghan flatbreads, called naan, are now common in most Indian households and restaurants. These breads may bear some influence of Egyptian and Mesopotamian breads [ 12 , 15 , 30 ]. Koftas in Afghanistan are meatballs of minced beef flavoured with onions and other spices. Indian koftas are very similar to their Afghan counterparts, but beef is often replaced with other meat. In the southern and eastern regions of India, koftas are made from fish and shrimps. The meatballs are then served in gravy made of broth, milk, and cashew paste. A vegetarian version of the dish replaces the meat with grated vegetables or paneer. Jalebi (see the details in Fig.  3 ) is an Afghan dessert that has placed itself so well in Indian cuisine, that most people often forget its origin. Jalebi is made by frying a batter of refined flour in ghee or oil and then soaking it in sugar syrup, which is often infused with saffron giving jalebi its colour and fragrance. It is served simply with rose petals or alongside rabri . Phirni is one of the recent additions of an Afghan dish into Indian cuisine and is made by boiling soaked rice in milk along with nuts and khoa .

Jalebi is a dessert made by deep frying flour batter in concentric shapes and later dipping in sugar syrup infused with saffron. It is enjoyed across the Indian subcontinent and is known by different names in different places. Souce : Shutterstock https://www.shutterstock.com/image-photo/jelebi-cafe-458778184

Mughlai cuisine, with its rich and spicy curries, and fascination with meat has been influential in shaping modern Indian cuisine. It is important to note that most of the ingredients present in the dishes which masquerade themselves as Mughlai dishes, such as chillies, tomatoes, and potatoes, were unknown to kitchens in India until the late seventeenth or early eighteenth century [ 28 ]. This is the effect of modernization and innovation in the culinary world. Memoirs of rulers, like the Baburnama and the Akbarnama , hold a lot of information about food and eating habits [ 48 ]. Manuscripts of what seem to be recipe books written in Persian have also been found.

Since the Mughals practised Islam, pork was forbidden in royal courts and to other Muslims. Similarly, Hindus frowned upon the consumption of beef and refrained from eating it. The meat of a buffalo seemed the appropriate replacement for beef. Mutton and goat meat were widely consumed, mutton being more common than the latter. Various fowls such as chicken, quail, and ducks were also consumed [ 50 ]. Even today, mutton and fowl meat are the most common meats eaten in India along with seafood like fish and shrimp though politics has not left food alone. In Mughal times, Akbar, the most powerful and influential among Mughal emperors, avoided repeating the mistake of using cows as food which his father Humayun did. Humayun’s men ate beef and that incited Rajputs, the Hindu royal class. Akbar went to the extent of becoming a virtual vegetarian [ 40 ] to please Hindus and maintain his supremacy. However, the emperors that succeeded Akbar could not play the politics of food as wisely as Akbar did.

Mughlai food has a distinct bouquet associated with it. This aromatic aspect of food is brought about by the spices used. Spices were traded between Mughals and other rulers in India. Cumin and coriander were and still are common across India and were probably grown locally. Saffron, a very expensive spice, known for its sweet floral scent was imported from Kashmir. Other spices that added heat like cardamom, pepper, nutmeg, and ginger were brought from Southern India near the Malabar Coast. Turmeric was also brought from there. Interestingly, these regions still maintain a very high quality in producing the spices.

One of the earliest accounts of Mughal cuisine is recorded in the Ni’matnama (the Book of Delights). It was a cookbook compiled by Ghiyath Shah, the Sultan of Malwa, in the fifteenth century. The manuscript is believed to have found its way into the Mughal kitchens in 1562, when Akbar conquered the Malwa Sultanate. The book has detailed recipes, accompanied by miniature illustrations where the king is the central figure of the drawings and notes following dishes, classifying them as being worthy to be served to a king, a poor man’s food, or a king’s favourite. In the Ni’matnama , there are recipes for a variety of sambusas . Sambusas are made with a pastry of refined flour, then filled with a savoury, spicy filling, and finally fried. The filling of sambusas was made from venison [ 49 ]. In samosas (see Fig.  4 ), a modern-day favourite, the filling is made up mainly of potatoes, peas, and onions. Samosas continue to be a crowd-favourite tea snack in modern India.

Souce : Shutterstock https://www.shutterstock.com/image-photo/samosa-sambusa-triangle-fried-baked-dish-1145803784

Sambusas or Samosas are snacks made by frying pastry filled with a savoury filling such as potato, peas, onions, chicken, or lentils. They are one of the most popular tea time snacks in India.

Karhi is a preparation of chickpea flour with yogurt and sour milk which has been mentioned in the Ni’matnama and is still made in a lot of Indian houses. It is served with fried vegetable fritters, called pakoras . The Guajarati variation of karhi is sweetened by adding sugar or jaggery.

A number of dishes mentioned in the Ni’matnama point to Central Asian influence in the food. Sambusa is an example of that. Other such dishes include sikh , kabab , yakhni , shurba , and kufta . Sikh is a cylinder of spiced minced meat on a skewer which is roasted on a tandoor . Yakhni is broth made with spiced meat, while shurba is soup.

Several dishes mentioned in the Ni’matnama , sambusa , sikh , kabab , yakhni , shurba , and kufta (refer to Fig.  5 a) , point to Central Asian influence. Sikh (refer to Fig.  5 b) is a cylinder of spiced minced meat on a skewer which is roasted on a tandoor (refer to Fig.  5 d). Yakhni is broth made with spiced meat, while shurba (refer to Fig.  5 c) is soup. Apart from the common spices, a variety of exotic aromatics were used, such as musk (nutmeg), camphor, rosewater, and ambergris. The use of exotic produce was not limited to aromatics [ 49 ]. Recipes included in the book called for the use of a variety of unique meats, like partridges, rabbits, goats, mountain sheep, pigeons, and quails. Some of these meats are not common in modern Indian kitchens. The use of nutmeg is still prominent in Indian cuisine, but ambergris is obsolete.

a Malai Kofta is a dish made by adding fried balls of potatoes or meat in a rich spiced tomato gravy, topped with fresh cream. Souce : Shutterstock https://www.shutterstock.com/image-photo/malai-kofta-mughlai-speciality-dish-served-1248493381 b Sikh kebab is an Asian preparation where ground lamb or beef meat is wrapped around skewers and roasted on an open flame. Souce : Shutterstock https://www.shutterstock.com/image-photo/seekh-kabab4-154602104 c Shurpa or Shorba is a soup made with meat, vegetables, and spices in broth. Souce : Shutterstock https://www.shutterstock.com/image-photo/shurpa-shorpa-soup-rich-lamb-vegetables-1875768625 d Tandoor is a traditional Indian oven, used to cook naan and tikkas. Souce : Shutterstock https://www.shutterstock.com/image-photo/tandoor-oven-tikka-masala-chicken-naan-1854698554

Rice became more popular than wheat during Mughal times. Khichri is one of the many preparations of rice mentioned the Ni’matnama [ 49 ]. It is a salty porridge made from rice and lentils, usually without spices. The preparation and ingredients used in khichri vary with region, and a lot of varieties of khichri can be found in homes all over India. Since it is nutritious and easy to digest, it is often fed to the ill. The Ni’matnama was not the first historical account of khichri . Selecus, when he was the infantry general to Alexander the Great, revealed that rice with pulses was extensively prepared in India, around 300 B.C.E. Further, it is also mentioned by Ibn Battuta as kishri from his stay in India in approximately 1350. In his description, kishri was a rice dish with mung beans (green gram). Afanasy Nikitin, a Russian merchant and one of the earliest Europeans to travel to India, has described khichri in his narrative— Khozheniye za tri moray (The Journey Beyond Three Seas). Khichri (refer to Fig.  6 a) was popular during the Mughal period, especially under Jahangir [ 47 ]. Ain-i-Akbari , by Abu’l-Fazl ibn Mubarak, mentions seven variations for khichri . There is also mention of the dish along with biryani in the letters of Aurangzeb to his son, Bahadur Shah I.

a Khichdi is a savoury rice porridge with the addition of lentils, vegetables, and spices. Source : Shutterstock https://www.shutterstock.com/image-photo/famous-indian-food-khichdi-ready-serve-1629680485 b Paneer do pyaza is a made of Indian cottage cheese and rich onion-based gravy. Source : Shutterstock https://www.shutterstock.com/image-photo/paneer-do-pyaza-popular-punjabi-vegetarian-1263154453 c Mutton do pyaza is made of lamb meat and seasoned with lots of onions. https://www.shutterstock.com/image-photo/mutton-do-pyaza-two-onions-1226282581

Nushka-i-Shahjahani or Nushka u Namak is another cookbook that has recorded the recipes prepared in the kitchens of the emperor Shah Jahan—from breads and stews to sweetmeats. The ten chapters on food describe the varieties of breads, pottages, meat dishes, and other rice preparations like pulao and zerbiryans . Among the breads, there are recipes for naan (leavened oven-baked flatbread) and roti (round flatbread made from wholemeal flour), but no mention of paratha , a flatbread made of layers of cooked dough (hence, paratha was a later addition to Indian breads). Meat dishes were of two categories: qaliyas and do pyazas . Do pyazas (refer to Fig.  6 b and 6c) got their name from the preparation, where piyaz (onion) is added twice ( do ) in the process. Kebabs, porridges, and omelettes are also mentioned. The end of the collection features preserves like jams and pickles, fried breads, sweets, and puddings. Finally, a few basic techniques for making yogurt and cheese curds are included. Paneer (or Panir ) is an example of a cheese that is extensively used in Indian cooking, especially in vegetarian dishes as a protein source. The cheese curd for Paneer is made by curdling milk with a fruit- or vegetable-based acid (like lime juice).

Khulasat-i Makulat u Mashrubat or Alwan-i Ni’mat or Khwan-i Alwan-i Ni’mat is a cookbook from the times of Aurangzeb’s reign. It has forty chapters, each describing a category of a course, including nan-ha (breads), qaliya and do pyaza (meat curries), sag (greens, refer to Fig.  7 a), bharta (mashes, see Fig.  7 b), dal (pulses and lentils), zerbiryan (layered rice dish), khandawi (savoury cakes made from pulses or gram flour, refer to Fig.  7 d), salan-ha-i hindi (sauced-based dishes of India), khichri , shola (dish with rice, meat, pulses and vegetables), kulthi (sweet sticky rice), thuli (sweet thick preparation with milk and flour), tahiri (rice and meat dish), halim (meaty stew made with wheat and barley, refer to Fig.  8 a), kashk (gruel with wheat and meat), ash (pottages), bara (fried cakes), jughrat wa sikharn (yogurt based dishes), shirbirinj (rice pudding of Iranian origin, refer to Fig.  8 b), firni , faluda (a cold dessert made with milk, rose syrup, vermicelli and basil seeds, refer to Fig.  8 c), panbhatta (a dessert made from fried soaked rice in sherbet), sambusa , puri (fried bread), gulgula and khajur (sweet dumplings), malida (sweet powdery mixture made of dough), shirni (sweetmeats), murabba (jams), and achar (pickles). In addition, there are chapters on basic activities, like shelling coriander. A lot of the dishes are drawn from the earlier cookbooks, notably the breads, meat curries, rice dishes, khichri , and jams and pickles. From the unique preparations mentioned in this book, sag , khandawi , salan , shirbirinj , and phirni are common preparations in Indian kitchens today. Dal has become a staple, especially in vegetarian households as a protein source. Salan , specifically mirchi ka salan (a curry of chillies in peanut gravy, refer to Fig.  7 c), is served with biryani. Faluda has also been popularized and is made on special occasions. It is usually topped with ice cream, and a particular variation calls for the addition of jelly cubes in the sherbet.

a Sarson ka sag with makki ki roti is a special preparation in the north of India, eaten during winters. The curry is made of mustard greens with sauteed onion and garlic. Makki ki roti is basically a flatbread made with ground corn. Source : Shutterstock https://www.shutterstock.com/image-photo/makki-ki-roti-sarson-ka-sag-774826045 b Baingan ka bharta is mashed roasted eggplant that is then sauteed with onions, chilli and spices. Source : Shutterstock https://www.shutterstock.com/image-photo/baingan-bharta-roasted-mashed-eggplant-black-1837782358 c Mirchi ka salan was a popular dish in the Mughal kitchens, eaten with rice preparations. Even today, mirchi ka salan is served with biryani across the country. Source : Shutterstock https://www.shutterstock.com/image-photo/hyderabadi-mirchi-ka-salan-green-chilly-1494080111 d Khandavi is a snack from Maharashtra and Gujarat made by rolling sheets of steamed batter of gram flour and yogurt into bite-sized pieces. Source : Shutterstock https://www.shutterstock.com/image-photo/khandvi-gujarati-patuli-dahivadi-suralichi-vadi-1460691638

a Halim is a slow-cooked stew made up of grains, meat and lentils. It is eaten to break fasts during the month of Ramadan owing to its high nutritional value. Source : Shutterstock https://www.shutterstock.com/image-photo/haleem-spices-herbs-1488278681 b Sher Birinj is a dessert originating in central Asia. It is a rice pudding flavoured with rose water, almonds, cinnamon and cardamom. Source : Shutterstock https://www.shutterstock.com/image-photo/pudding-shir-berenj-afghan-delicious-dessert-1956784180 c Falooda is an Indian dessert made with rose syrup, milk, vermicelli noodles and sweet basil seeds, topped with ice cream. It is commonly sold on the streets by vendors in India. Source : Shutterstock https://www.shutterstock.com/image-photo/pink-sweet-strawberry-falooda-1779708683

Today, cities like Delhi, Lucknow, Kolkata, and Hyderabad boast the best Biryanis . There are a lot of similarities in its preparations across the country—the use of nutmeg, cinnamon, cardamom, and cloves for aroma, adding saffron to colour the rice, and the flavour developed by cooking meat and vegetables with rice. Despite similarities, the differences are evident. The Hyderabadi Biryan i is a spicy preparation, made with Basmati rice, which adds to the aroma, and the flavours of saffron and coconut dominate. It is served with brinjal (eggplant) gravy. The Kolkata Biryani also uses Basmati rice and saffron, but it is the use of meat and ghee (clarified butter) that defines the taste. Potatoes and boiled eggs are also added. The Lucknowi Biryani does not use Basmati rice and uses a paste made from curd and cashew nut along with the aromatics like star anise and nutmeg. The flavour of saffron is not as pronounced, and the Lucknowi Biryani is served with meat gravy.

Mughals ruled the majority of Northern and Northwestern India, but their dominion was not as powerful in Southern India. However, this did not affect their cuisine from spreading into the south. Nobles from courts of Nawabs and other rulers of Southern India travelled to cities under the Mughal rule as diplomats and brought back with them accounts of food eaten and, sometimes, the recipes. The Mughal dishes made in Southern India often had a different set of ingredients or different methods of preparation.

Modern Indian cuisine has incorporated many dishes from Mughal kitchens. With the Mughal invasion, there was an increase in the consumption of rice, which has led to a decrease in dependency on wheat. As a result, rice has become the staple crop in India and Indian cuisine lacks identity without it. Mughals also introduced a wide variety of desserts and sweetmeats. India boasts a wide range of milk-based sweets and desserts, and most of these dishes originated in the Mughal times. Hence, the Mughal era was arguably the time that shaped Indian cuisine as it is today.

European colonization

When Mughals were reigning most of India, the Portuguese slowly started building their colonial empire. Unlike Mughals who intended to own the land and live here, the Portuguese wanted to send profits to their homeland. Spices were one of the main reasons that the Europeans sought a sea route to the Indian subcontinent. It was a powerful product in the trade market, and establishing trade with India was deemed necessary. However, the opulence and wealth of kings and the royal class changed their intentions and colonisation became a priority over trade [ 9 ].

In 1498, Vasco da Gama reached the Malabar Coast of Calicut, a bustling port where the trade of spices, cloth, and a variety of extravagant goods took place between Hindu, Arab, and Chinese merchants [ 12 ]. As the Portuguese Empire expanded, trade of plants, animals, and ideas became common at ports. This was called the Columbian Exchange and was paramount in shaping the cuisine of various regions [ 5 ]. In 1510, after seizing Goa, they made it their capital of Estado de India (State of India). Soon after, spices were grown all along the Malabar Coast for trade. A lot of customs and traditions of Hinduism and Islam were questioned and eradicated [ 31 ]. Those that could not be eliminated were banned. They questioned the refusal of eating pork and beef and introduced a variety of dishes cooked with those meats. They also did away with the Hindu tradition of cooking rice without salt. Growing tulsi (holy basil) was banned.

Cheese was introduced to India by the Portuguese. Bandel cheese is a smoked semi-soft cheese made from cow’s milk. It has a dry, crumbly texture and is one of the very few Western-style cheeses indigenous to India. Another creation of the Portuguese in India is the cheese curd called chhana [ 26 ]. However, it has been argued that the practice of curdling milk had been practised earlier. There is little doubt that a Portuguese cheese similar to chhana was the inspiration for a variety of Bengali sweets like rasgulla and Sandesh (refer to Fig.  9 a and b) [ 41 , 42 ]. Rasgulla is a ball-shaped dumpling made of semolina dough and chhana , which is boiled in light sugar syrup till the syrup steeps into the dumpling. They are then stored in sugar syrup, often flavoured with saffron strands or rosewater [ 45 ]. Rasgulla is known as Rasagola in Odisha, another state in the eastern part of India, and recent research also indicates that Rasagola was prepared in Jagannath Temple in Odisha in the thirteenth century AD. Sandesh , a sweet dish, has been mentioned in medieval Bengali literature, including Krittibas’ Ramayana , but the original dish was most likely very different from the current version. The modern-day Sandesh is presumably made with chhana cheese, but there are other preparations of sandesh without chhana , with just milk and sugar. Normally, sandesh is made by tossing chhana in sugar over low heat. In some cases, use of khejur gur [ 3 ] as a sweeter has been mentioned.

a Rasgulla is a Bengali/Odia sweet made of dumplings of chhena and semolina flour and cooked in sugar syrup. Source : Shutterstock https://www.shutterstock.com/image-photo/bowl-full-rasgulla-pistachios-food-table-1338243359 b Sandesh is a Bengali sweet made by moulding chhena and sugar in different shapes. It can be flavoured with saffron and cardamom and topped with chopped almonds. Source : Shutterstock https://www.shutterstock.com/image-photo/assortment-sandesh-traditional-bengali-sweets-prepared-279675779

The greatest gift to Indian cuisine by the Portuguese was perhaps the Columbian Trade which introduced crops like potatoes, okra, chillies, pineapples, papaya, cashew, peanuts, maize, guava, custard apples, and sapodilla. Tomatoes were not native to India and are believed to have been brought in through trade with other Asian empires, who in turn acquired them through trade with Spain. Most gravies in Indian curries today are tomato-based. Potatoes have been integrated into modern Indian cuisine as if it were a native plant [ 5 ]. India was the third largest producer of potato in 2007 at 26,280,000 tonnes and the third largest consumer at 17,380,730 tonnes in 2005, according to FAOSTAT, Food and Agriculture Organization of the United Nations [ 18 ]. Maize has also been adopted in Indian cuisine. Corn roasted on the cob is a popular street food seasoned with lime, salt, and red chilli powder. Certain west-Indian dishes call for kernels of corn in the recipes and cornflour is used to make a flatbread called makki ki roti (refer to Fig.  7 a) in Punjab.

There is a strong Portuguese influence in Goan cuisine, and many preparations are adaptations of Portuguese dishes. The dish carne de vina d’alhos transformed to vindaloo (refer to Fig.  10 a), which is a sweet and sour, but very spicy pork curry. Unlike the Portuguese preparation, vindaloo was made dry, so that it could be carried on long journeys. Caldo Verde , a Portuguese soup of potato and kale, is made in Goa with the same name, but is spicier, with the addition of ginger and black pepper. Also, kale is replaced by spinach in the soup. Xacuti Chicken (refer to Fig.  10 b) is a Goan curry made with a paste of crushed peanuts, grated coconut, and a complex spice mix. A similar Portuguese dish is Chacuti de Galinha . The Portuguese also introduced a few types of breads, for example, the pau (oven-baked bread rolls). Many street foods across the country use pau today, like the vada pau (fried balls of mashed potato and spices served with different relishes in a bread roll, see Fig.  11 a) and bhaji pau (tomato and potato-based tangy spicy curry served with bread roll roasted in butter, see Fig.  11 b). Bibinca is a Portuguese baked dessert, where seven layers of cake made with flour, sugar, ghee, egg yolk, and coconut milk are stacked over each other to make a pudding. It is Goan tradition to make Bibinca at Christmas [ 42 ].

a Chicken Vindaloo is a spicy, aromatic Goan curry adapted from the Portuguese Carne de vinha d'alhos. Source : Shutterstock https://www.shutterstock.com/image-photo/chicken-vindaloo-spinach-black-bowl-on-1933658453 b Chicken Xacuti is another Goan curry made with chicken, poppy seeds, toasted coconut shredding, black pepper, chilli, and other spices. Source : Shutterstock https://www.shutterstock.com/image-photo/chicken-xacuti-classic-dish-goa-region-1639569403

a Vada Pau is a famous Maharashtrian snack. It is a fried ball of spiced mash of boiled potatoes served in a bread roll with green chilli chutney, garlic chutney and fried green chilli. It is often eaten alongside chai (tea). Source : Shutterstock https://www.shutterstock.com/image-photo/masala-cutting-chai-bombay-vada-paav-1909642918 b Bhaji Pau is another Maharashtrian street food made up of a spiced tomato and potato curry, served with bread rolls toasted on a pan with butter. Source : Shutterstock https://www.shutterstock.com/image-photo/pav-bhaji-fast-food-dish-india-396513787

Coloquios dos simples e drogas da India by Garcia de Orta, who served as the physician to the Portuguese viceroy to India, describes a lot of ingredients used in India. There are notes on pepper, banana, galangal, turmeric, betel, bhaang (preparation of cannabis, see Fig.  12 ), camphor, and asafoetida. He describes the excess use of asafoetida in India, especially in Hindu kitchens, and notes the extremely pungent smell of the dried latex of a Ferula herb [ 2 ]. The preparation of bananas involved roasting them and then serving them with wine and cinnamon. In another dish, bananas were cut in half, fried in sugar, and served with cinnamon sprinkled on top. The widespread use of bhaang was also noted adequately.

Source : Shutterstock https://www.shutterstock.com/image-photo/hindu-priests-prepare-bhang-milk-on-653747197

Bhaang being prepared by Hindu priests for consumption during the festival of Holi. It is prepared by crushing cannabis leaves with sweetened milk.

Following the Dutch entry into the trade market of the East Indies, the French established trade with India [ 50 ] and made Pondicherry their capital. Their legacy is visible in the local cuisine there. Ragout (a stew flavoured heavily with garlic and aromatics) and rum-soaked Christmas fruitcake are some of the French dishes incorporated in the cuisine of Pondicherry. Meen puyabaisse is a local preparation of the French dish Fish Bouillabaisse .

Like the previous colonisers, the British also started with trade intentions. They first docked at the port of Surat and this became their first trading establishment in India. This trade mainly involved the export of silk, sugar, salt, opium, and tea and the import of English products like ham, cheese, beer, and wine to supply the English population in India. After the decisive Battle of Plassey in 1757, the British managed to drive the French away from Bengal and laid a strong foundation for their future supremacy [ 14 ]. The British also overthrew the Dutch from Bengal in the mid-eighteenth century, and the thriving Dutch trade under Mughal patronisation came to an end [ 37 ].

The most notable effect of the British on Indian cuisine is found in the field of brewing. Taverns opened by the British served wine, beer, rum, and other British spirits. Punch was a popular drink served at such places. It was made by mixing five ingredients – arrack, rosewater, citron juice, sugar, and spices, resulting in the name paanch (means five in Hindi).

Beer, especially ale, was a popular beverage among the British in India. Later, the antimalarial properties of quinine were discovered and it was added to soda water, giving birth to tonic. It was paired with gin and served as gin and tonic at clubs. There are speculations that gimlet (gin with lime juice) was first made in India. The first brewery in India was established in the Solan District of Himachal Pradesh in 1830 and twelve breweries were established in India in the following fifty years.

The British were fond of tea and most of it was exported to England from the plantations in China. The British discovered tea growing as a wild plant in Northeastern parts of India where the locals used it in pickling and infused it in dishes. Tea plantations were then set up by the British in Assam, which spread to Darjeeling in the Himalayas, Nilgiri Hills in Southern India, and even to Sri Lanka. However, the drink was not common among the Indian population. It was only after the forced departure of the British that officials realized the surplus in fields that were previously farmed to export to Britain [ 19 ]. The Indians took their tea with milk and spices, which is now consumed today under the name chai . A similar story follows the cultivation of coffee in Southern India, but its growth was not as pronounced as that of tea, because coffee was mainly planted to meet the needs of locals.

The British also grew crops like oranges, carrots, cauliflower, spinach, and cabbage, which have found their way into Indian kitchens. Omelettes became famous as breakfasts served in clubs, where they would be made with minced onions and chillies.

As time passed and the British ruled over the country, Christian missionaries found a true mission in converting the Hindu population to Christianity. Several internal problems like the caste system, practices of untouchability, oppression of certain sections of the population, lack of access to health care, and so on opened the doors for conversion to Christianity. Along with educational and health care facilities, the converted Christians also started imitating English food habits. On the other hand, there evolved a community of Anglo-Indians who adopted most of the English practices and cultures. They have been attributed a distinct cuisine that involved preparing English ingredients in Indian ways [ 9 ]. Roasted beef, meat and potato curry, pepper water or rasam (spicy beef broth), jalfrezi (meat or fish stir-fried in a dry sauce along with vegetables), and dry fry (beef cooked with onions, tomatoes, and spices in very little oil) are some common dishes of the Anglo-Indian cuisine. Rasam and jalfrezi are popular in India even today. They also cooked Portuguese inspired Goan dishes like the vindaloo , balchow (meat or seafood cooked in spicy tomato chilli sauce), and foogath (vegetables sautéed with onion, garlic and mustard seeds) and celebrated Christmas with roast turkey or duck and plum pudding. In certain ways, Anglo-Indian cuisine was an introduction to pan-Indian cuisine.

The paper attempted to present a historical review of the diversity of food cooked in India. The paper is a small attempt at bringing together several scattered historical pieces of information related to how food evolved in India. While the pressure of not adequately providing ‘gastrosemantics’ [ 25 ], i.e. how a culture orchestrates the centralisation of food through a well-organized attempt to bring together the philosophy, consumption, beauty and transmission of food cultures, cannot be undermined, the satisfaction of making a shot at it is nevertheless immense. Not many mainstream journal articles are available on Indian Cuisine, and this study is by no means an exhaustive study of the History of Indian Cuisine. It is a fine line between risking oversimplification and focusing only on a certain era or a region, but that has been overcome here by focusing on a few decisive aspects of every era while keeping a note of developments elsewhere in the subcontinent. A few historical and philosophical aspects of the cultures associated with the history are highlighted to explain some of the culinary preferences and food habits. Future researchers can investigate some of the areas this paper has opened. As languages of the past begin being understood, it will be worth closely looking at manuscripts from the Vedic eras for culinary culture. From the understanding that religious practices often had a culinary aspect, studying the religion would inevitably teach us about the food and the ingredients available to the population. It would also be helpful to break the subcontinent down to meaningful regions and confine the study of the development of cuisine to particular regions. Lastly, since food politics in India has recently gained some research interest, it will be worth exploring how historically food politics has been played out in the country.

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Antani, V., Mahapatra, S. Evolution of Indian cuisine: a socio-historical review. J. Ethn. Food 9 , 15 (2022). https://doi.org/10.1186/s42779-022-00129-4

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Cultural History Research Paper Topics

Exploring cultural history research paper topics opens the door to understanding humanity’s diverse societal heritage. This comprehensive guide, presented by iResearchNet, is a valuable resource for students tasked with writing a research paper on this rich and wide-ranging subject. Through this guide, you will delve into an extensive list of cultural history research paper topics categorized into ten distinctive areas. We provide a deep-dive into what cultural history encompasses, offering you a range of exciting research paper topics. Moreover, we guide you on selecting the most suitable topic, as well as tips on writing an exceptional research paper. iResearchNet takes a step further to offer professional writing services, presenting an array of features that guarantee top-quality, custom research papers. Ultimately, we aim to equip you with the tools to excel in your academic journey. This guide extends an invitation for you to uncover the vibrancy of cultural history.

100 Cultural History Research Paper Topics

Cultural history is a fascinating field of study that delves into the various aspects of human culture, including beliefs, customs, traditions, arts, and social practices. Researching cultural history provides valuable insights into the development and evolution of societies and helps us understand the complexities of human civilization. In this section, we present a comprehensive list of cultural history research paper topics, covering a wide range of categories. These topics will inspire students to explore different dimensions of cultural history and uncover intriguing research possibilities.

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Cultural Identity and Representation

• The construction of cultural identity in colonial societies.
• Depictions of cultural diversity in art and literature.
• The influence of cultural identity on political movements.
• Cultural appropriation and its impact on marginalized communities.
• Cultural heritage preservation and its significance in contemporary society.
• Cultural symbols and their role in shaping collective identity.
• Cultural assimilation and the preservation of indigenous traditions.
• Representations of gender and sexuality in popular culture.
• The role of language in cultural preservation and identity.
• Cultural memory and its portrayal in museums and exhibitions.

Rituals and Festivals

• The significance of religious rituals in ancient civilizations.
• The cultural meanings behind traditional wedding ceremonies.
• The evolution of holiday celebrations and their cultural implications.
• Folklore and storytelling traditions in different cultures.
• Rituals of passage and their cultural significance.
• Cultural festivals as a means of cultural expression and community bonding.
• Indigenous rituals and their survival in modern society.
• The role of music and dance in cultural celebrations.
• Symbolism and symbolism in traditional ceremonies.
• The transformation of rituals and festivals in the modern world.

Cultural Exchange and Cross-Cultural Influences

• The impact of globalization on cultural homogenization.
• Cultural exchange and trade routes in ancient civilizations.
• The influence of colonialism on indigenous cultures.
• Cultural diffusion and the spread of ideas and practices.
• Intercultural communication and its role in promoting understanding.
• The cultural exchange between East and West in the modern era.
• Diaspora communities and their cultural contributions.
• Cultural fusion in contemporary art, music, and fashion.
• Transnationalism and its effects on cultural identity.
• The role of technology in facilitating cross-cultural interactions.

Art, Literature, and Culture

• The portrayal of cultural values in visual arts.
• Literary movements and their reflection of cultural shifts.
• Architecture as a representation of cultural identity.
• The impact of cinema on cultural perceptions.
• The role of music in cultural expression and preservation.
• Theater and performance arts as cultural showcases.
• Cultural artifacts and their significance in museums.
• The evolution of fashion and its connection to cultural trends.
• Cultural icons and their impact on popular culture.
• The intersection of art, politics, and cultural movements.

Cultural Transformation and Resistance

• Cultural responses to social and political revolutions.
• The role of cultural institutions in challenging power structures.
• Cultural movements and their impact on social change.
• Cultural resistance against colonial rule.
• Cultural preservation as a means of resistance.
• Indigenous cultural revitalization and its impact on communities.
• The role of cultural leaders in promoting social justice.
• Artistic expressions of protest and dissent.
• Cultural memory and resistance in post-conflict societies.
• The impact of digital media on cultural activism.

Language and Cultural Communication

• The role of language in shaping cultural identity.
• Language revival and its impact on cultural preservation.
• Multilingualism and its effects on cultural interactions.
• Translating cultural nuances and challenges in intercultural communication.
• Sign language as a cultural mode of expression.
• The influence of language on cultural perception and worldview.
• The evolution of slang and its cultural implications.
• Language policies and their impact on cultural diversity.
• The role of storytelling in preserving cultural heritage.
• Language barriers and their impact on cross-cultural understanding.

Social Movements and Cultural Change

• The impact of the civil rights movement on cultural attitudes.
• Feminism and its influence on cultural norms and gender roles.
• LGBTQ+ rights movements and their effect on cultural acceptance.
• Anti-war movements and their impact on cultural consciousness.
• Cultural responses to environmental activism and sustainability.
• Disability rights and the challenge to cultural perceptions.
• Cultural movements for racial equality and social justice.
• Youth subcultures and their influence on cultural trends.
• Countercultural movements and their impact on mainstream culture.
• The role of social media in facilitating cultural mobilization.

Material Culture and Everyday Life

• The cultural significance of food and culinary traditions.
• The impact of technology on material culture.
• Housing and architectural styles as reflections of cultural values.
• Clothing and fashion as expressions of cultural identity.
• The significance of religious artifacts and symbols.
• Tools and technology in ancient civilizations.
• Cultural meanings of body adornments and jewelry.
• Sports and games as cultural practices.
• Transportation and its influence on cultural development.
• The role of consumerism in shaping cultural practices.

Cultural History of Specific Regions

• Cultural history of ancient Egypt.
• The impact of colonialism on African cultures.
• Indigenous cultures of the Americas before European contact.
• Cultural history of medieval Europe.
• Cultural transformations in Asia: From feudalism to modernity.
• The cultural legacy of ancient Mesopotamia.
• The influence of religion on cultural practices in the Middle East.
• Cultural developments in pre-Columbian Mesoamerica.
• Cultural history of the Pacific Islands.
• Cultural diversity in contemporary Australia.

Cultural Heritage Preservation and Museums

• The role of museums in preserving cultural heritage.
• Challenges in repatriating cultural artifacts to their countries of origin.
• Cultural heritage sites and their preservation.
• The impact of tourism on cultural heritage preservation.
• Digital technologies and their role in cultural heritage conservation.
• Cultural heritage and indigenous rights.
• The ethical considerations in curating cultural artifacts.
• Museums as spaces for intercultural dialogue.
• Cultural heritage and sustainable development.
• The role of education in promoting cultural heritage awareness.

This comprehensive list of cultural history research paper topics provides students with a diverse range of possibilities for their research endeavors. Whether exploring cultural identity, rituals, art, language, social movements, or material culture, there is an abundance of fascinating topics to delve into. By delving into these subjects, students can gain a deeper understanding of the intricate tapestry of human culture and its impact on societies past and present.

Cultural History: Exploring the Range of Research Paper Topics

Cultural history is a vibrant field of study that examines the ways in which societies have shaped and been shaped by their unique cultural practices, beliefs, values, and expressions. It delves into the rich tapestry of human experiences, exploring everything from art, literature, and music to language, rituals, and social movements. Researching cultural history allows us to better understand the complexities of human existence and the diversity of human cultures throughout time. In this section, we will embark on a journey through the vast range of research paper topics within cultural history, showcasing the diverse aspects of human culture that you can explore in your own research.

• Art and Culture : Art has always been an integral part of human expression, reflecting cultural values, beliefs, and societal norms. Within this category, you can explore a multitude of cultural history research paper topics, including the influence of art movements on cultural perceptions, the role of art in social and political commentary, the representation of cultural identity in visual arts, and the impact of technology on artistic practices. You can also investigate specific artists or art forms that have significantly contributed to the cultural landscape.
• Literature and Culture : Literature provides us with a window into the past, offering insights into the thoughts, emotions, and experiences of people from different cultures and time periods. Within this category, you can delve into various aspects of literature, such as the representation of cultural values in literary works, the influence of literature on cultural and social movements, the role of storytelling in cultural preservation, and the exploration of cultural identity in literary texts. You can also examine the impact of translation and cross-cultural literary influences.
• Music and Culture : Music has the power to transcend language barriers and convey cultural expressions. Within this category, you can explore cultural history research paper topics such as the role of music in cultural rituals and ceremonies, the influence of music on cultural identity formation, the impact of globalization on music and cultural fusion, and the relationship between music and social movements. You can also examine specific genres or musicians that have made significant contributions to the cultural history of a particular region or era.
• Language and Culture : Language is a fundamental aspect of culture, shaping our worldview and serving as a medium for communication and expression. Within this category, you can explore topics such as the relationship between language and cultural identity, the impact of language policies on cultural diversity, the role of language in cultural preservation, and the influence of language on social and political movements. You can also delve into the study of dialects, slang, and language variations within specific cultural contexts.
• Rituals and Traditions : Rituals and traditions play a vital role in shaping cultural practices and beliefs. Within this category, you can explore topics such as the significance of religious rituals in different cultures, the evolution of cultural traditions over time, the role of rituals in cultural identity formation, and the impact of globalization on traditional practices. You can also investigate the cultural meaning behind specific rituals or explore the preservation of indigenous rituals in contemporary society.
• Gender and Sexuality : The study of gender and sexuality within cultural history offers insights into the ways in which cultural norms and expectations have shaped the experiences of individuals throughout history. Within this category, you can explore topics such as the representation of gender and sexuality in art, literature, and popular culture, the impact of cultural beliefs on gender roles, the history of LGBTQ+ movements and their influence on cultural acceptance, and the intersectionality of gender and other aspects of identity within different cultural contexts.
• Social Movements and Cultural Change : Cultural history is intertwined with social movements that have challenged the status quo and brought about cultural transformations. Within this category, you can explore topics such as the impact of civil rights movements on cultural attitudes, the role of cultural resistance against colonial rule, the influence of countercultural movements on mainstream culture, and the cultural responses to environmental activism and sustainability. You can also investigate the role of social media in facilitating cultural mobilization and the cultural legacy of specific social movements.
• Material Culture and Everyday Life : Material culture refers to the physical objects and artifacts that reflect the values, practices, and beliefs of a particular culture. Within this category, you can explore topics such as the cultural significance of food and culinary traditions, the influence of technology on material culture, the relationship between clothing and cultural identity, and the impact of consumerism on cultural practices. You can also investigate housing and architectural styles as reflections of cultural values, the significance of religious artifacts and symbols, and the role of transportation in shaping cultural development.
• Colonialism and Postcolonial Studies : The legacy of colonialism has had a profound impact on cultural history. Within this category, you can explore topics such as the cultural implications of colonial encounters, the influence of colonialism on indigenous cultures, and the processes of decolonization and cultural revitalization. You can also investigate the representation of colonial experiences in literature and art, the role of museums in preserving and interpreting colonial history, and the impact of postcolonial theories on cultural studies.
• Globalization and Cultural Exchange : The era of globalization has facilitated the exchange of ideas, practices, and cultural expressions across borders. Within this category, you can explore cultural history research paper topics such as the impact of globalization on cultural homogenization and hybridization, the role of cultural diplomacy in fostering international relations, the influence of transnational media on cultural values, and the cultural implications of migration and diaspora. You can also investigate the challenges and opportunities that arise from cultural interactions in the globalized world.

The field of cultural history offers an expansive range of research paper topics that allow students to explore the intricate connections between culture, society, and human experiences. By delving into the diverse aspects of cultural history, students can gain a deeper understanding of the complexities of human culture and its impact on societies throughout time. Whether you are interested in art, literature, music, language, rituals, social movements, material culture, colonialism, or globalization, there is a wealth of fascinating topics waiting to be explored. So, embark on your research journey and unravel the captivating stories that cultural history has to offer.

Choosing Cultural History Research Paper Topics

Choosing a research paper topic in cultural history can be an exciting yet challenging task. With the vast array of cultural practices, beliefs, and historical periods to explore, it’s essential to narrow down your focus and select a topic that aligns with your interests and academic goals. In this section, we will provide you with expert advice on selecting cultural history research paper topics that are engaging, relevant, and offer ample opportunities for exploration and analysis.

• Follow Your Passion : When choosing a research paper topic, it’s important to select a subject that genuinely interests you. Consider your personal passions, curiosities, and areas of expertise. Think about the cultural aspects that intrigue you the most—whether it’s art, literature, music, social movements, or any other cultural facet. By selecting a topic that aligns with your passion, you will be motivated to delve deeper into the subject and produce a well-researched and engaging paper.
• Conduct Preliminary Research : Before finalizing your research paper topic, conduct preliminary research to familiarize yourself with the existing scholarship in the field of cultural history. Read scholarly articles, books, and research papers to gain an understanding of the current debates, gaps in knowledge, and emerging trends. This will help you identify potential research areas and refine your topic to ensure its relevance and originality.
• Consider Chronological and Geographical Scope : Cultural history encompasses a wide range of historical periods and geographical regions. Consider the chronological and geographical scope that interests you the most. Do you prefer to focus on a specific time period, such as ancient civilizations, the Renaissance, or the 20th century? Are you drawn to a particular region, such as Asia, Europe, Africa, or the Americas? Narrowing down the chronological and geographical scope will help you create a more focused and manageable research paper topic.
• Explore Understudied or Emerging Areas : One way to contribute to the field of cultural history is by exploring understudied or emerging areas of research. Look for topics that have received limited attention or have recently gained significance in cultural studies. For example, you can investigate the cultural history of marginalized communities, explore the impact of technology on cultural practices, or analyze the cultural dynamics of globalization in a specific region. By delving into these uncharted territories, you can make a unique and valuable contribution to the field.
• Interdisciplinary Approaches : Cultural history is a multidisciplinary field that intersects with various disciplines, including anthropology, sociology, literature, art history, and more. Consider incorporating interdisciplinary approaches into your research paper topic. For example, you can examine the intersection of cultural history and gender studies, explore the relationship between cultural practices and environmental history, or analyze the cultural significance of technological advancements. By embracing interdisciplinary perspectives, you can offer a more comprehensive and nuanced understanding of cultural phenomena.
• Analyze Primary Sources : To enhance the authenticity and depth of your research, incorporate primary sources into your analysis. Primary sources can include historical documents, artworks, literary works, music recordings, oral histories, and archival materials. Analyzing primary sources allows you to directly engage with the cultural artifacts and voices of the past, providing firsthand insights into the cultural practices and beliefs of a specific time and place. Incorporating primary sources can add richness and authenticity to your research paper.
• Consider Ethical and Social Implications : Cultural history research often raises ethical and social implications, especially when studying sensitive topics or marginalized communities. It’s important to consider the ethical dimensions of your research and approach your topic with sensitivity and respect. Take into account the potential impact of your research on communities, ensure that your research adheres to ethical guidelines, and consider ways to present diverse perspectives and voices in your analysis.
• Consult with Your Advisor or Instructor : Don’t hesitate to consult with your advisor or instructor during the topic selection process. They can provide valuable guidance, suggest relevant sources, and offer insights based on their expertise. Discuss your research interests, proposed topics, and research goals with them to receive feedback and refine your ideas. Their support and expertise can greatly enhance your research paper.
• Be Open to Revision : As you delve deeper into your research, be open to revising your research paper topic if necessary. Sometimes, new insights or challenges may arise that require you to adjust or refine your focus. Embrace this iterative process and remain flexible in adapting your topic to ensure its coherence and relevance throughout your research journey.
• Stay Organized and Manage Your Time : Researching and writing a cultural history research paper requires careful organization and time management. Create a research plan, establish a timeline, and allocate sufficient time for conducting research, analyzing sources, and writing. Break down your research paper into manageable tasks, set deadlines for each stage, and make sure to leave ample time for revisions and proofreading.

Choosing a cultural history research paper topic requires careful consideration, passion, and a solid research plan. By following these expert tips, you can select a topic that aligns with your interests, engages with the current scholarship, and offers opportunities for original analysis and contribution to the field of cultural history. Remember to stay organized, consult with your advisor, and approach your research with intellectual curiosity and sensitivity to the cultural contexts you explore. With the right topic and diligent research, you can produce a compelling and impactful research paper in cultural history.

How to Write a Cultural History Research Paper

Writing a cultural history research paper requires a systematic and thoughtful approach to ensure a comprehensive and engaging analysis of your chosen topic. In this section, we will provide you with guidance on how to write a cultural history research paper, from developing a strong thesis statement to effectively analyzing primary and secondary sources. By following these steps, you can craft a well-structured and compelling paper that contributes to the field of cultural history.

• Define Your Research Question : Before diving into the writing process, it’s crucial to define a clear and focused research question. Your research question will guide your investigation and provide a framework for your paper. Consider the specific aspects of cultural history you want to explore and formulate a research question that encapsulates the key issues or themes you aim to address. A well-defined research question will ensure the coherence and direction of your research paper.
• Conduct In-Depth Research : A successful cultural history research paper relies on thorough and comprehensive research. Explore a range of primary and secondary sources, including books, scholarly articles, archival materials, oral histories, artworks, and other relevant cultural artifacts. Analyze and critically evaluate these sources to gather evidence and support your arguments. Take comprehensive notes and organize your research material to facilitate the writing process.
• Develop a Strong Thesis Statement : Crafting a strong thesis statement is essential to guide your research paper and present a clear argument. Your thesis statement should reflect your research question and articulate the main argument or perspective you intend to explore in your paper. It should be concise, specific, and capable of generating insightful analysis and discussion. Your thesis statement will serve as the foundation upon which you build your research paper.
• Outline Your Paper : Creating an outline is an effective way to structure your cultural history research paper and ensure a logical flow of ideas. Start with an introduction that provides background information on your topic, introduces your thesis statement, and outlines the main points you will address in your paper. Divide the body of your paper into sections or paragraphs, each focusing on a particular aspect of your research. Finally, include a conclusion that summarizes your findings, restates your thesis, and offers insights for further research.
• Analyze Primary and Secondary Sources : In cultural history research, the analysis of primary and secondary sources is crucial to support your arguments and provide a deeper understanding of the cultural phenomena you are studying. When analyzing primary sources, consider their historical context, authorship, intended audience, and cultural significance. Use these sources to provide direct evidence and insights into the cultural practices, beliefs, and values of a specific time and place. Secondary sources, such as scholarly articles and books, offer critical perspectives and interpretations that enrich your analysis.
• Engage with Theory and Scholarship : Cultural history research benefits from engaging with relevant theoretical frameworks and scholarly debates. Familiarize yourself with the key concepts and theories in cultural history that are applicable to your research. Analyze the work of prominent historians and cultural theorists in the field and consider how their ideas inform your analysis. Engaging with theory and scholarship will provide depth and context to your research paper and demonstrate your understanding of the broader intellectual discourse.
• Structure Your Arguments and Evidence : Organize your arguments and evidence in a coherent and logical manner. Present your ideas in a way that supports your thesis statement and contributes to the overall narrative of your research paper. Use topic sentences to introduce new sections or paragraphs and provide smooth transitions between ideas. Incorporate evidence from your research, such as direct quotations, statistical data, or visual representations, to support your arguments and add credibility to your analysis.
• Provide Context and Analysis : While presenting facts and evidence is important, a cultural history research paper also requires critical analysis and interpretation. Contextualize your findings within the broader historical, social, and cultural contexts. Explain the significance of the events, practices, or phenomena you are examining and consider their impact on the culture and society of the time. Analyze the motivations, beliefs, and ideologies that underpin the cultural practices you are studying. This analytical approach will add depth and nuance to your research paper.
• Use Clear and Concise Language : Write your research paper using clear and concise language to ensure your ideas are effectively communicated. Avoid jargon or overly complex language that may hinder understanding. Use precise terminology and define key terms to facilitate reader comprehension. Maintain a consistent writing style and ensure your sentences and paragraphs flow logically from one to another.
• Revise and Edit : Once you have completed the initial draft of your research paper, take the time to revise and edit your work. Review your paper for clarity, coherence, and logical organization. Check for grammar, spelling, and punctuation errors. Trim any unnecessary or repetitive content. Consider seeking feedback from peers or instructors to gain fresh insights and identify areas for improvement. Revision and editing are crucial steps to ensure your research paper meets high academic standards.

Writing a cultural history research paper requires careful planning, thorough research, and effective communication of ideas. By defining a clear research question, conducting in-depth research, developing a strong thesis statement, and engaging with primary and secondary sources, you can produce a compelling and insightful research paper in cultural history. Remember to analyze and interpret your findings, provide context, and revise and edit your work for clarity and coherence. With these guidelines, you will be well-equipped to craft a research paper that contributes to the understanding of cultural history.

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• Expert Degree-Holding Writers : Our writing team consists of highly qualified professionals with advanced degrees in history and related fields. They possess in-depth knowledge of cultural history and are well-versed in conducting thorough research and analysis. Our writers are dedicated to delivering top-notch papers that reflect their expertise and passion for the subject matter.
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• In-Depth Research : Cultural history research papers demand extensive research to uncover relevant sources, analyze primary and secondary materials, and gain a comprehensive understanding of the topic. Our writers are adept at conducting in-depth research using credible sources and scholarly databases. They will gather a wide range of materials to support your arguments and provide a well-rounded analysis of cultural history.
• Custom Formatting : We understand the significance of adhering to specific formatting styles in academic writing. Whether your paper requires APA, MLA, Chicago/Turabian, or Harvard formatting, our writers are well-versed in these styles and will ensure that your research paper meets the required standards. Proper formatting enhances the professionalism and readability of your paper.
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• Customized Solutions : We recognize that every research paper is unique, with its own set of requirements and objectives. Our writers provide customized solutions to address your specific research needs. Whether you require an in-depth analysis of a particular cultural phenomenon, a comparative study of multiple cultures, or an exploration of cultural interactions, our team will tailor their approach to meet your research goals.
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American Research Journal of History and Culture

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American Research Journal of History and Culture is an international, peer-reviewed, open access online journal. Research of History and Culture combines the approaches of anthropology and history to look at popular cultural traditions and cultural interpretations of historical experience. It examines the records and narrative descriptions of past matter, encompassing the continuum of events (occurring in succession and leading from the past to the present and even into the future) pertaining to a culture. This journal publishes research work on topics like Anthropology, Cultural history, Early modern discourses, Historiography, Museum Studies, Sociology, World history. The journal welcomes and publishes insightful global history and various cultures related articles in the form of original articles, review articles, short reviews, short notes etc.

Given below are some of the key (but not limited) topics of this journal.

• Anthropology
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ARJ @ SOCIAL

Editorial Board

Angela Bellia

Prof. Alisson Eugênio

Dr. Francisco Villacorta Baños

Dr. Antonio Sustaita

Dr. Grant Rodwell

Dr. Asyraf Hj Ab Rahman

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“The End of Everything,” with Victor Davis Hanson

Classicist Victor Davis Hanson is the Martin and Illie Anderson Senior Fellow at the Hoover Institution and the author of hundreds of articles, book reviews, and newspaper editorials on Greek, agrarian, and military history and essays on contemporary culture.

“The End of Everything,” With Victor Davis Hanson

Classicist Victor Davis Hanson is the Martin and Illie Anderson Senior Fellow at the Hoover Institution and the author of hundreds of articles, book reviews, and newspaper editorials on Greek, agrarian, and military history and essays on contemporary culture. He has written or edited twenty-four books, the latest of which is The End of Everything: How Wars Descend into Annihilation . The book—and this conversation—charts how and why some societies choose to utterly destroy their foes and warns that similar wars of obliteration are possible in our time. Hanson provides a warning to current societies not to repeat the mistakes of the past.

To view the full transcript of this episode, read below:

Peter Robinson: It may not happen often, but sometimes, sometimes, entire civilizations die in a single day. Historian Victor Davis Hanson on Uncommon Knowledge Now. Welcome to Uncommon Knowledge.

Peter Robinson: I'm Peter Robinson, a fellow at the Hoover Institution here at Stanford. Victor Davis Hanson is a classicist and military historian. Dr. Hanson has published more than two dozen major works of history, including A War Like No Other, his classic work on the Peloponnesian Wars. Victor Davis Hanson's newest book, The End of Everything, How Wars Descend into Annihilation. Victor, thanks for joining me.

Victor Davis Hanson: Thank you for having me, Peter.

Peter Robinson: First question, The Destruction of Thebes by Alexander the Great, The Obliteration of Carthage by the Romans, The Defeat of Constantinople by the Turks, and The Destruction of the Aztecs by Cortez. Those are your four case studies in this book. All those happened a while ago. Why write this book now?

Victor Davis Hanson: I was curious, most of my career, I've been curious why Thebes, or I can go into the details, but why these...

Peter Robinson: We'll come to it.

Victor Davis Hanson: Yes, we'll come to it.

Victor Davis Hanson: Why these civilizations were not just defeated, but were annihilated. And there were others. I had, there's a wide array in the ancient world, the island of Milos, towns in the Peloponnesian war like Schioni, et cetera. And this is very different than natural disasters like the Mycenaeans, et cetera. But I was wondering if there was a typology, a repeating pattern, and if it would be applicable to any of the value. And I found that there was, both on the part of the attacker, certain a mindset, and on the part of the defender, and that those situations that we think could not happen today, because we're supposedly a postmodern moral world.

Peter Robinson: We're more advanced than they were, Victor.

Victor Davis Hanson: That's what we think. And it's there...So in the epilogue, I just did a brief survey. Well, not a brief survey, but I did a survey of countries that are very vulnerable as described, either in the nature of their enemies and the intent of their enemies, or the neighborhood in which they reside, or their size, or their limits. So for example, there's only 12 million Greeks in the world.

Peter Robinson: Right.

Victor Davis Hanson: There's Cypriots, but Greeks, and they have a lot, they have a bad neighborhood, and they have been existentially threatened by the Turks, especially the present government. Israel is another example. The Kurds are an example. The Armenians are still an example. And all of them have had a history where at times people thought they would be existentially gone, because that was the intent. And yet, we feel that today when somebody threatens to wipe somebody out, either with nuclear weapons or with conventional weapons, we discount that. That can't happen.

Peter Robinson: It's mere hyperbole.

Victor Davis Hanson: In the epilogue, I think I mentioned maybe a half a dozen, or maybe even a dozen direct threats by various Turkish figures, Russian, Chinese, where they actually threaten to destroy and wipe out, whether it's Ukraine or Taiwan, or the Armenians, or Greeks, or Israel.

Peter Robinson: And the argument is, take that possibility seriously, because every so often it really does happen.

Victor Davis Hanson: Maybe so often the exception that nobody thinks, the unimaginable, or what people think it can't happen here does happen here.

Peter Robinson: The end of everything presents almost 300 pages of your usual approach, which is meticulous, thorough, and engrossing historical writing.This is television.We can't go into it that deeply.But I would like to touch on these case studies at least briefly, because even put in some reform, my feeling was as I went through the book, even in some reform, every one of them is just fascinating and surprising in some way. All right. Thebes, the end of everything, I'm quoting you. In 335 BC, the Thebans not only revolted against the Macedonian occupation of Greece, but defiantly dared Alexander the Great to take the legendary city, that is to take Thebes itself. He did just that. All right. Briefly, the significance of Thebes, it was a major city. Who were the Macedonians? Set it up. Who were the Macedonians? And who is this brilliant figure who arises as a very young man, Alexander the Great?

Victor Davis Hanson: Well for 20 years prior to 335, Philip II of Macedon...

Peter Robinson: Alexander's father.

Victor Davis Hanson: Alexander's father had taken a backwater area that was deprecated as uncivilized by Greeks.

Peter Robinson: The northern mountainous region.

Victor Davis Hanson: The mountainous region of today is parts of northern Greece and the autonomous state of so-called Macedon, Macedonia. And he had forged a imperial power. He borrowed...he was a hostage at Thebes when he was a young man himself and he learned from the great master of Pamanondas about Greek military tactics. He lengthened the Sarissa. He did all of his military war.

Peter Robinson: The Sarissa is..

Victor Davis Hanson: Pike. So he innovated and improved on Greek phalanx warfare, fighting in Colum. And it was a juggernaut and he came from the north and he conquered at the Battle of Carinea three years prior to this. He destroyed Greek freedom by this...basically it was an army of Thebans and Athenians and a few other city states and they were conquered and they were occupied and there was no longer a truly consensual government in these cities, 1500 city states.

And he had a plan or an agenda that said, "I will unite you and even though you think I'm semi-barbarous..." Macedonian, it was sort of hard to understand. You could understand it, the language and the tradition. But it had no culture, the Greeks thought, but we're going to unite and take Persia and pay them back for a century of slights and get rich in the process.

And the Greeks revolted in 335. He died, he was assassinated and he had his 21-year-old son who had been at the Battle of Carinea at 18 and had been spectacular in defeating the Theban and they didn't take him seriously.

Peter Robinson: The Thebans or the Greek city states in general?

Victor Davis Hanson: Yes, they thought, "You know what?"

Peter Robinson: He's a kid.

Victor Davis Hanson: And who's going to take over from Philip II? He was a genius and he's got bastard children here and concubines there and he's got this one guy named Alexander and it's, "Don't take him, we're going to revolt." And everybody said, "Well, we hear about him and he's kind of fanatic, be careful, but we're willing to revolt if you revolt first." And Thebes was at this time legendary because it's the legendary home of Oedipus and Antigone. It's the fountain of Greek mythology. It has kind of a dark history because bad things happen at Thebes like Oedipus kills his father or Antigone is executed.

Peter Robinson: Not a lot of cheerful stories.

Victor Davis Hanson: In Euripides' Bacchae, it's under the shadows of Mount Cthyrum. But the point is that it had been under a Pamanondas, a Pythagorean, enlightened society. The first really expansive democracy was trying to democratize the Peloponnese. So it was the moral leader at this point. It happened to be...

Peter Robinson: Roughly how big a population is.

Victor Davis Hanson: It was small. It was somewhere between 15 and 25,000 citizens and maybe at most 5 or 10,000 residents. But it was the capital of what we would call today in English a province called Boeotia. And that probably had somewhere around 150 to 250 and it was the capital that subjugated that.

Peter Robinson: Okay.

Victor Davis Hanson: But it has separate dialect, Theban dialect was different than the Boeotian dialect and it was the stellar city. So Alexander then says, "If you revolt, we're going to come down." So he eliminates his enemies. He starts to march. The Athenians are egging the Thebans on and said, "Don't worry, we'll come." And the Spartans are going to come, both of them in decline. And the long and the short of it is he arrives there. The Thebans mock him. They think we can replay the Battle of Carinea, we'll win. And all of a sudden, when he shows up, they have no idea who he is. They don't know what he's intending. Had they studied his career, they would see he's a killer and he's a genius and he's about ready to conquer the Persian Empire. And he needs to have a solid home front and he means business and he doesn't play by the rules. And the rules of Greek warfare, except for the Peloponnese, you don't destroy your enemy. You don't, even Athens as it lost the Peloponnesian War, they did not destroy Athens. The Spartans and the Thebans. So Spartans say they're going to come, the Athenians are going to help them, they egg them on, they revolt, they kill the Macedonian garrison, are they imprison them? And Alexander pulls up with this huge army. You can't get 200 miles from the north in 10 days. You can if you're Alexander. You march at 20 plus miles a day. He pulls up, they build siegecraft and...

Peter Robinson: Is it fair to say he's a little bit like Napoleon?

Victor Davis Hanson: Yes.

Peter Robinson: He's shocking.

Victor Davis Hanson: He's quick, or Caesar, quickness of Caesar and Napoleon, audacity, it's like Donton. And the Spartans dissipate and the Athenians dissipate.

Peter Robinson: You're on your own.

Victor Davis Hanson: You're on your own and they think, this is the seven gates of Thebes, the magnificent walls of Thebes. We've only been broached once after the Persian War. We can endure, we're on the defensive, we've got this wonderful army, we'll go out in front of the...and they're defeated. And they think...

Peter Robinson: But not just defeated.

Victor Davis Hanson: Not defeated. They think they can negotiate, I think. And he says, "I'm gonna kill every single person that's over the age of 16. I'm gonna enslave every woman and child." But you know what? I will save the descendants of Pindar, the poet, his house, and maybe some religious shrines and he levels the city down to the foundations and there are no more Thebans. Later the Macedoians will take the site and bring in other people, other Greeks.

And so there is no longer a Theban who have been there for two millennia. They're gone.

Peter Robinson: They have their own culture, their own history. It is recognized as such by the entire Greek-speaking world and they even have their own, not quite their own language, but their own dialect. And it just ends.

Victor Davis Hanson: And some of the surrounding Vyoshan villages, of course, don't like them so they join Alexander and they haul off the marble columns, they haul off the roof tiles, they level it. After Alexander's death, some two decades later, they think it's be good propaganda to refound Thebes and they call it Thebes, which is the modern city today, but it's not the same culture.

Peter Robinson: Okay, that's example number one. By the way, do we have, from contemporary sources, who would have written about that? What effect did that event have? It shocked all the other Greek city states into total submission?

Victor Davis Hanson: Yes, they could not believe it. They completely folded and it was...

Peter Robinson: So he got the stable home base he wanted that permitted him to advance into...

Victor Davis Hanson: Yes. And it became, even among the Macedonians, it became shameful that Alexander had destroyed this legacy city, the fountain, as I said, of Greek mythology and of Paminondas, the great liberator, his legacy Pythagoras, the Pythagorean group there, and he'd wipe them out and they regretted it later. But at the time, nobody came to their aid. They were very confident. They didn't think anybody would ever do that and they were shocked. It was something that had not happened before.

Peter Robinson: Carthage.

Peter Robinson: Rome and Carthage, The End of Everything, your book, the three centuries long growth of the Roman Republic, this is BC, we're not at Caesar, we're certainly not close to Augustus, we're seeing Rome grow from a city to the dominant force in the Mediterranean. The three centuries long growth of the Roman Republic was often stalled or checked by its formidable Mediterranean rival Carthage on the other side of the Mediterranean, at the northern tip of, northern edge of Africa. The competition between Rome and Carthage involved antithetical civilizations.

Peter Robinson: Explain that.

Victor Davis Hanson: Carthage was founded about the same time as Rome was, but it was... We use the word, they use, it's an ancient word, Punic, and all that is, is a Phoenician transliteration for Phoenician culture that would be today where Gaza is along that area. This was a colony, colonists founded under the mythical Dido at what is modern Tunisia, right? Just 90 miles opposite, it's the narrowest point of the Mediterranean. 90 miles opposite Sicily.

Peter Robinson: 90 miles..thats Sicily, yes.

Victor Davis Hanson: And they were a Punic-Semitic culture, so their language was not linguistically related to Greek or Latin. They did things that classical culture abhorred such as child sacrifice. However, they did, were heavily influenced by Greek constitutional history, so they actually had a constitutional system. They learned about Western warfare from Spartan taskmasters. And so they fought these series, what we call the Punic War, first and second.

Unfortunately for Rome, they were confronted with an authentic Alexander Napoleon-like figure in Hannibal who took the war home.

Peter Robinson: Second Punic War, he goes across into what is now Spain.

Peter Robinson: And goes behind the Roman line, so to speak, famously taking elephants up over the Alps and then wreaking havoc.

Victor Davis Hanson: From 219 to 202, this war went on.

Peter Robinson: In Italy itself.

Victor Davis Hanson: In a series of battles at the river Caecanius, Trebia, Lake Trasimone and Canai, he killed or wounded a quarter million Italians. And he ran wild for over a decade in Italy until Scipio Africanus invaded Tunisia and forced him to come home. But when I'm getting it.

Peter Robinson: To defend his home.

Victor Davis Hanson: Yes, and he lost the Battle of Zama. He was exiled. But that was such a trauma or wound in the Italian mind. It was always Hannibal ad Portis. They scared little kids with, "Hannibals at the gates." And they were traumatized. So they had given a very punitive piece to the Carthaginians and they said, "You're going to pay this huge fine and you can never make war without our permission. You're going to surrender all of your European and Sicilian colonies. You're going to have it and you're going to be largely confined to the city of Carthage and some satellite villages."

Peter Robinson: So the Romans, I'm thinking now of a phrase that was used by Madeleine Albright to describe what we had done to Saddam Hussein.The Romans had Carthage in a box.

Victor Davis Hanson: Yes. That was the idea.

Peter Robinson: So may I set up the third Punic War here, which brings us to the event to which this chapter is dedicated. I'm quoting the end of everything. After the first two Punic Wars, there was no call at Rome to level a defeated Carthage and yet Rome attacks Carthage again. Why?

Victor Davis Hanson: Well, it's very ironic and tragic because they paid the identity off early.

Peter Robinson: The Carthaginians did.

Victor Davis Hanson: Yes. They were Carthaginians and they discovered that without these overseas colonies and given their prime location in North Africa. I've got to remember that this time North Africa was the most fertile part of the Mediterranean, much more fertile than the shores of Europe, southern shores. And so they sent a delegation three years earlier to Carthage to inspect what was going on and how did they pay the fine off and they were astounded. The city had somewhat 500,000 to 600,000 people in it. It was booming. It was lush. The countryside was lush. They were confident and unfortunately for them...

Peter Robinson: And they had one of the great ports of the ancient world.

Victor Davis Hanson: Yes, it was the part of Carthage. It's about 20 miles from modern Tunis today. Was starting to rival Rome again and yet they professed no bellicosity at all. They said, "You know, we have no problem with you."

Peter Robinson: We've learned our lesson.

Victor Davis Hanson: We learned our lesson. We're just a mercantile. They were sort of re-fashioning themselves from an imperial power to something like Singapore or Hong Kong.

Victor Davis Hanson: And Rome unfortunately was in this expansionary mood. They had now consolidated Spain. They had consolidated Italy. They consolidated much of Greece and soon would conquer all of Greece and Macedon and they had Cato the Elder and he got up, you know, legendarily and say, "Carthage must be destroyed as the epithet of every speech." So there was...

After the inspectors came back, they said, "These people are insidious. They may not have Hannibal but they're going to rival us again."

Peter Robinson: They're doing too well.

Victor Davis Hanson: They're doing too well and we've got all... There were people in the Roman Senate that said, "No, no, don't do that." They don't pose a threat and actually they're good for us because the more that they're there they put us on guard and they don't...we don't get luck. The Romans had this idea that affluence and leisure make you decadent. So just the fact that they're right across the Mediterranean means it will always be vigilant.

Peter Robinson: The competition is good for us, Cato.

Victor Davis Hanson:  Yes. Kind of what Americans used to think, 19th century. So unfortunately they decided that they would present Carthage with a series of demands that could not possibly be met and still be autonomous. So they sent a group from the Senate down to consuls. Consular army was rare but they brought two consuls in an army and they landed them there and they said to them, "You're going to move your city at least 15 miles from the ocean. You're not going to be a sea power." If you get mad about it, we're the same way. We're Rome, we have Ostia, we're from the end. No problem. But you're going to destroy this ancient city and then you're going to have to move lock, stock and barrel. And by the way, we want all of your arms. We want your elephants, your famous elephants. They have personal names even. We want your elephants, we want your siege craft, we want your armor, we want everything.

Peter Robinson: You’ll live.

Victor Davis Hanson:  And if you're willing to do that, we'll consider that the city can live.And they were willing to do that. Not to move. They sent a delegation. They said, "Okay, here's our catapults, here's our body armor and we'll negotiate about the rest." They went back and they think, "I think we're okay." And then they went back the next time and the Romans who were camped away with this huge army said, "You know..."

Peter Robinson: You said that the Romans took an army across the Mediterranean.

Victor Davis Hanson: It's in Utica, right near them, about 20 miles away.

Peter Robinson: That was bigger than the landing force in the invasion of Normandy.

Victor Davis Hanson:  Yes.

Peter Robinson: It was a vast force.

Victor Davis Hanson: Our sources are somewhat in disagreement but it could have been anywhere from 70 to 90 to 100,000 people. It took us all day to land 135,000, us being the British and the Americans. But the Americans themselves did not have as many people as the Romans landed at Utica. And so the Romans then told all of the Carthaginian allies on the North Coast, "Are you with us or against us? Because if you're with us, we're going to destroy them and you're going to be a favored colony. You're going to get to share in the spoils. We won't tax you. You'll be the guys that run North Africa for it." If you're with them, we're going to do to them what you...And so most of them, not all defect. And then the legates come back and they tell the Carthaginians, "We blew it. They're going to kill us. And now we have no weapons because they're going to make us move." We thought if we turned in our weapons, they might not make us move. So they bring out of retirement, Hasrbal, who's this fanatic, not the famous Hasrbal, father of Hannibal, but another named Hasrbal. And he's a complete maniac and they had not trusted him. And he says, "Kill all the legates. Anybody who was an appeaser, we're in full moor. We're going to rearm." And they do. They get all the women's hair, they make catapults and they go crazy and then they put a siege around the city. The problem the Romans have is these walls are, until Constantinople, they are the greatest walls in the ancient world, 27 miles of fortifications. Carthage is on a peninsula and it's kind of like a round circle with a corridor. And they've got that all area walled and they have a fleet still and it's very hard to take that city and the Romans are not known for their siege craft and they can't take it. And they lose, lose, lose and they get the Numidian allies to join them. And suddenly, after two years, they've lost probably 20 or 30,000 Romans. Sometimes they break into the suburbs but not the main walls and it looks like it is an ungodly disaster. And they are very confident and then just in the case of Alexander, they don't know who they're dealing with. They bring out of this obscurity, Scipio Emilianus and he is the adopted nephew, grand nephew of Scipio Africanus, the famous one. And he is a philosopher like Alexander the Great. He's a man of letters. He wouldn't do such a thing. He has a Scipionic circle, playwrights, terrains. He's a friend of Polybius the Great historian just like Alexander has the student of Aristotle. So he comes and he's a legate and he's been there and he keeps saying the consuls are incompetent and they don't know what they're doing and I should be it but he's a lowly young guy. And they said, "You take over." So he comes, he gives a big lecture and says, "You guys are pathetic, his soldiers. You're lazy. This is what's going to happen." He has discipline. They build a counter wall and over the next year he turns out to be an authentic military genius. He cuts off the city. He cuts off the corridor. He cuts off all the allies supplying them and he besieges them and they will not surrender but they still have a hope that he's a man of principle and he will negotiate with them and he will give them terms and he is a killer. And he does not give them terms and he systematically breaks for the first time and only time in history the great walls of Carthage. He gets into the city and then over a two-week period he systematically kills every single person that the Romans. In fact, the descriptions are horrific.

Peter Robinson: Now, are we still dealing with half a million people or have men haven't fled by now?

Peter Robinson: No, no, it's still densely possible.

Victor Davis Hanson: They have nowhere to go. They're stuck and they're starving now. And he's...

Peter Robinson: So this is an act of butchery. Like Slaughtering and cattle or sheep.

Victor Davis Hanson: Well, our sources, we have accounts in Diodorus and somewhat in Libya, Polybius fragments here and there. We're told that the Roman army has to scrape off the bodies because they've killed so many people because they're in...it's like Gaza or Fallujah or Mosul.

It's fighting in block by block and they're destroying...to get rid of the Carthaginian defenders, they're destroying the buildings and they topple and then the bodies are there and then the army can't move. So they go, go, go until they get to the pinnacle, the capital. And there is Hasrbal and his wife and of course he flips and cuts a deal with...

Peter Robinson: And on your side now, boys.

Victor Davis Hanson: He leaves his wife and they burn themselves up. And then he goes...he ends up in retirement in Italy, one of the few people who is...endures a Roman triumph and humiliated in the parade and they let him live.

Peter Robinson: and they let him live. 

Victor Davis Hanson: And then they wipe it out. I don't think it's accurate to say they sowed the ground with salt as myth goes, but they did completely declare it an inhospitable place and it was sacrosanct to even get near it. They took it down to the foundation. There is no more formal Punic center of knowledge. They had a very rich agriculture, agronomy literature. It's gone. What happens? It's remnants of people who in Augustine's time in the fifth century AD, there are still people who they claim speak Punic, very few of them. And Romans under Caesar then they make something called Carthago Nova, a new city, but it's a Roman city built on the...somewhat near the old city.

Peter Robinson: So it's gone.

Victor Davis Hanson: It's gone.

Peter Robinson: It's just disappeared.

Victor Davis Hanson: Gone. Caput.

Peter Robinson: All right. I want to get two more stories, but probably don't have time to go into as much detail.

Victor Davis Hanson: Yeah, I won't break it. Yeah, I won't go into it.

Peter Robinson: But well, these become a little bit more modern, so maybe we know a little bit more about them. I mean, maybe your listeners will know more. Constantinople. The end of everything. Quote, "The most infamous of wartime extinction was the destruction of Byzantine Constantinople on May 29, 1453." Let me just set this up to go to so I can do this set up, kind of condense the material a little bit. We have the Emperor Constantine in the very early fourth century moves the capital of the empire from Rome to this city in what is now Turkey.

It's been called Byzantium. He refounds the capital as Constantinople. Walls get erected.

It remains, it becomes a Christian, Greek speaking empire that lasts a thousand years.

Victor Davis Hanson: Beyond the West, when the West falls. A thousand years.

Peter Robinson: A thousand years after Rome itself falls. Eighth century AD, we have the rise of Islam. And now pressure begins to be brought on the Christian Byzantine empire century after century after century after century. And in 1453, on May 29th, what happens?

Victor Davis Hanson: Black Tuesday. I lived in Greece and on Black Tuesday I was awakened by my landlord to make sure that I went to mass or Greek Orthodox services to lament that the Emperor Constantine had been marbleized and saved by the archangels. And he was going to come one day and free Constantinople. So around noon, the Ottomans under Met that had brought this.

Peter Robinson: Sultan.

Victor Davis Hanson: Yes, Sultan. And he had, his father and everybody had said they're declining. The empire of 20 million is now shrunk down to about a million. And there's only the city of a million people because of the fourth crusade where Christians, Western Christians sacked it. It's in decline. All we have to do is wait. And it's very lucrative because it's still a beacon of Western culture in our area. So Venetians come in, Genovese come in, they bring in crossbows, they bring in gunpowder. It's very—

Peter Robinson: It's in trade with these people.

Victor Davis Hanson: Exactly. So most Sultans had let it live. And there was actually Turkish people within the city. This amendment, the great says, no, no, no, no, no, no. I am 19. I'm going to destroy this. And he systematically fortifies the Hell's Point, the entrance in the Black Sea. And he takes the Dardanelles, you can't go in or out. And he squeezes it. And when he surrounds the city, there's only about 7,000 to 8,000 actual combatants of a city that's no more than 50,000. And he thinks it's going to be easy. But the walls of Constantinople, the so-called built by the emperor Theodosus, are, they remain the most impressive walls in the world. They were a tripart system.

Peter Robinson: When you say remain, you mean today.

Victor Davis Hanson: Yes. You can vote you— Yes, you can see them today. Yes, you can see it.

Peter Robinson: There was a-- Istanbul, and go ahead.

Victor Davis Hanson: There's a mound, there was a moat, another mound, and then the so-called outer wall. And then with turrets, 20 to 30 feet high. And then in between a killing space of a plaza where there's nowhere to hide, and then the massive inner walls of 40 feet, and gates where they could retreat in. And no one had ever taken that. The Fourth Crusade came in through a fluke on the seaside. But no army had ever. It was like a triangle. So there was sea on the Golden Horn, sea on the sea of Marmara. And then the exposed land had the Theodosian walls, five to six miles. And they camp out there, and they cannot take it. And with this reduce, they have these brilliant Genovese that are fighting for them, some mercenary. They call to Christendom, "Help us. We're your Christian brotherhood." And they said, "Nah, you're orthodox, and we're not going to help you." They said they were.

Peter Robinson: How many forces has Mehmet got?

Victor Davis Hanson: It's debatable, but he's probably got 250,000 and probably at least 100,000.

Peter Robinson: On land?

Victor Davis Hanson: Yes. The whole force is 250,000 and probably 100,000 on land. And he's got at least 10,000 cracked Janissaries, the mercenary elite. And they can't take the city.

Peter Robinson: So it says something about those walls, that 7,000 defenders can hold off 100,000.

Victor Davis Hanson: Yes. And they have a massive, the so-called Hungarian cannon that was built by a Hungarian. And they have these huge cannon, and they knock down holes on the walls. At night, the civilians are all mobilized. And then Gustiani, the famous Genovese merchant, gets wounded. And he is the—

Peter Robinson: The leader of the defenders.

Victor Davis Hanson: And he, even though he's Italian, he's the spiritual anchor. And he, for some reason, they withdraw the contingent. And people say, "Oh my God, he's withdrawing." And they panic. And when they leave the outer wall, they don't do it in order to get into the inner wall. They leave the gates open. And then it's every man for themselves. And it's one of the most heart-wrenching descriptions in Byzantine literature. We have about 11 different sources in Italian, Turkish, Byzantine Greek. And it's a free-for-all. And they slaughter everybody. And 7,000 go into the great church that you can go to today at Hagia Sophia. They think the archangel is going to come down and save them. He doesn't. The Janissaries break in. And it's three days of absolute slaughter, wreckage, sort of like the Fourth Crusade. But the—

Peter Robinson: More deadly. These are being-- Civilians are being slaughtered.

Victor Davis Hanson: Yes. And so at the end, after three days, Mehmet and the Sultan, his entourage, realized that we need somebody to run this city because we're not going to destroy it like Carthage or Thebes. We're going to take a new DNA and use it because it's a beautiful city. And we'll put Menoroth.

Peter Robinson: We could use those walls. We could use that church.

Victor Davis Hanson: We could use those walls. It's got the best location in the world. We need menorets on Hagia Sophia. We'll turn it into a mosque. So they get a few there. And within 50 years, they have wiped out what had one time been 20 million Byzantine Christian Greek speakers. In the ancient home, the Seljuk Turks who became Ottomans were not indigenous to that area. Something Mr. Erlian today does not understand. This was from time memorial a Greek-speaking area way back. Now it never would be again.

Peter Robinson: Cardinal-- St. John Henry Newman referred in this history, he referred to the Turks as the people who had destroyed half the civilized world. He was very conscious that Byzantine culture represented half of Christendom up until it was gone. Okay. But what is the legend that you referred to at the very beginning?

Victor Davis Hanson: Constantine didn't die.

Peter Robinson: That the last emperor—

Peter Robinson: - was turned into marble.

Victor Davis Hanson: Yes, he's marbleized. And he went into a secret chamber in Hagia Sophia. And he was lifted up into heaven. And he's in suspended animation. And in 1928-21, there was the Megala Iida, the idea that after World War I, Greeks had bet on the winning side and Turks had been on the losing side and they were going to reform. And they got almost to Ankara. And then—

Peter Robinson: The Greeks did.

Victor Davis Hanson: Yes. And maybe he was going to come out of marbleization and they would have Constantinople as the spiritual, political, religious home of Hellenism again. And then, of course, they were betrayed by the Europeans. They cut off and they were slaughtered.

Peter Robinson: So we have-- and that is an extinction. Well, I suppose the most dramatic way of describing how completely it ended, we still have Orthodox. We still have the Orthodox Church.We still have devout Greeks who remember that day and shake young Victor awake and saying, you must remember this day with us. You must go to Orthodox Mass. But Hagia Sophia, which was for centuries the largest church in Christendom, is now a mosque.

Victor Davis Hanson: Yeah. And today, if you or I were to walk along Ionia and see the ancient-- the richest part of Hellenism, the ancient-- or the pre-Socratic philosophers, the lyric poets were if we went to Didama or Pergamum or Ephesus or Miletus, it's Ottoman Muslim culture, Turkish. If we went to Constantinople, especially under Mr. Erdogan, it's—

Peter Robinson: There's no current leader of Turkey.

Victor Davis Hanson: There are Christian churches are being shut down. And this was a UN historical site, Hagia Sophia, the great cathedral. And now it is a mosque again under his leadership.

Peter Robinson: Well, it was a museum and now it's back to being a—

Victor Davis Hanson: He took it away from its UN status and made it into a mosque. And there are no Greeks to speak of who speak Greek and openly are Christian in all of what was a 20 million person. There is no such thing as Hellenism or Greek speakers outside of Cyprus and Greece except the diaspora, say, in Europe, the United States. He extinguished it.

Peter Robinson: This brings us to the last of these four, which I'm going to try to-- this is like turning an ox into a bullion cube here. We're engaging in an act of compression victory. Hernán Cortés lands in Mexico in 1519. He has 500 soldiers, about 100 sailors. The sailors climb out of their ships and march with him. He has 600 men, some horses, some guns. And two years later, he destroys the Aztec capital, 1521. And well, let me quote you. "Although they had become familiar with Aztec civilization over the prior two years, the Spanish almost immediately sought to obliterate its religion, race, and culture. In their view, they had more than enough reasons to destroy the Aztec empire." Close quote. Now, the book describes the way Cortés does-- he's a politician as well as a general. And he discovers that many of the subject tribes in the area hate the Aztecs. So he assembles a force that he can use against the Aztecs. The history here is rich and fascinating. It is an astonishing story of how this small Spanish force conquers Mexico.

But what I want to get to-- the Spaniards had more than enough reasons to destroy the Aztec empire. Could you explain that a little bit? This was not just raw hunger for land. It wasn't just gold lust. What else was going on?

Victor Davis Hanson: Unfortunately, for the Az-- Tenochtitlán, the historic capital, they had an empire of four million people. And they-- this was 1492 to 1519 was only 30 years, not even that. So they didn't really know what-- the Westerners didn't know what was in Mexico. They'd heard of this legendary—

Peter Robinson: 1492 being Columbus' first encounters, the new one.

Victor Davis Hanson: And Cortés was a minor official. He wasn't a general. He wasn't-- he was just-- he was an entrepreneur. And he got it in his head that he got temporary permission from the governor of Cuba to form this tiny force and go explore. But he knew that he wanted to do more than that. So he goes in, he marches, and he's entertained. They cannot-- they think he's-- it's debatable whether they really think he's a god. But they've never seen people with white skin. They've never seen people with armor. They think the horses and the man are one person. They're centaurs. They think the dogs are-- they've never seen these mass-steeped dogs before. They've never heard gunpowder before. They have no idea what steel is. Steel. They use obsidian blades. They don't know what Toledo steel is. They have no idea what the wheel is. They've never seen-- except in toys. So these guys come and they think they're gods. And then the more they see, they like to eat, they like to drink, they like to have sex. They bleed and they start to get wary. So there's a faction. And on the Nochetriste, the sad night of sorrows, they almost get completely slaughtered. And they're chased out. They come back with more soldiers. Never at one time did he ever have in one place more than 1,500 soldiers. Unfortunately for the Aztecs, they were not dealing with Plymouth Rock and pilgrims. They were dealing with the most warlike, deadly Europeans in the world. They were dealing with the Spanish who had just finished the Reconquista and fought for 300 years against Islam. They had been fighting during the Reformation field. They had been in Italy.

Peter Robinson: By the way, both of those are religious wars.

Victor Davis Hanson: Yes. And the people who came with Cortez were some of the most brilliant-- Pedro Alvarado, people like that-- some of the most skilled soldiers. And they had horses. And they loved to fight. And they were accompanied by a zeal that was in reaction. It was just the very beginning of the Counter-Reformation. And they felt that their religion was going to be questioned unless they got souls. They come here and they say, oh my God, 25,000 people are being sacrificed, human sacrifices, on the Great Pyramid.

Peter Robinson: Now this, by the way, this has become a matter that intrudes into political correctness.

Peter Robinson: And Lord knows I'm not an expert on this. But in my little lifetime, we were taught that the Aztecs engaged in widespread human sacrifice. Then we have a revisionist school that says, well, no, wait a minute. The only authority we have on that is the Spanish documents. And they clearly overstated what was taking-- And now it's my understanding that the archaeologists are discovering more and more and more evidence that it was indeed not just occasional human sacrifice, but a regular feature, a daily feature of-- I mean, it's almost as if in the island of Manhattan every single day there were some human sacrifices at the top of the Empire State Building. Is that right?

Victor Davis Hanson: No, it's absent.

Peter Robinson: And you insist on that in this book.

Victor Davis Hanson: You look at contemporary archaeological reports that confirm what Bernal Diaz said or Prescott said in the 19th century great historian. But more importantly, there are pictorial skull racks that can be-- that they have numerical records. And we know that the lake that surrounds-- it's an island civilization, Tenochtitlán, was polluted because of a festival where they may have sacrificed 20,000 or 30,000 or 40,000 within a four-day period. It was something like Auschwitz. And they threw the bodies into the-- so I don't think anybody-- what they challenge is there are anthropologists that say, well, there was no large herbivores. So there was no source of protein for the sophisticated, systematic, centrally planned economy. And they were very architecturally advanced. And they needed, in this very urban society of a quarter million people. And the Spanish said it was more impressive than Venice. Venice at its height. It had a very sophisticated system of law.

Peter Robinson: But the Spanish are not saying these people are barbarians.

Victor Davis Hanson: No, they're not. They're saying that they are very sophisticated people, which means that we have-- they're even more dangerous because they have spread three or four things that we think are terrible. They're cannibals, and we know that they did eat portions of the sacrificial. They engage in ritual sodomy, which we don't think is permissible. And they sacrifice humans. And so it's our duty as emissaries of Christianity, defined as Catholicism of the Spanish Reconquista period, to kill people to save them. We've got to kill the people who are doing this. And then we're going to surround all of the people around the lake of the empire who resent being harvested. When they lose-- war is not fought to take land. War is fought to get captives, so-called flower wars. So you don't try to kill somebody. You knock them down and tie them up. And you drag them. And then the guy that has the most captives is very famous. And then they're brought up to Tempo Major, and they're sacrificed. And the Spanish find this horrific, but they also discover that it's in their interest, because they're not-- when you get Spanish Toledo steel, male helmet, especially if you're mounted, Toledo blade, arquebus, gunpowder—

Peter Robinson: They can't capture you.

Victor Davis Hanson: And they're trying to capture you. They're not trying to-- if they would just swarm them and cut their throat, they might have won. But they're trying to knock them down and tie. And then the final thing about it is they don't know who Cortez is. He's more gifted than Alexander in some ways. He's more gifted than Mehmet. He's probably more gifted than even Scipio. And nobody knows that he's never had any experience in this. And it turns out he is a natural military genius. And every time they should have been extinguished, they lose, they've got-- they're sick. He comes out.

Peter Robinson: He finds a way out.

Victor Davis Hanson: And he finds a way out. He's a military genius. And he's ruthless. And he—

Peter Robinson: So, Victor, on the Spaniards, wipe out the Aztecs. To what extent-- I'm just trying to think, to what extent is that an annihilation of the kind that takes place in the other examples? We have-- I have a Mexican friend who said, well, just look around Mexico City. You see very few people of Spanish descent and millions of people of Indian or Aztec descent. They didn't destroy them.

Victor Davis Hanson:  Aztec is a key word.

Peter Robinson: Oh, is that so? And then the other thing is there is-- I checked on this. I went online. There is still a duke de Montezuma in Spain today. They took the grandchildren of the last emperor of the Aztecs back to Spain.

Victor Davis Hanson:  They did.

Peter Robinson: And honored them by making-- ennobling them. OK, so what gets destroyed? What is ended?

Victor Davis Hanson:  Nahutul, the language exists in Mexico today. I have been in my hometown where people who have come from Mexico and Spanish speakers cannot understand them. So-- but Aztecs as a city, as a unique culture among indigenous people is destroyed. So when you talk to somebody and you say, well, if somebody says to you, the San Diego Aztecs, they never say to San Diego-- you know, the sports team, college team, they never say to San Diego Tlaxcalans or Toltecs. Why do they not do that?

Peter Robinson: They couldn't spell it apart from anything else.

Victor Davis Hanson:  Because first of all, we have a chronicle of this majestic civilization and how advanced they were, as I said, in architecture, town planning, sanitation, very sophisticated. But they don't want to talk about the downside of cannibalism, human sacrifice. But the point is, if we were transported to 1521, Cortes would have never been able to defeat them without the help of the Tlaxcalans. He had-- and their allies, he must have had at least-- he drew on an army of-- over that two and a half year period of over 200,000. And they probably lost—

Peter Robinson: Because the surrounding tribes are tired of being raided for captives who would be sacrificed.

Victor Davis Hanson: As soon as the word got out that Cortes was back again, that he was not annihilated, he came back. And this time he had brigandines or boats. And he would navigate in a combined amphibious and land attack on the causeways and by land. And he figured out how to beat them. And he gave them an ultimatum that I want to save your city and make it the capital of New Spain. But if you don't, I'm going to destroy it. That word went out. And all of a sudden, these fickle allies that sometimes had helped them, they-- they masked. I said, my God, you're going to destroy—

Peter Robinson: We have a moment here.

Victor Davis Hanson: And I can pay them back for all the things they did. And they ran wild. And in fact, he says in his letters and so does contemporary sources that he regretted that once he unleashed these people, they butchered and butchered and butchered and they destroyed Aztec's central civilization. So yes, there are people who survived the Holocaust, and that's what it was, but centrally planned civilization with a precinct class, an urban center, an-- no, it's now Spain and sp-- the Spanish build on top of it, right on top of Tenochtitlan. They used the very foundations of the destroy-- they destroyed it down to the foundations. It's gone. And there is no formal Aztec culture. There are indigenous people, of course, because they form about 1% of the population in what is Mexico, the Spanish.

Peter Robinson: And you can go to the Socoló in Mexico City today. And on the very side of the Templo Mayor, the great Aztec pyramid is a gigantic Spanish cathedral which says among any-- among other things what it says is, we won, we lost.

Victor Davis Hanson: Yes. They were-- they were actually given special-- there were concessions given from the Spanish to the Flaxcawans, and they honored them. In other words, that you are not going to be subject to the same degree of subservience, of the scattered remnants of anybody who fought for the Aztecs.

Peter Robinson: I see. Last questions. You've got a number of themes of relevance to us today. One theme here is the capacity of the doomed for self-delusions. The Thebans failed to grasp the military revolution that's taking place under Philip. They failed to grasp, even though they have some intelligence, they have reasons to question their own judgment of Alexander's ability, they say no. The Carthaginians failed to grasp the change in Roman power and determination over two centuries. The Byzantines cannot bring themselves to imagine that a city that has lasted a thousand years could fall, let alone fall in a day. The end of everything, I'm quoting you, the gullibility and indeed ignorance of contemporary leaders about the intent, hatred, ruthlessness, and capability of their enemies are not surprising given unchanging human nature. At the beginning of the program, you talked about the plight of the Greeks, you talked about threats against Israel. What are Americans to make of this?

Victor Davis Hanson: Well, I think we should take these lessons very seriously, both from the point of the attacker and the attack, because you mentioned some of the commonalities. At the end of the book, I give you a kind of a common denominator blueprint. People who have not been defeated or accustomed to a position of superiority culturally, militarily, they think that they're invulnerable forever and they're not aware of insidious decline. The things that Alexander took is not the themes of the Pamanandas and yet the walls look as stout as they ever were and the people are the same, they think. Same thing with Carthage, the same thing with Constantinople. Nobody's ever, they said nobody's ever going to get through the walls. They tried just early, you know, 50 years early, they couldn't do it. We're invulnerable and people said, "Well, we're not the same people." They don't think that anybody would ever dream of extinguishing them. We've been here a thousand years. We're the children of Oedipus. To note, Shitlan, we've been here, this is the pinnacle of our civilization, etc. Constantinople, this is the city of Constantine and Justinian. We can't fall. So there's an unreality and then they have no idea who they're facing. They have no idea what's in the mind of Cortez. They have no idea.

Peter Robinson: Do we have any idea what's in the mind of Xi Jinping, of Vladimir Putin?

Victor Davis Hanson: We have no idea. We think that he, we think that Xi thinks as I think George H.W. Bush, George W. Bush, Bill Clinton, Barack Obama, all bipartisan. They all thought that he is so impressed with Western civilization. He's globalizing, he's changed his economy. Yes, he's rough around their edges but our leisure, our affluence, globalization will acculturate them and China will take their place among the family of nations.

Peter Robinson: Because of course they want to be like us.

Victor Davis Hanson: They don't understand that that is exactly what the Byzantines said about the Ottomans, that's exactly what people said about Alexander, that's exactly what they said about the Carthage. And these people don't understand, so in our, whether it's Putin, so when Putin says I'm going to use nuclear weapons if I lose. And there's been about 17 threats from high members of the Russian military, high members of the Russian parliament such as it is and Putin himself, tactical, nuclear, even. We say this is crazy. They would never do that. We never say, well if I was going to lose and be humiliated or if I wanted Ukraine the breadbasket of the old Soviet Union and ports on the Black Sea and a window right under Europe I'd be willing to do a lot of stuff for it. And I've done, so there's an unreality and then on the part of the attacker they need to understand what the attacker is capable of. I just add one quick thing. I also mentioned the serial threats that China has given Taiwan. And they even made a brief film about nuking Japan if it interfered, called in the war criminal.

Peter Robinson: The Chinese have.

Victor Davis Hanson: Chinese commerce.

Peter Robinson: And they've distributed that in China.

Victor Davis Hanson: And just like Er And just like Er Israel, he said I'm going to do the same thing to the Armenians and he just ethnically cleansed 100,000 of them. I'm going to do the same thing as my grandfathers. They had the solution. So these people are serious when they say this. But I had one line in the epilogue to that effect of what China and that's a very big market, the Chinese market. And I've had a lot of success with other books.

Peter Robinson: Victor, one other of the... But anyway, just to... Yeah, go ahead. I'm sorry.

Victor Davis Hanson: I was given notice by the Chinese publisher. I had to take out that line or there would be no book sales and everything would be cancelled, no Chinese translation in the epilogue.

Peter Robinson: So they are serious.

Victor Davis Hanson: So I didn't do it and there's not going to be a book in China. That book will never be in China.

Peter Robinson: Oh, well, all right. Yes. You don't think somebody will... Okay. That's a good conversation. Another of the themes that strikes me here, war changes things. I'm quoting you again, the end of everything. Once Alexander grasped the full extent of Theban hatred, he concluded that only destroying the city rather than merely capturing it would end Greek opposition to Macedonia. Cortes decided there was no way to root out the imperial system without knocking the Aztecs' infrastructure down upon them. Such revised decisions are common throughout military history. Near the end of World War II, US Army Air Corps General Curtis LeMay decided the only way to destroy Japan's dispersed manufacturing, which was deeply embedded within the neighborhoods of Tokyo, was to ignite the city and we get the fire bombing of Tokyo. Again, war changes things. So we are supplying the Ukrainians with weapons and materiel. We have two carrier groups in the Eastern Mediterranean to support the Israelis and we have our forces disposed, our naval forces disposed in the Pacific. We don't know where our attack submarines are because there's no reporting on that and there shouldn't be, but we're concerned about Taiwan. We're very concerned about Taiwan. What are the lessons of the end of everything for Americans as we face trouble, military challenges on three fronts?

Victor Davis Hanson: If we would look at ourselves dispassionately and not say we're Americans, we're always numbered countries. We would say the following. We've never had in terms of the percentage of GDP debt or in actual numbers except for a brief period in World War II, $35 trillion we owe and we are borrowing $1 trillion every hundred days. It's completely unsustainable. We've never had the military admit to us that it is short 40,000 troops and they don't know where to get them at a time when the American population has never been larger. We have ...

Peter Robinson: The Army missed its recruiting goals last year by 10%.

Peter Robinson: And that was not the first year.

Victor Davis Hanson: We have had a porous border. We have never had no border at all. It ceased to exist. We've had 10 million people walk across without audit. We've never seen anything like it. We have the largest number of foreign-born residents, both in numbers, 50 million and in percentages of the resident population, 13. We've ever had at a time when we haven't lost confidence in the melting pot. Okay. We've had high crime areas before, high crime periods. We've never had a period in American history where our elites say that crime is not crime. It's a social construct and you have to let somebody, a violent criminal out the same day that he's arrested. That's a new theory, critical legal theory. Okay. We're a multiracial society and we're the only successful multiracial democracy. We know that it depends on relegating your tribal affiliations to the general idea of being an American. We are regressing into tribalism. So when you look at all of these challenges and you look at the symptoms, we have never done anything like Afghanistan. Just completely flee and leave $50 billion in weaponry to the terrorist organization that's selling all over the globe. Never had that before. We have never had since World War II a Verdun. We have passed the numbers of dead and wounded in Verdun. We're above 700,000 wounded, missing or killed Russians and Ukrainians and we're headed to Somme territory and nobody has any idea how to stop this. Russia is not going to be able to take all of Ukraine and we are not going to be able to get back the Donbass in Crimea. So it's going to continue. Nobody has an answer. Nobody takes serious that the Chinese would be crazy enough to go across the Taiwanese Strait and try to take that city. They say they can do it. So my point is, never have we been faced with such existential challenges in the post-war period. At a period when we are so weak, or at least we're not naturally weak, our constitution is there, our natural resources, we lead the world. But when you look at crime, when you look at debt, when you look at the border, when you look at our universities, which were the envy of the world, they were the engine that drove American culture and power and technology and they're in crisis. Science is in crisis.

Peter Robinson: So we're like the Thebans. We're not the same people.

Victor Davis Hanson: We're not the same people. We're not the same people that maybe we have it in us. We're not the same people who stormed Omaha Beach when the first 2,000 people were mowed down. They just kept coming.

Peter Robinson: Victor, I want to play a brief video excerpt.

Video Excerpt:

-You both work here.

-You're among the intellectuals who are offstage, the members of Congress are on stage, but they're always turning around saying, Blumenthal, did I get that right?

-Bridge, what about this?

-Is this town serious?

-Do you feel a sense of seriousness descending that is adequate to the moment?

-Absolutely not.

-Absolutely.

-We're in a world of warfare and we're not on a war footing.

Peter Robinson: Victor, Washington is, there's no sense of seriousness in Washington.

Victor Davis Hanson: No. We think that the most important thing is counseling student debt or inaugurating new woke programs, but it's going to take us seven years to replace the javelin anti-tank weapons. We're short 155 millimeter. That was our signature. We were the biggest producer of shells in the world.

Peter Robinson: Victor, can I ask one, the book is called The End of Everything and you describe four episodes, all of which take place, the most recent of which takes place about four centuries before the invention of nukes. So let me quote if I may, here's a quotation from Clausewitz, who saw the Napoleonic Wars as a young Prussian officer and meditated on military theory and the rest of his life. Here's Clausewitz. This has always bothered me. “If one side uses force without compunction, that side will force the other to follow suit. Even the most civilized of peoples can be fired with passionate hatred of each other. The thesis must be repeated. War is an act of force and there is no logical limit to the application of that force.” Thebes wiped out. Carthage leveled.

Constantinople civilization blotted out. His text gone. And now we have nuclear weapons.

Victor Davis Hanson: Among other things, we have AI, we have bio weapons apparently, an accidental release from the Wuhan lab.

Peter Robinson: So what I'm desperate to do here is to end on an upbeat if I can find one anywhere. Should we take encouragement from the long period of the Cold War? When we had nuclear weapons but managed to defeat Soviet communism without any use, without warfare, without a major war, without a major confrontation, should we be cheered by that?

Victor Davis Hanson: We should learn—

Peter Robinson: Or are we doomed?

Victor Davis Hanson: No, we're not doomed. We need to learn from wise men like Franklin Roosevelt, Harry Truman, Dwight Eisenhower, John Kennedy, even to an extent Lyndon Johnson, Richard Nixon and the rest of them.

Peter Robinson: They all had one thing-- You mentioned Ronald Reagan?

Victor Davis Hanson: Yeah, I'm getting to him.

Peter Robinson: Oh, all right.

Victor Davis Hanson: And in a period of doubt where people had questioned their so-called Neanderthal approach to human nature that they believed that deterrence and not dialogue or the UN kept the peace, along came Ronald Reagan. And he said-- he basically said the degree which we are safe is the degree to which we help our friends and tell our enemies to be careful because we will defend us and we're going to have the capability to do it. Deterrence, deterrence, deterrence, which is just a Latin word to scare somebody off from doing something stupid. And if you don't believe in deterrence, then as Vegeta said, if you want peace, prepare for war. If you want war, prepare for peace.

Peter Robinson: Victor, will you close our conversation by reading a passage from-- Victor Davis Hanson, the author of The End of Everything, reading a passage from The End of Everything.

Victor Davis Hanson: Well, thank you. I'm going to give you the degree that I can read well.

“The fate of the Thebans, Carthaginians, Byzantines, and Aztecs remind us that what cannot possibly happen can indeed on occasion occur when war unleashes timeless human passions and escalation rather than reduction in violence becomes a role of conflict.”

“In this regard, we should remind ourselves that we really do not know the boundaries of what may follow from the dispute in the Ukraine or a standoff over Taiwan or strikes on nuclear facilities in Iran.”

“Like their predecessors, modern attackers will on occasions insist on impossible turn.

They will sometimes become further enraged by prolonged and toxic resistance.”

“The targeted will believe that doom resistance may not be so impossible, that their defenses are underestimated while their enemies' powers are exaggerated, and that reason rules war.”

“And so they will hope that even their own defeat cannot possibly entail the end of everything.”

Peter Robinson: Victor Davis Hanson, author of The End of Everything. Thank you.

Victor Davis Hanson: Thank you very much for having me.

Peter Robinson: For Uncommon Knowledge, the Hoover Institution, and Fox Nation, I'm Peter Robinson.

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• v.28(1); 2015 Jan

Current and Past Strategies for Bacterial Culture in Clinical Microbiology

A pure bacterial culture remains essential for the study of its virulence, its antibiotic susceptibility, and its genome sequence in order to facilitate the understanding and treatment of caused diseases. The first culture conditions empirically varied incubation time, nutrients, atmosphere, and temperature; culture was then gradually abandoned in favor of molecular methods. The rebirth of culture in clinical microbiology was prompted by microbiologists specializing in intracellular bacteria. The shell vial procedure allowed the culture of new species of Rickettsia . The design of axenic media for growing fastidious bacteria such as Tropheryma whipplei and Coxiella burnetii and the ability of amoebal coculture to discover new bacteria constituted major advances. Strong efforts associating optimized culture media, detection methods, and a microaerophilic atmosphere allowed a dramatic decrease of the time of Mycobacterium tuberculosis culture. The use of a new versatile medium allowed an extension of the repertoire of archaea. Finally, to optimize the culture of anaerobes in routine bacteriology laboratories, the addition of antioxidants in culture media under an aerobic atmosphere allowed the growth of strictly anaerobic species. Nevertheless, among usual bacterial pathogens, the development of axenic media for the culture of Treponema pallidum or Mycobacterium leprae remains an important challenge that the patience and innovations of cultivators will enable them to overcome.

INTRODUCTION

As proposed by Robert Koch, a pure culture is the foundation of all research in infectious diseases ( 1 , 2 ). The first isolation of a bacterium enables the design of experimental models to analyze virulence and to complete Koch's criteria, thereby establishing a link between microorganisms and infectious diseases ( 3 ). Bacterial culture also enables the study of the antibiotic susceptibility of bacteria and is the first step in establishing recommendations for effective treatment ( 4 , 5 ). Obtaining a pure bacterial culture also enables genome sequencing of these strains ( 6 , 7 ) and proteomic studies to highlight specific proteins and analyze their antigenicity by immunoproteomic techniques, eventually facilitating the production of these proteins, which serve as antigens for serologic tests ( 8 ). Finally, pure bacterial culture enables manipulation and transformation by adding or deleting genes to analyze the cause of virulence and antibiotic resistance and the invasive potential of bacteria. However, over the last 30 years, the same progress observed with molecular biology has not emerged with culture in clinical microbiology ( 9 ).

Bacterial culture is frequently more difficult and often requires more training than molecular techniques. Consequently, the number of microbiologists specializing in anaerobic bacteria has declined steadily for 30 years, and currently, there are few specialists compared with the number of specialists during the 1970s. Renewed interest in bacterial culture was initiated in large part by clinical microbiologists ( 10 , – 12 ) specializing in intracellular bacteria. They have developed axenic media, which are sterile media containing no living organism except the one being cultivated, to culture extremely fastidious bacteria ( 11 , 13 , 14 ). We propose here, after a brief report of early strategies of culture, a comprehensive review regarding past and current culture techniques used for the culture of fastidious bacteria. In an additional review , we elaborate on the progresses allowed by new identification methods and the application of all these advances through the example of the study of human gut microbiota by culturomics ( 15 ).

EARLY STRATEGIES AND GENERAL PRINCIPLES

The first culture media were developed empirically, using environmental components. Overall, the choice of nutrients, atmosphere, temperature, and time of incubation are the 4 primary elements that determine the growth of bacteria ( 16 ).

Nonselective Culture Media

Nonselective culture media contain no inhibitors and should permit the growth of most of the microorganisms present in the clinical samples studied. Meat infusions or heart or brain extracts were the initial substrates used empirically. Yeast extracts remain among the major components of several culture media ( 17 ). Vegetable components can also be used ( 18 ).

Peptones, which are carbohydrate-free sources of nutrients, defined as soluble products from the enzymatic hydrolysis of proteins, are more often used as nutrient additives in culture media. Diverse enzymes can be distinguished, and the diverse substrates are meat, casein, soya, and gelatin.

Solid agar and coagulated serum.

Clinical microbiology was revolutionized by the invention of the petri dish ( 19 ), which, because of the use of a transparent lid, has allowed us to observe colonies and to limit contamination. The addition of kitchen components, such as gelatin or agar, has led to the design of solid culture media ( 20 ) and, consequently, the possible description of bacterial species in pure culture. The use of solid culture media and petri dishes was probably comparable to the progress by Koch and Pasteur in modern clinical microbiology. Other solidifying components can be used, such as coagulated eggs, as in Lowenstein-Jensen medium, which is used for Mycobacterium culture ( 21 ). Coagulated serum can also be used, notably in Loeffler medium, which was designed for performing Corynebacterium culture ( 22 ).

Enriched media.

Enriched media are designed to facilitate the growth of fastidious microorganisms. The primary enrichment component is blood, which provides hemin and other nutrients. This component is frequently added to agar media in several commercial media to notably increase the growth of anaerobic bacterial species ( 23 ). Blood agar, which was initially accidentally used for Mycobacterium tuberculosis culture, was also shown to be a cost- and time-effective method, with better growth than with the egg-based agar reference medium ( 24 , 25 ).

Selective Culture Media

In clinical microbiology, a challenge has been to isolate the pathogenic microorganisms from complex microbiota in pure culture ( 26 ).

Organic and inorganic components and minerals.

Deoxycholic acids are frequently used as Gram-positive bacterial inhibitors. Bile salts are also used as Gram-positive inhibitors, while crystal violet inhibits the growth of Gram-positive bacteria ( 26 ). Bismuth sulfite agar uses the ability of bismuth to inhibit most Gram-positive and Gram-negative commensal organisms for the culture of enteric bacilli ( 26 ). Finally, Chapman agar is based on the ability of staphylococci to grow in culture media with a high NaCl concentration (7.5%) ( 26 ).

Antibiotics and antiseptics.

Antibiotics are not specific to bacterial species and generally inhibit the growth of many bacterial genera or species. As examples, ANC (nalidixic acid and colimycin) medium promotes the specific growth of Gram-positive bacteria, and Campylobacter blood agar is an enriched selective medium that contains 5 different antimicrobial agents. Antibiotics are used in Mycobacterium culture media primarily as a cocktail that includes polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin ( 27 ). Antiseptics, such as bromocresol purple, were used previously in agar culture media to select Enterobacteriaceae in particular, with different uses, such as the isolation of injured coliforms from drinking water ( 28 ).

Sample Decontamination

Another strategy is to decontaminate the samples to decrease the rapid overgrowth common in commensal bacteria ( 21 ). The N -acetyl– l -cysteine–NaOH method was initially used for culturing Mycobacterium spp. ( 29 ). Chlorhexidine ( 30 ) was used to decontaminate the sputum of cystic fibrosis patients, with the aim of culturing nontuberculous mycobacteria ( 31 ). El Khechine et al. used chlorhexidine to decontaminate stool samples before Mycobacterium species culture ( 32 ). This noninvasive method obviates the requirement for gastric aspiration without a difference in pulmonary tuberculosis diagnosis ( 33 ). Finally, lytic phages have been used to decontaminate the normal flora from sputum before M. tuberculosis culture to replace the use of antibiotics ( 27 , 34 ).

Temperature and Atmosphere Control

Temperature..

Most of the species implicated in clinical microbiology are mesophilic bacterial species, and these species grow at medium temperatures of 25°C to 45°C. In clinical microbiology, one of the most famous examples of the requirement for the use of an adapted temperature for growth was highlighted by Rickettsia felis ( 35 ). After failing to grow the bacterium in human embryonic lung (HEL) cells at 37°C, researchers suspected that temperature growth was critical because this factor had been reported for many arthropod-borne microorganisms ( 36 , – 39 ). Finally, the first cell culture of R. felis was performed, using XTC2 cells obtained from Xenopus laevis oocytes growing at 28°C, which were usually used for arboviruses ( 39 , 40 ).

Atmospheres.

(i) aerophilic and anaerobic conditions..

Various atmospheres can be used in clinical microbiology. To describe microbes from human feces, roll tubes (defined precisely in the section on anaerobic methods, below) were previously designed ( 41 ). Nottingham and Hungate first used a nonselective medium but a stringent atmosphere consisting of 80% H 2 and 20% CO 2 to isolate nonidentified methanogenic Archaea from humans ( 42 ).

(ii) Microaerophilic conditions.

When the concentration of oxygen required to obtain growth is relatively low, the bacterial species are considered microaerophiles, such as Campylobacter spp., which can cause human infections involving primarily the gastrointestinal tract ( 43 ). Most Campylobacter species require a microaerobic atmosphere containing ∼5% O 2 , 10% CO 2 , and 85% N 2 for optimal recovery. Recently, a microaerophilic atmosphere demonstrated better efficiency than aerobic conditions in promoting Mycobacterium culture and was proposed as a routine condition for laboratories performing these cultures ( 44 ).

Incubation Time

Most clinical pathogens grow easily over 24 to 48 h in plate media ( 45 ), but several bacterial species require a much longer time, whereas most routine laboratories maintain cultures within 5 days. As a common example, Helicobacter pylori , the bacterium causing most gastrointestinal ulcers, requires a longer incubation period. Petri dishes with bacterial cultures were inadvertently left by B. Marshall in the incubator for 5 days ( 46 ). Thereafter, he observed small colonies constituting the first successful culture and isolation of a bacterium from the stomach of a patient suffering from gastritis. The association between the bacterium and gastritis was met with great skepticism by the scientific community, and to confirm this association, Marshall inoculated himself by drinking a solution containing vast amounts (10 9 ) of the bacterium. He developed acute gastritis, confirming Koch's postulates that the bacterium was the causative agent ( 3 ).

As another example, human infections caused by Bartonella species can cause trench fever; cat scratch disease; and, under particular conditions, bacillary angiomatosis, peliosis hepatitis, endocarditis, or chronic lymphadenopathies ( 47 , – 49 ). Usually, the growth of Bartonella spp. is slow, requiring 12 to 14 days when blood agar is used, with certain isolates sometimes requiring longer incubation periods of >45 days ( 48 ). Bordetella pertussis colonies are visible in 3 to 4 days, ( 18 ), and Legionella pneumophila colonies usually appear on day 3 ( 50 ). Fecal samples should be incubated for at least 3 days to culture Campylobacter spp. ( 43 ). For the isolation of aerobic actinomycetes, such as Nocardia spp. and Actinomyces spp., an incubation time ranging from 2 to 3 weeks is recommended ( 51 ). Finally, for routine clinical microbiology, anaerobic species cultures should be incubated for at least 5 days ( 45 ).

Improving Collection and Transport Time to the Laboratory

The viability of organisms depends on several factors, such as transport time, storage period, temperature ( 52 ), as well as specific storage systems to ensure that the microorganism's viability is maintained, although clinical or research samples should ideally be cultivated immediately after sampling. The use of an inefficient sampling device will lead to a misidentification of pathogens due to contaminant growth resulting from oxygen exposure or from an extremely small fraction of bacteria being recovered. Because of their low cost, utility, ease of use, and availability, swabs are still used to collect and transport various sample types ( 53 , 54 ). New swab types have been designed ( 55 ) to improve specimen collection, such as the new nylon-flocked swab with Amies liquid medium, which is more effective in bacterial recovery and which provides for more efficient release than classic swabs ( 54 ).

SPECIFIC STRATEGIES FOR FASTIDIOUS BACTERIA

Mycoplasma spp. do not have cell walls ( 56 ) and do not stain by Gram coloration. In 1898, Nocard and Roux reported the first culture of Mycoplasma mycoides subsp. mycoides , which is the agent of bovine pleuropneumonia ( 57 ), 15 to 20 days after inoculation of a semipermeable collodion sac. In 1960, Mycoplasma mycoides was successfully cultured in a medium that included a heat-stable defatted serum protein fraction, cholesterol, both saturated and unsaturated fatty acids, serum albumin, glycerol, and high concentrations of dl - or l -lactate and glucose ( 58 , 59 ). For diverse strains of Mycoplasma (known at that time as pleuropneumonia-like organisms [PPLOs]) ( 60 ), a variety of substances were used as supplements, including blood serum or ascitic fluid ( 61 ), lipid extracts of egg yolk, cholesterol ( 62 ), lipoprotein, lecithin, and acetate ( 63 , 64 ). The Eaton agent ( Mycoplasma pneumoniae ) was first cultured in a cell-free medium consisting of 70% Difco PPLO agar, 10% of 25% boiled yeast extract, and 20% unheated horse serum ( 65 ). M. pneumoniae was successfully isolated directly from patients with atypical pneumonia by using this medium and adding penicillin, amphotericin, and thallous acetate ( 66 ). Chanock et al. proposed an effective medium ( 65 ), and Tully et al. then developed the refined SP4 medium in 1979, which became the most widely used broth and agar medium for culturing of M. pneumoniae from clinical samples ( 67 ). This medium contains tryptone, peptone, PPLO broth, heat-inactivated fetal bovine serum, yeast extract, Yeastolate, and CMRL 1066 medium ( 67 ). Ureaplasma urealyticum (also called T-strain mycoplasma for tiny colonies) causes genital infections in humans. The total growth of Ureaplasma urealyticum was directly correlated with urea concentrations, with a maximum yield of organisms being observed with a 32 mM urea concentration. Finally, the addition of SP4 medium, glucose, urea, or arginine, depending on the suspected Mycoplasma species, was proposed. Indeed, common human mycoplasmas were then distinguished by 4 biochemical tests: glucose oxidation, arginine deamination, urea hydrolysis, and methylene blue reduction or growth inhibition.

Anaerobes are generally widespread, can be found in the environment, and are members of the normal human flora ( 68 , – 70 ), but only a few species are frequently encountered in significant human infections ( 71 ). The sensitivity of anaerobes to oxygen differs depending on the species ( 72 , 73 ). Loesche et al. ( 72 ) classified bacteria into three different categories according to their oxygen sensitivity: strict (bacteria cannot grow on medium with a partial O 2 pressure [pO 2 ] of >0.5%) ( 73 , 74 ), moderate (bacteria can grow in the presence of oxygen levels of between 2 and 8%) ( 72 ), and microaerotolerant (growth occurs in the presence or absence of oxygen in the medium; however, maximal growth occurs at intermediate oxygen levels) ( 72 ).

Culturing of strictly anaerobic bacteria in the absence of oxygen requires specific bacteriological techniques, which could explain the low frequency of isolation in many laboratories ( 75 , 76 ). Therefore, strictly anaerobic bacteria require complex media with many supplements for growth ( 77 , – 81 ). Hungate revolutionized the culture of anaerobic species by cultivating extremely oxygen-sensitive microorganisms, such as sulfate-reducing bacteria and methanogenic Archaea ( 76 ).

Brief history of earlier methods.

The equipment required for maintaining reduced oxygen tension ( 77 , 78 ) is simple and inexpensive. First, most of the oxygen is removed and is replaced by a suitable gas source.

(i) Physical reduction of oxygen tension.

The more commonly used technique for obtaining free O 2 medium consisted of using carbon dioxide or hydrogen through or over the surface of the medium to replace oxygen ( 82 ). Additionally, the candle technique was commonly used to cultivate anaerobes ( 83 , – 85 ). This technique consisted of burning a candle inside a sealed jar to replace the oxygen with CO 2 , which resulted in the production of 3% CO 2 under standard conditions ( 84 ).

(ii) Chemical reduction by reducing agents.

Strictly anaerobic bacteria grow in an almost total absence of oxygen, which is often toxic ( 70 ). These bacteria must be grown in a reducing atmosphere, in which energy is produced by fermentation or anaerobic respiration ( 86 ). Chemical techniques were based on reducing oxygen tension by adding reducing agents such as thioglycolate, glutathione, cysteine-HCl, sodium sulfide (Na 2 S) ( 82 ), or sodium carbonate-oxalic acid ( 84 ). Ascorbic acid-supplemented medium can also be used as an anaerobic medium. Recently, La Scola et al. successfully performed an aerobic culture of 6 strictly anaerobic species, including Fusobacterium necrophorum , using Schaedler agar supplemented with high-dose ascorbic acid or glutathione and with pH adjustment to 7.2 ( 87 ), suggesting substantial perspectives in routine bacteriology.

Constituents of anaerobic media.

Freshly prepared, highly enriched, and properly stored medium has been essential to enhance anaerobic bacterial growth ( 88 , 89 ).

(i) Major and minor constituents of suitable anaerobic media.

Anaerobic media must contain carbon sources, electron acceptors, and donor elements ( 90 ). The major constituents of suitable growth medium are represented by macroelements and metals in sufficient quantities ( 90 ), because the typical composition depends on a percentage of microbial dry mass ( 91 ). The addition of minor constituents (trace elements) is not required because most microorganisms use a unique carbon source to grow. Moreover, it is difficult to demonstrate which growth factor is required for an organism to allow better growth and which factor is essential ( 90 ). Nevertheless, a few bacteria require specific focus and particular constituents, such as many Bacteroides strains, which require the addition of vitamin K 1 and hemin to the medium for growth ( 92 ).

(ii) Growth factors.

Growth factors of undefined composition, such as yeast extract and pyrimidines ( 93 ), peptone ( 93 ), Casitone, Casamino Acids, or clarified rumen fluid, are still preferably added to culture media ( 90 ). These fluids contains volatile fatty acids and heme, which are not commonly found in the extracts or hydrolysates added to classical media ( 94 ). These fluids have been supplemented with glycerol, Trypticase, hemin, and mineral solutions 1 and 2 (modified 98-5 medium) ( 95 ) and sometimes with sterilized fecal extract ( 92 ) to allow higher percentages of anaerobe recovery than with other analyzed media ( 95 , 96 ). Further studies were performed in humans, and human sterilized fecal samples have been used as a nutrient source to provide specific anaerobe growth ( 42 , 97 ).

Anaerobic incubation systems used to increase the ability to cultivate anaerobes.

Clinical laboratories may prefer to use anaerobic jars and anaerobic chambers rather than roll tubes because of the delay and because of the complexity of this method. The Hungate technique may be used particularly for research activity ( 98 ).

(i) Anaerobic jars.

Currently, the GasPak system produces an atmosphere containing ∼10% CO 2 with sachets containing a dry powder or pellets of sodium borohydride and sodium bicarbonate, which react with water to produce hydrogen gas and carbon dioxide. The hydrogen produced then reacts with oxygen gas on a palladium catalyst, allowing greater water production to remove the oxygen gas ( 74 ). Anaerobic jars do not allow a continuously anaerobic atmosphere from sample reception to seeding to be established, and their use is inadequate for cultivating certain strictly oxygen-sensitive bacteria ( 72 ).

(ii) Roll tube method.

The Hungate method, which is based on the use of roll tubes, was introduced to prepare an anoxygenic medium for methanogen cultivation ( 99 , 100 ). The principle is based on replacing atmospheric oxygen with other gases, such as N 2 , CO 2 , H 2 , or other mixtures with adjusted pH ( 101 ), using an anaerobic glove box. After incorporation of all the elements, the medium is mixed and then boiled in an oxygen-free nitrogen atmosphere in the presence of resazurin as an indicator of the presence of oxygen. After resazurin reduction, the medium is cooled under nitrogen at room temperature. The flask is then capped and transferred to the anaerobic glove box, where the medium is distributed into serum vials or Hungate tubes. The containers are sealed with plastic stoppers, and the media are then sterilized in an autoclave before inoculation ( 76 ). The isolated colonies then form on the walls of the tubes after several days or weeks of incubation ( 101 ). The roll tube method greatly improves anaerobic growth ( 102 ) and remains the reference method for methanogens and archaea; however, this technique remains time-consuming and rather complex for large-scale studies due to the requirement for the use of roll tubes instead of petri dishes and cannot be used in clinical laboratories ( 99 ).

(iii) Anaerobic chambers.

The best method to ensure anaerobe viability is to incubate anaerobic organisms directly in an anaerobic chamber ( 103 , 104 ), never allowing exposure of the sample to air ( 105 ). This method, which is usable in both research and clinical laboratories because it does not require special training ( 106 ), is inexpensive. A prereduced medium with low redox potential is used to seed the samples.

Perspectives on anaerobic culture.

The human gut microbiota is the human site that contains a higher concentration of anaerobes ( 9 ). In future research projects, it may be important to continue to characterize human anaerobic microflora by high-throughput sequencing, to identify anaerobic clusters, and to design new anaerobic strategies that use antioxidant agents ( 87 ) that allow the cultivation of uncultivated anaerobic bacteria. This approach will clearly revolutionize the culture of anaerobic bacteria in routine bacteriology and in culturomics studies.

Spirochetes

Although most spirochetes are free-living chemoorganotrophs, spirochetes of the genera Borrelia , Treponema ( Spirochaetaceae ), and Leptospira ( Leptospiraceae ) are major human pathogens ( 107 ). Many members of the genus Brachyspira are important veterinary pathogens but occasionally have been reported to be associated with human pathology ( 108 , – 110 ). Spirochaetes are often not visible by Gram staining, requiring dark-field microscopy or special coloration ( 111 ). Molecular identification is usually based on group-specific flaB , ospA , ospB , and ospC genes and rrf (5S)- rrl (23S) intergenic spacer amplification ( 112 ). Among conservative housekeeping genes, rpoB could be a useful tool ( 113 ).

All borreliae are fastidious organisms and chemoheterotrophic and use carbohydrates, amino acids, long-chain fatty acids, or long-chain fatty alcohols as carbon and energy sources. Depending on the species, growth occurs under anaerobic, facultatively anaerobic, microaerophilic, or aerobic conditions ( 114 ). Because of the lack of or limited biosynthetic potential (the ability to elongate long-chain fatty acids to synthesize most amino acids, enzyme cofactors, and nucleotides), complex nutritional requirements are needed for spirochete cultivation.

Leptospira interrogans was previously considered the only species of this genus that is pathogenic in humans; however, a phylogenetic analysis of all isolated strains showed that among 22 currently recognized species ( http://www.bacterio.net/leptospira.html ), leptospirosis-causing bacteria belong to at least 11 different species ( 115 , 116 ) (see Table 2 ). Leptospirae can be isolated by inoculating ∼100 μl of a patient's blood in solid or semisolid oleic acid, which is an albumin-containing medium. Several media are available, namely, EMJH ( 117 ), polysorbate medium ( 118 ), and LVW agar ( 119 ). The presence of rabbit serum, polysorbates, and vitamins is essential.

Growth characteristics of obligate and opportunistic mycobacteria frequently encountered in clinical microbiology, including optimal temperatures a

Four different Treponema spp. are human pathogens, including 3 subspecies of T. pallidum ( T. pallidum subsp. pallidum [syphilis], subsp. pertenue [yaws], and subsp. endemicum [nonvenereal epidemic syphilis]) and the pinta agent T. carateum . None of these pathogens has yet been successfully cultivated in axenic medium, and isolation in pure culture is not a diagnostic option. Treponema pallidum subsp. pallidum can be propagated only in laboratory animals (rabbits) by intratesticular, intradermal, intravenous, or intracisternal inoculation ( 120 ). T. pallidum grows slowly, with a doubling time of 30 to 33 h ( 107 ), and a mean of 10 10 bacteria has been harvested from the testis of a rabbit. Treponema paraluiscuniculi , which is the causative agent of rabbit venereal spirochetosis, is genetically closely related to T. pallidum ( 121 ) and easily infects laboratory rabbits. The closely related agents of yaws and nonvenereal epidemic syphilis share similar features; however, their culture has been even less studied. T. carateum has also never been isolated; moreover, there are no successful animal model. These organisms are among the last few as-yet-uncultured major human pathogens. Similarly, among the numerous Treponema phylotypes found in the oral cavity, only 10 different species have been cultivated, with Treponema denticola being the most extensively studied ( 122 ). The development of an axenic culture of pathogenic Treponema remains a challenge for microbiologists.

The genus Borrelia is essentially composed of the Lyme disease group and the relapsing fever group ( 123 , – 125 ). Many Borrelia species, particularly those species of the Lyme disease group, are routinely cultured in liquid Barbour-Stoenner-Kelly II (BSK-II) medium under microaerophilic conditions ( 126 , 127 ) or its variations, such as BSK-H ( 128 ), at 30°C to 34°C. Despite the existence of other media such as MPM ( 129 ), inoculation of clinical samples (blood or skin lesion biopsy specimens) or triturated ticks in BSK medium remains the most reliable approach for isolating Lyme disease borreliae. Successful culture of these fastidious organisms has also been achieved in BSK medium solidified with 1.5% agarose ( 130 ). Contaminants can be eliminated by adding nalidixic acid and 5-fluorouracil ( 126 , 131 ). These organisms grow slowly, dividing every 8 to 12 h during the exponential growth phase. Primary strains from clinical samples and ticks can take as long as several weeks to grow; however, culture-adapted isolates can reach cell densities of 10 7 to 10 8 cells per ml after cultivation in vitro for 5 to 7 days.

Isolation by animal inoculation was the first and most reliable method for the isolation of relapsing fever borreliae. BSK medium incubated at 35°C was also used to isolate Borrelia recurrentis from the serum of a patient with louse-borne relapsing fever ( 132 ). One bacterium is sufficient to produce infection in laboratory animals, including mice, rats, rabbits, guinea pigs, and monkeys ( 133 ), but the mouse is the most susceptible animal, producing high-level borreliemia (reaching the maximum at 3 to 6 days) ( 134 ). However, within 10 to 14 days postinoculation, when bacteria disappear from the blood, the bacteria can still be found in the reticuloendothelial system or in other organs of infected animals, primarily in brain, where borreliae persist for a longer time ( 133 , 135 ). The genome of Borrelia miyamotoi was obtained from Borrelia cells present in the blood of infected mice ( 136 ).

Similar to its use on Lyme disease borreliae, BSK-II medium was also used for isolating and culturing Borrelia duttonii from patients' blood specimens ( 137 ) and for culturing Borrelia crocidurae from ticks in Mali ( 138 ). Successful isolation was achieved by inoculating blood specimens at room temperature, with subsequent blind subculture every 3 days in fresh medium at 33°C. Bovine serum albumin fraction V complement seems to be an extremely important component of this medium. Borrelia hispanica is particularly difficult to isolate in axenic medium ( 124 ), and animal inoculation remains the method of choice ( Fig. 1 ). For Borrelia hermsii , a combination of both methods is applicable: inoculation in mice followed by cultivation of isolated Borrelia in BSK medium ( 139 ) ( Table 1 ).

Thick smear of mouse blood showing Borrelia hispanica strain OM003 (Giemsa staining). Magnification, ×900.

Culture strategies for isolating spirochetes related to human pathology

The development of optimized culture medium for borreliae and axenic culture of spirochetes remains a challenge; thus, increasingly, new methods are appearing in the literature. Recently, the growth of Leptospira and Borrelia was shown to be supported by a versatile, axenic medium composed of Vero cell extracts ( 140 ).

Mycobacteria

Mycobacteria are environmental organisms that can act as opportunistic pathogens, with a few notable exceptions that are host adapted and responsible for severe infections, including tuberculosis ( M. tuberculosis complex), leprosy ( Mycobacterium leprae ), and Buruli ulcers ( Mycobacterium ulcerans ). Mycobacterial cultures have long been regarded as requiring specialized laboratories. Recent data have indicated that mycobacteria are in no way particular regarding their manipulation in routine clinical microbiology laboratories. Concerning isolation, routine sheep blood medium sustains the isolation and growth of the vast majority of mycobacteria encountered in clinical microbiology laboratories, including M. tuberculosis , Mycobacterium avium , and Mycobacterium intracellulare ( 24 , 141 , 142 ), with the notable exception of M. leprae , which cannot be propagated outside animals ( 143 ) but can be cultured in the footpads of immunocompromised mice. In addition, sheep blood agar and sheep serum agar media have also been used to analyze in vitro susceptibility to antibiotics ( 144 ). Incubation temperature is another key factor for the successful culture of mycobacteria. Although 37°C is the standard incubation temperature used for most human pathogens, the ability of M. leprae to be cultured in the footpads of mice indicates that this species requires a low temperature of 28°C for growth. This finding is consistent with the clinical observation that leprosy develops in the nasal mucosa, superficial nerves, and the skin, which are body regions with temperatures of <37°C. Other pathogens, including Mycobacterium marinum and its derivative, M. ulcerans , as well as M. haemophilum , require a lower temperature of 30°C ( 145 ), and others, such as Mycobacterium genavense ( 146 ) and Mycobacterium xenopi , grow better at temperatures as high as 45°C. Similarly, the atmosphere of incubation warrants further evaluation. Mycobacteria have been reported to be aerobic; however, we recently observed that M. tuberculosis grew better in microaerophilia, which is defined as 2.5 to 5% oxygen tension, than in a conventional 5% CO 2 atmosphere ( 44 ). The fastidiousness of M. tuberculosis complex mycobacteria has further challenged growth detection techniques. In liquid medium, growth is routinely detected with automated instrumentation by significant changes in oxygen and carbon dioxide concentrations; in solid medium, colonies are routinely monitored by the naked eye, with difficulties in distinguishing colonies from inoculation debris. The natural autofluorescence of some mycobacteria can help in the detection of microcolonies. We observed 943- ± 51-μm-diameter colonies containing 6.4 × 10 5 ± 3.5 × 10 5 mycobacteria by the naked eye, whereas by autofluorescence, we detected 103- ± 19-μm-diameter microcolonies containing 7.9 × 10 3 ± 5.3 × 10 3 mycobacteria. Such microcolonies are identifiable by using the matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) technique, thus allowing culture-based diagnosis of mycobacterial infections as a routine task ( 147 ).

We also observed that most opportunistic mycobacterial pathogens are indeed amoeba-resistant organisms ( 148 ), as described comprehensively below. Interestingly, various amoebae are cultured at various temperatures, which allows the modulation of the incubation temperature to isolate mycobacteria such as M. xenopi ( 149 ). We further showed that M. tuberculosis complex mycobacteria are also intra-amoebal organisms ( 150 ), as previously shown for the other two major obligate pathogens, M. leprae ( 151 ) and M. ulcerans ( 152 ). Therefore, almost all the opportunistic and obligate mycobacterial pathogens are, in fact, intra-amoebal mycobacteria, suggesting that this system could be used in cases of as-yet-uncultured opportunistic pathogens such as Mycobacterium tilburgii ( 153 ) ( Table 2 ).

Automatic systems using Middlebrook broth have revolutionized the routine culture of mycobacteria, yet parallel inoculation of solid media is still recommended to fill the gaps with automatic detection. Rapid progress in colony imaging, coupled with MALDI-TOF MS ( 147 ), could render broth culture obsolete. Solid media in miniaturized formats adapted to automated scanners could become the standard for culture and first-line antibiotic susceptibility testing (M. Drancourt, unpublished data). Axenic culture of as-yet-uncultured pathogens is another exciting challenge, as is the discovery of new opportunistic pathogens using the extended spectrum of axenic cell extract-containing media, such as that used for Mycobacterium bovis ( 120 ), and cell-based systems, including protozoa ( 150 ), offered by culturomics ( 154 ).

Phylogenetic classification based on the sequence of the 16S rRNA gene divide archaea into four phyla: the Euryarchaeota and Crenarchaeota ( 155 , – 157 ), which include most cultivated archaea, as well as two new phyla, the Nanoarchaeota and Korarchaeota , which have a single cultivated species each. Methanogenic Archaea belonging to the phylum Euryarchaeota showed great adaptation to the human gut ( 158 , – 164 ). Euryarchaeota and Thaumarchaeota are parts of the skin microbiota ( 165 , – 167 ) and the mucosa-associated microbiota of the oral and vaginal cavities ( 168 ). Methanobrevibacter species, Methanosphaera species, and Sulfolobus species sequences have been retrieved from coprolites dating from AD 180 to AD 600 ( 169 ). However, metagenomic studies, including the Human Microbiome Project, have most likely underestimated the diversity and the density of Archaea in microbiota ( 165 ) due to limitations of the currently used PCR primers and of archaeal DNA extraction protocols ( 170 ). Several studies have consistently observed an age-dependent increase in the prevalence and diversity of gut Archaea and methanogens ( 171 , – 174 ). An increased prevalence of Methanobrevibacter spp. correlated, albeit nonsignificantly, with short-term and long-term carbohydrate ingestion ( 160 ), as was the case in anorexic patients ( 175 ), whereas the prevalence of Nitrososphaera spp. correlated, albeit nonsignificantly, with protein and amino acid ingestion ( 160 ). Methanogens in the oral cavity have been associated with periodontitis ( 176 , 177 ); however, their role, if any, remains unknown.

Among the 20 currently described species among the human microbiota, 3 Archaea have been enriched but not obtained in pure culture. Halophilic Halobacteriaceae archaea were enriched in colonic mucosal biopsy specimens collected from one patient with inflammatory bowel disease ( 178 ). Fluorescence in situ hybridization (FISH) confirmed the presence of viable archaeal cells, but this archaeon has not been isolated in pure culture or deposited in public collections ( 178 ). Similarly, the use of methanol as the substrate led to the enrichment in stool microbiota of “ Candidatus Methanomethylophilus alvus” and “ Candidatus Methanomassiliicoccus intestinalis,” a Thermoplasmatales -related lineage distantly related to its most closely related species, Methanomassiliicoccus luminyensis ( 179 , 180 ) and Aciduliprofundum boonei ( 159 , 174 ). M. luminyensis appears to be the sole cultured representative of a new, seventh order of methylotrophic methanogens ( 181 ). Finally, since the seminal isolation of uncharacterized methanogens 45 years ago ( 42 ), only 6 human-associated archaeal species have been isolated in pure culture and deposited into collections, all of which are strictly anaerobic. Methanogenic Archaea have also been isolated from the digestive tract ( Fig. 2 ). After Methanobrevibacter smithii ( 97 , 182 ), Methanosphaera stadtmanae ( 183 ) was isolated from the human gut, and Methanobrevibacter oralis was isolated from the oral cavity ( 184 ). In addition, our laboratory isolated M. luminyensis ( 179 ), Methanobrevibacter arboriphilicus , Methanobrevibacter millerea , and M. oralis from stool specimens ( 177 ). A versatile medium, combined with incubation at 37°C under an atmosphere of 2 × 10 5 Pa consisting of 80% H 2 and 20% CO 2 , permitted the isolation and culture of these methanogens ( 98 ). This medium consisted of trace elements, including tungsten ( 185 ). Primary isolation was attempted by using the liquid formulation, with cultures being monitored by the CH 4 concentration, which was measured by chromatography, and by microscopic examination of the broth. The colonies were then obtained by subculturing of positive broth onto an agar-based formulation of the same medium and by using the roll tube technique of Hungate ( 98 , 186 ). Methanogens are autofluorescent at 420 nm and can be identified by using matrix-assisted laser desorption ionization–time of flight mass spectrometry ( 187 ). The availability of colonies allowed antibiotic susceptibility testing, which demonstrated the high natural resistance of Archaea , except against imidazole, its derivatives, and fusidic acid ( 188 , 189 ), and genome sequencing ( 174 , 180 , 190 ). Several archaeal species remaining uncultured have been detected only by molecular tools ( 159 , 161 , 162 , 165 , 178 , 191 , 192 ). Thus far, only strictly anaerobic methanogenic Archaea have been isolated from humans. Specific strategies could be designed to enlarge the archaeal spectrum from human microbiota, such as halophilic or thermophilic Archaea . In addition, in the future, it will be interesting to determine the potential pathogenic roles of Archaea in clinical microbiology, particularly in polymicrobial abscesses. We suggest that large archaeal cultures applied to various human samples will partially fill the gap of “microbial dark matter” among the gut microbiota ( 193 ).

Archaeal species detected in or cultured from the human gut ( 97 , 98 , 159 , 170 , 181 , 183 , 185 ).

INTRACELLULAR BACTERIAL CULTURE

Intracellular bacteria are responsible for human diseases with significant morbidity and mortality ( 194 , – 196 ). Rickettsia spp. and Orientia tsutsugamushi , the causative agent of scrub typhus, have tropism for endothelial cells ( 195 ). Coxiella burnetii , which is the agent of Q fever, replicates in the acidic phagolysosomes of macrophages and monocytes ( 196 ). Anaplasma phagocytophilum and Ehrlichia chaffeensis , which cause tick-borne febrile illnesses, replicate preferentially in neutrophils and monocytes, respectively ( 195 ). Chlamydia trachomatis is the agent of trachoma, lymphogranuloma venereum (LGV), and other sexually transmitted diseases, and Chlamydophila pneumoniae and Chlamydophila psittaci can cause pneumonia. These cultures, which require suitable techniques, have been performed only in specialized laboratories. Some intracellular bacteria with high potential for infectivity require biosafety level 3 laboratories for their culture. At the beginning of the 20th century, the culture of intracellular bacteria developed considerably by adaptation of the systems used for virus isolation. For a long time, intracellular bacteria were isolated in embryonated eggs and/or with animal inoculation. Thus far, the successful isolation of intracellular bacteria has been based primarily on cell culture systems ( 197 ). Recently, the development of axenic medium led to a revival of intracellular pathogen culture ( 140 , 198 ).

Culture in Embryonated Eggs

In 1931, Goodpasture and colleagues described how fertilized chicken eggs could be used to cultivate some viruses ( 199 , 200 ). Several years later, Cox injected intracellular bacteria into the yolk sacs of eggs and demonstrated the ability of bacteria to multiply extensively ( 201 ). The infections were confined to the yolk sacs and did not extend to adjacent embryonic tissues. For the growth curve of C. burnetii in yolk sacs, we observed a lag phase until the fourth day and a period of exponential growth, with maximal infection on the seventh day, followed by a rapid decrease ( 202 , 203 ). Consequently, the strains were transferred from egg to egg on the third or fourth day, while the embryo was still living or during the 24 h following its death. The incubation temperature and the age of the eggs were critical. The optimal temperature was 35°C, and a higher or lower temperature was deleterious for the survival of the embryo. Embryos aged 5 or 6 days were preferentially used; the yield was less elevated in older embryos, and younger embryos died prematurely. Virulence and infective titers were maintained through at least several dozen passages in the yolk sac, and this culture system provided higher infective titers than did mammalian tissues. The major inconvenience of this system is its high susceptibility to contamination and variations in its sensitivity according to egg susceptibility and host factors. Moreover, this procedure requires antibiotic-free-diet embryonated eggs from 5 to 8 days of age, which are often difficult to obtain. This culture system was extensively used to isolate and propagate C. burnetii and Rickettsia spp. as well as to study antibiotic susceptibility ( 204 , 205 ). Currently, this culture system has been replaced by cell culture systems, which are more convenient to use. Today, only influenza viruses are routinely cultivated in eggs for vaccination production, and Chlamydia spp. and L. pneumophila are routinely cultivated for producing antigens for immunofluorescence assays.

Animal Inoculation

The susceptibility of animals is dependent on the bacterial species from which the infection originated. Animals for which bacterial multiplication is more important and which present symptoms similar to those of humans are preferentially selected. Despite improvements in culture media, some bacteria remain uncultivable except in animal models, such as M. leprae and T. pallidum . This technique is expensive, time-consuming, and technically difficult and is applied only in specialized laboratories.

M. leprae differs from other mycobacteria in that this species cannot be cultured in vitro , despite many attempts. M. leprae propagation has been restricted to animal models, and several attempts have been undertaken in different animals with low body temperatures ( 206 ). First, M. leprae was propagated in mouse footpads, where 10 6 bacteria were harvested; however, no leprosy-like lesions were produced ( 207 , 208 ). This yield could be increased by growing the bacteria in immunosuppressed mice ( 208 ). The animal most susceptible to M. leprae was a natural host for the bacterium, the nine-banded armadillo ( Dasypus novemcinctus ), which exhibited manifestations of leprosy comparable to those in humans ( 209 , 210 ). In 1971, Kirchheimer et al. successfully cultured M. leprae in this exotic-looking house-cat-sized animal, which is present only on the American continents ( 209 ). Their low body temperature (32°C to 35°C) and long life span (12 to 15 years) make these animals preferred for propagating M. leprae ( 211 ). Dasypus novemcinctus is the only animal to develop regularly disseminated infections following intravenous inoculation; however, 30% of the infected animals do develop systematic infections. The organs of the reticuloendothelial system are the most affected ( 208 ), with bacterial concentrations reaching 10 9 to 10 11 M. leprae bacteria/g ( 211 ). The growth of M. leprae is extremely slow, with a doubling time of 14 days. Consequently, cultivation in armadillos requires a minimum of 18 months to obtain a sufficient number of bacilli.

T. pallidum culture is possible only after animal inoculation, which also remains the preferred method for isolating some species of Borrelia that are difficult to cultivate in cell culture or in artificial medium, as described above ( 134 ).

Inoculation of animals was also used for cultivating intracellular bacteria before the use of cell culture systems. Guinea pigs and mice are the preferred animals for C. burnetii and Rickettsia ( 196 , 212 ). Moreover, some bacteria have been isolated from animals before being isolated by using cell culture systems. Rickettsia felis from fleas was propagated in male Sprague rats before cell culture ( 213 ). Similarly, “ Candidatus Neoehrlichia mikurensis,” which was first described in Rattus norvegicus rats and in Ixodes ovatus ticks, was cultured successfully in rats after intraperitoneal inoculation but has not yet been isolated in cell culture ( 214 ). Two major inconveniences of animal inoculation for culture is cross-contamination between infected and uninfected animals and low reproducibility. However, animal models are still useful when attempting to isolate intracellular bacteria from specimens contaminated with other bacteria and when removing contaminated mycoplasmas or microsporidia from cell cultures of intracellular bacteria ( 215 , 216 ).

Coculture with Amoebae

Some bacterial species are capable of resisting digestion after internalization by free-living amoebae and apply this amoeba-resisting property as a survival strategy in nonfavorable environments. These amoeba-resisting microorganisms (ARMs) consist of bacteria that belong to various phylogenetic clades dispersed throughout the prokaryotic tree ( 217 ), and some are human pathogens.

T. J. Rowbotham was a pioneer in the isolation of ARMs ( 218 ), using an amoebal enrichment method which allowed the isolation of L. pneumophila and other nonculturable, protozoonotic, Gram-negative bacilli, called “ Legionella -like amoebal pathogens” (LLAPs), from diverse environmental and clinical origins ( 218 , – 220 ). Other investigations were performed to investigate the relationship between Legionellae species and other protozoa, primarily the ciliate Tetrahymena sp., in which L. pneumophila is able to survive and acquire high-virulence factors ( 221 , 222 ).

The investigation of ARMs continued and resulted in the isolation of other microbial types, such as several Mycobacterium species ( 223 , 224 ) and Chlamydiales or Chlamydia -like species ( 225 ). Several viruses were also isolated by using amoebae as culture tools, such as the giant amoeba-associated Mimiviridae ( 226 ) and their occasionally associated Sputnik virophages ( 227 ), Marseilleviridae ( 228 ), and, more recently, Pandoravirus ( 229 ).

Notably, protozoa have also been used in the past to isolate and/or to analyze the pathogenicity of strains of bacteria such as Pseudomonas aeruginosa ( 230 , 231 ) and Cryptococcus neoformans ( 232 , 233 ).

Two types of strategies have been used to isolate these ARMs ( Fig. 3 ).

Amoeba culturing methods for recovering amoeba-resisting microorganisms (ARMs). On the left, the method is performed by using laboratory axenic amoebal strains for recovering ARMs from diverse samples. On the right, the amoebae are wild species isolated from the samples, and the ARMs are recovered after amoebal isolation. The circles represent agar plates coated with bacteria, the line is the inoculation point of the sample, and the squiggles represent the growth of the amoebae, following a migration front. BCYE, buffered charcoal-yeast extract.

(i) Coculture with amoebae.

The most commonly used method for the isolation of ARMs consists of inoculating samples onto a monolayer of amoebae, usually of the genus Acanthamoeba . This method has been largely used and described for isolating bacteria and even giant viruses ( 234 , 235 ).

(ii) Isolation of wild amoebae with their symbionts.

The second strategy consists of isolating the amoebae present in the sample by using culture on nonnutrient agar layered with a bacterium used as food (usually Escherichia coli or Enterobacter sp.). After isolation, the amoebae are analyzed for association with eventual ARMs. This method is efficient in isolating strict ARMs ( 236 , 237 ).

(iii) Protozoa used for ARM culture.

The amoebal species used for isolating ARMs are commonly Acanthamoeba , although other species have also been found to harbor ARMs. Amoebae and protists from other clades, which are presented in Table 3 , have been found to be involved in symbiotic or parasitic associations with bacterial and/or viral ARMs.

Protists associated with amoeba-resisting microorganisms

Bacteria from many different clades have been isolated from and/or associated with protozoa ( 217 ).

(i) Alphaproteobacteria .

The species Bartonella rattaustraliani and Rhizobium radiobacter were shown to survive in Acanthamoeba polyphaga ( 238 ). Bradyrhizobium sp. or Methylobacterium sp. strains were found by using an amoebal coculture with A. polyphaga ( 235 ) and Acanthamoeba castellanii ( 223 ) and had a pathogenic effect on the amoebae ( 235 , 239 ). Bacteria of the genera Bosea and Afipia have frequently been isolated from Acanthamoeba sp. in hospital networks and could possibly be related to human pathogenicity ( 219 , 231 , 235 , – 238 ).

The species Sphingomonas koreensis was found to be associated with Vanella sp. In this case, the amoebae were also harboring a new genus of Chlamydia -like bacterium, called “ Candidatus Mesochlamydia elodeae,” pathogenic for Vanella sp. and maintained in culture in another amoebal species, Saccamoeba lacustris ( 240 ).

Rickettsia bellii was found to enter and remain stable for 6 weeks within the species A. polyphaga. Coinfection with L. pneumophila shows that both bacteria colocalize in the vacuoles of the amoebae, and together with the observation of sequences similar to those of L. pneumophila and Protochlamydia amoebophila in the genome of R. bellii , this feature suggests communication and gene exchanges between bacteria inside the amoeba. Moreover, R. bellii has the ability to process gene exchange by conjugation with sexual pili ( 241 ).

The bacterium Odyssella thessalonicensis is a new, strictly intra-amoebal genus of the Alphaproteobacteria , which was isolated from environmental water samples and A. polyphaga .

(ii) Betaproteobacteria .

Several Betaproteobacteria have been found to be associated with Acanthamoeba sp., for example, Burkholderia sp., Delftia sp., and Acidovorax sp. strains isolated from environmental samples ( 235 , 242 ).

(iii) Gammaproteobacteria .

Two species of the genus Stenotrophomonas were isolated by using coculture, S. acidaminiphila ( 242 ) and S. maltophilia ( 235 ). S. maltophilia was also found in association with Dictyostelium discoideum , causing infection, invasion, and growth inside the amoeba ( 243 ). More recently, two bacterial strains related to the Stenotrophomonas maltophilia complex were isolated from two different amoebae, Acanthamoeba sp. and Naegleria sp. Both amoebae were recovered and infected by the endosymbiont. These bacteria are pathogenic for the amoebae, and coculture leads systematically to the lysis of the amoebae ( 244 ). The genus Aeromonas has been associated with Acanthamoeba sp. ( 235 , 242 ), has the capacity to multiply in the amoeba, and has cytopathic effects. The species Aeromonas hydrophila was found to interact actively with A. castellanii and to be able to survive within cysts ( 245 , 246 ).

Some Enterobacteriaceae are also cultivable in several amoeba species. One study focusing on environmental water samples led to the isolation of several strains of Acanthamoeba -resisting Enterobacteriaceae , which are able to multiply within the amoeba but are nonpathogenic, including Klebsiella sp., Enterobacter sp., Escherichia coli , Morganella morganii , Pantoea sp., Providentia sp., and Serratia sp., etc. ( 235 , 247 ); Yersinia pseudotuberculosis , Salmonella enterica serovar Typhimurium, and Klebsiella pneumoniae are able to infect the amoeba D. discoideum ( 243 ), and some strains of Enterobacter aerogenes and E. coli also multiplied within A. castellanii and survived within cysts ( 246 , 248 ).

Some Acinetobacter species were found in coculture with A. castellanii and A. polyphaga , with cytopathic effects ( 235 , 242 ). Additionally Acinetobacter baumannii was shown to interact actively with A. castellanii ( 249 ). For the genus Pseudomonas , environmental coculture studies with Acanthamoeba led to the isolation of P. fluorescens ( 242 ) and P. aeruginosa ( 223 ). P. aeruginosa also interacts with the amoeba D. discoideum ( 243 ). Moreover, this amoeba is now established as a culture model not only for P. aeruginosa but also for L. pneumophila and Mycobacterium sp. ( 232 ).

Historically, the genus Legionella was among the first to be associated with amoebae due to the ecology of these bacteria. L. pneumophila can multiply in not only Acanthamoeba sp. but also Hartmanella and Dictyostelium ( 243 ) and has the ability to stay in vacuoles of the ciliate Tetrahymena . Several other Legionella species have been found in association with different amoebae, and some of these species were new, such as the LLAPs Legionella drozanskii , L. rowbothamii , L. fallonii ( 250 ), and L. drancourtii ( 251 ). However, some other previously known Legionella species showed abilities to infect and multiply within amoebae ( 252 ). In 2004, an Amoeba proteus strain was isolated, which harbored a Legionella -like endosymbiont, named “ Candidatus Legionella jeonii,” closely related to Legionella sp. ( 253 ).

Finally, other strictly intracellular Gammaproteobacteria have occasionally been shown to be associated with protozoa, such as C. burnetii , which is able to survive within A. castellanii ( 254 ), and Francisella tularensis , which is able to survive within cysts of A. castellanii ( 255 ).

(iv) Epsilonproteobacteria .

Within the Epsilonproteobacteria , only Campylobacter jejuni has been demonstrated to have the capacity to multiply within protozoa, specifically Acanthamoeba sp. ( 256 , 257 ).

(v) Firmicutes .

Among the Firmicutes group, Bacillus licheniformis was shown to be pathogenically associated with Naegleria fowleri , with the effects depending on the bacterium/amoeba ratio ( 258 ). These different effects were further investigated by using several bacterial strains of the genus Bacillus and the amoebae A. polyphaga and D. discoideum ( 259 ).

Strain RN4220 of methicillin-sensitive Staphylococcus aureus was shown to be able to be internalized by A. polyphaga and to multiply within the amoeba ( 260 ), and the species Staphylococcus pasteuri and S. pneumoniae were isolated in a coculture with Acanthamoeba sp. ( 261 ). The pathogen Streptococcus suis was studied for its pathogenesis and interactions with the amoeba Dictyostelium discoideum ( 262 ).

(vi) Bacteroidetes .

Flavobacterium johnsoniae , Flavobacterium succinicans , and Flexibacter canadensis were isolated by amoebal coculture with Acanthamoeba sp. or as symbionts of Acanthamoeba sp. ( 261 ). The two periodontopathogenic species Porphyromonas gingivalis and Prevotella intermedia were able to enter and multiply within A. castellanii ( 263 ). Several species of Chryseobacterium were isolated by coculture with Acanthamoeba sp. ( 235 , 242 ), and two strains were described as new species: Chryseobacterium massiliae ( 239 ) and C. massiliensis ( 235 ). Other Proteobacteria representing new species or genera were isolated for the first time by amoebal coculture: Cytophaga massiliensis ( 235 ), “ Candidatus Amoebinatus massiliae” ( 239 ), and “ Candidatus Amoebophilus asiaticus” ( 264 ).

(vii) Actinobacteria .

Several Microbacterium species and other Actinobacteria , such as Rhodococcus equi and Rhodococcus erythropolis ( 235 , 242 , 261 ), have been isolated by coculture with Acanthamoeba sp. The primary group of Actinobacteria studied for their interactions with protozoa is Mycobacterium . Indeed, many rapidly growing mycobacteria were able to be isolated by coculture with Acanthamoeba sp. ( 223 , 224 , 235 , 252 ) or as natural symbionts in amoebae isolated from contact lens storage cases ( 265 ). In 1997, it was demonstrated for the first time that M. avium could enter and multiply in vegetative forms and also survive in cysts of Acanthamoeba sp. ( 266 , 267 ). In 2004, D. discoideum was demonstrated to be a good model for studying mycobacterial intracellular trafficking using M. marinum ( 268 ). One study investigated the ability of waterborne Mycobacterium to survive within A. polyphaga and demonstrated that these bacteria could enter and multiply in the vegetative form of the amoeba, survive in the cyst forms, and resist chlorine treatment while protected inside those cysts ( 142 , 148 , 149 ). Moreover, a link was made between the ability of Mycobacterium to survive and multiply within amoebae and the acquisition or enhancement of some virulence traits ( 269 , – 272 ). Some pathogenic or slow-growing mycobacteria were also shown to be associated with amoebae, such as the M. tuberculosis complex ( 150 ) and the pathogenic M. ulcerans ( 152 ). Another feature of the interactions between Mycobacterium and amoebae, the presence of several functional cellulose-targeting genes, was also elucidated recently ( 150 , 273 , 274 ). Another study showed the presence of the celA gene, which codes for a cellulose able to degrade carboxymethylcellulose and microcrystalline cellulose, in Legionella pneumophila strain 130b ( 275 ). The presence of these cellulose genes in the genomes of amoeba-associated bacteria suggests that these proteins could facilitate the growth of these bacteria within amoebae.

(viii) Chlamydia .

The association of Chlamydiales with amoebae was demonstrated in 1997 with Chlamydia pneumoniae , which was able to multiply in A. castellanii ( 276 ). Recently, Chlamydophila abortus was shown to enter and survive within A. castellanii but without multiplying ( 277 ). Coculture with several amoebae led to the discovery of several new genera, and these findings have extended much of the order Chlamydiales . Indeed, since the discovery and description of the new genus Parachlamydia in Acanthamoeba sp. ( 278 ), many other bacteria have been isolated from amoebal cultures, including Neochlamydia hartmanella ( 237 ), Protochlamydia amoebophila ( 279 ), Criblamydia sequanensis ( 280 ), Protochlamydia naeglerophila ( 281 ), Metachlamydia lacustris ( 282 ), Estrella lausanensis ( 283 ), and “ Candidatus Mesochlamydia elodeae” ( 240 ).

Some other Parachlamydia -related bacteria isolated in mammalian cells have demonstrated susceptibility to amoebae. Simkania negevensis was cultivated successfully in A. polyphaga ( 284 , 285 ), and Waddlia chondrophila was cultivated in A. castellanii , which enabled antibiotic susceptibility testing ( 286 ).

(ix) Endomicrobia .

The Endomicrobia group is represented by uncultivated bacteria within the candidate phylum termite group I (TG-1) ( 287 , 288 ). These bacteria were demonstrated to be associated with termite gut flagellates, primarily the species Trichonympha and Pyrsonympha , and each of the flagellate species harbors phylogenetically distinct species of Endomicrobia ( 288 , 289 ).

(x) TM6 phylum.

The TM6 phylum is a putative bacterial phylum represented by the single draft genome of a putative bacterium, JCVI TM6SC1, which was obtained by single-cell genomics and from numerous environmental sequences ( 290 ). Amoebal coculture with Acanthamoeba sp. recently enabled the isolation of the first effective strain of this phylum, a bacterium that we named Babela massiliensis . This bacterium harbors genome features that show high levels of degradation in the cell division machinery, and these features are introduced phenotypically by multiplication in the amoeba that resembles budding, different from classical binary fission (I. Pagnier, N. Yutin, O. Croce, K. S. Makarova, Y. I. Wolf, S. Benamar, D. Raoult, E. V. Koonin, and B. La Scola, unpublished data).

Use in clinical microbiology.

Some ARMs have already been implicated in human pathology, primarily Mycobacterium spp., Legionella spp., and some Chlamydia spp. and Parachlamydia spp. Amoebal culture and research into ARMs are alternative methods of clinical diagnosis. For example, some Legionella species were able to be recovered from clinical specimens only by using amoebal culture ( 291 ). Moreover, the first giant virus isolated from a patient sample was a member of the Marseilleviridae , called senegalvirus, which was isolated from a stool sample of an asymptomatic patient from Senegal ( 154 , 292 ). Later, the first human virus of the Mimiviridae , LBA111, was isolated from a pulmonary sample ( 293 ). Finally, a last giant virus of the Mimiviridae , called Shan virus, was isolated from a stool sample of a patient presenting with pulmonary disease ( 294 ). Amoebal culture could be useful for clinical diagnosis, primarily to target strictly intracellular ARMs, such as Chlamydiales or Parachlamydiales species. For example, the species Protochlamydia naeglerophila , which was isolated as a symbiont of the amoeba Naegleria sp., was found in a pathological sample of a patient with pneumonia by using molecular methods ( 281 ), and the species Waddlia chondrophila was shown to be able to survive and multiply within human macrophages, suggesting possible implications for human pathology ( 295 , 296 ).

Perspectives.

Culture of microorganisms with protozoa has facilitated several important steps in the comprehension of the microbiological world, and the relationships between protozoa and bacteria were able to be studied more deeply. Amoebal coculture led to the discovery of many new microorganisms, particularly new species such as L. drozanskii , L. rowbothami , L. fallonii ( 250 ), and L. drancourtii ( 251 ). New genera ( Chlamydia - and Parachlamydia -related microorganisms) ( 282 , 283 ) and new phyla ( Babela massiliensis ) (Pagnier et al., unpublished) or life domains (all amoeba-associated giant viruses) have also been recovered by amoebal coculture. The example of the isolation of Babela massiliensis (CSUR P554; GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"GQ495224","term_id":"258674431","term_text":"GQ495224"}} GQ495224 for 16S rRNA) is a good illustration of the increasing importance of microbial culture (Pagnier et al., unpublished). Indeed, many metagenomic approaches led to the discovery of entire unknown groups of microorganisms, and deeper study of these groups has been limited by the nonavailability of real bacterial strains. The use of coculture with amoebae allowed the recovery of a representative member of the putative TM6 phylum, and this finding will lead to other studies elucidating the intracellular way of life of the bacterium. The principle of isolation by coculture can be transferred to other types of protozoa, which could have other host-microorganism spectra. Indeed, culture assays have already been performed with the axenized protists Vermamoeba vermiformis , Dictyostelium discoideum , Tetrahymena hyperangularis , and Colpoda steinii . Recently, a high-throughput method was developed by using the amoeba Acanthamoeba sp. to isolate giant viruses from >1,000 samples ( 297 ) by observation of a lysis plaque on an amoebal monolayer resting on an agar plate ( 297 ). Using other protists, lysis due to ARMs can be observed by flow cytometry. These methods have already led to the isolation of new microbial species, such as Rubidus massiliensis (CSUR P942; GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"HG726047","term_id":"565666443","term_text":"HG726047"}} HG726047 for 16S rRNA), a new Parachlamydia -related bacterium, and a new giant virus, associated with Hartmanella vermiformis .

Cell Culture

Currently, the most common method for cultivating and isolating intracellular bacteria from clinical samples is cell culture. Two different strategies can be used. First, culture conditions can be standardized to allow the efficient growth of a broad range of bacteria. The second strategy consists of the specific isolation of an already known bacterium with specific adapted culture conditions for bacterial growth optimization. In fact, the type of cells and culture medium used should be adapted specifically, depending on the bacterium being cultivated. The susceptibility of bacteria to different cells lines varies according to their cell tropisms and optimal growth temperatures. Several eukaryotic cell lines can be used, including mammalian and arthropod cell lines. Two types of cells may be used: (i) cells cohering with each other, forming a cell monolayer, and (ii) cells circulating in suspension.

The temperature and atmosphere of incubation are critical and should be adapted. This culture strategy should also be used in the genomic and proteomic era, which requires a large amount of bacteria for large-scale investigations and for antigen production for diagnostic testing. Cell culture systems have also been used to evaluate the antibiotic susceptibility of intracellular bacteria by a plaque assay and a dye uptake assay ( 298 , 299 ) or with a combination of culture and a PCR assay ( 5 ).

Cell lines grown at 37°C.

Mammalian cells developing at 37°C are the most often used cells. C. burnetii can be grown with a doubling time of 20 h in mouse macrophage-like (P388D1 and J774), murine fibroblast (L929), and kidney epithelial (Vero) cell lines ( 196 , 300 , – 302 ). Human embryonic lung (HEL) fibroblasts have been preferentially used for routine culture because of their easy maintenance and high level of susceptibility to C. burnetii infection. The observed cytopathic effects were the occurrence of large vacuoles in the cytoplasm. C. burnetii infection does not destroy host cells, allowing the maintenance of C. burnetii for several months in persistent culture ( 196 , 303 ).

Culture of Rickettsia was first performed by using a primary monocyte cell culture ( 304 ). In the 1980s, Vero and L929 cells were used, allowing better and more rapid isolation of rickettsiae than with HEL and MRC5 (human fetal lung fibroblast) cells ( 305 ). In fact, Rickettsia conorii presented higher multiplication rates in L929 cells than in Vero or 3T3 cells ( 306 ). Most of the Rickettsiae belonging to the spotted fever group can polymerize actin from cells, to move into the nucleus of the host cell and promote cell-to-cell spreading, which generates rapid cytopathic effects and rapid plaque formation at an optimal growth temperature of 32°C ( 307 ). The optimal growth temperature of Rickettsia from the typhus group is 35°C. The bacteria invade adjacent cells only when their multiplication and accumulation in the cytoplasm cause host cell lysis. Consequently, cytopathic effects were less prominent, and the plaques formed were small ( 307 ).

L929 cells have been used for culturing O. tsutsugamushi , which grew slowly, and 1,000 bacteria were required to invoke cell disruption and propagation in adjacent cells ( 308 ). Consequently, a long incubation period is required, which involves difficulties in maintaining the integrity of the cell monolayer and contamination-free medium.

The establishment of T. whipplei in culture was reported in 2000, using HEL cells ( 309 ), and the bacterium was then cultured from various clinical samples ( 310 ). Antibiotics were added to the culture medium to cultivate samples that were naturally contaminated, such as duodenal biopsy specimens or stool specimens ( 311 , 312 ). The growth of T. whipplei was also observed in MRC-5 ( 313 ) and HeLa ( 314 ) cells.

DH82, which is a continuous canine macrophage cell line ( 315 ), is usually used to isolate E. chaffeensis and Ehrlichia canis ( 316 ). However, several other cell lines can be employed, such as mouse embryo, Vero, Buffalo green monkey kidney (BGMK), HEL, HeLa, and L929 cells ( 315 , 317 , 318 ). E. chaffeensis induced cytopathic effects on mouse embryo, L929, HEL, and Vero cells and caused macroscopic plaque formation in L929 and mouse embryo cells ( 318 ) and microscopic plaque formation in Vero cells ( 317 ). Anaplasma phagocytophilum has been cultured routinely and with a rapid development of cytopathic effects ( 319 ) in HL-60, a human promyelocytic leukemia cell line that grows in suspension culture ( 320 ). This cell line was highly susceptible to A. phagocytophilum infection. Fresh blood or the leukocyte fraction of EDTA-blood can be inoculated into a 25-cm 2 flask containing a density of 2 × 10 5 cells/ml. However, the ability of A. phagocytophilum to establish in vivo infection successfully decreased considerably when it was continually maintained in cell culture ( 321 ).

Several mammalian cell lines have been used to propagate Chlamydia spp., including mouse fibroblast (McCoy and L cells), human cervical carcinoma (HeLa 229), human laryngeal carcinoma (HEp-2), human epithelial (HL), BGMK, and Vero ( 322 ) cell lines. To permit the entry of Chlamydia spp. into host cells and the creation of a favorable intracellular environment, several steps in the process are critical for the success of Chlamydia species culture ( 323 , 324 ). A centrifugation step and/or the addition of polycationic stabilizing molecules to overcome the repellent forces between the membrane of the host cell and the bacteria was required, except for C. psittaci and the LGV agent. Cycloheximide, which is a selective inhibitor of eukaryotic protein and nucleic acid synthesis, was added to reduce the competition for nutrients between host cells and bacteria, thus enhancing the growth of Chlamydia ( 325 ). The cultures were incubated for 48 to 72 h at 35°C. McCoy and HeLa 229 cells are the cell lines most commonly used for C. trachomatis culture ( 322 , 324 ), and HL and HEp-2 cells seem to be more sensitive for the recovery of C. pneumoniae ( 323 , 326 ). The cytopathic effect occurs as small, rounded intracytoplasmic inclusions, visualized by immunostaining of cell monolayers.

Cell lines grown at 28°C.

Some bacteria require temperatures of <37°C to grow; consequently, the use of cell lines specifically tailored to lower temperatures is required, such as amphibian, tick, mosquito, and fish cell lines. These cell lines have been commonly used for arbovirus culture and allow the isolation of intracellular bacteria, such as Rickettsia felis . The first culture of R. felis , which was performed with Vero and L929 cells after incubation at 37°C, was in reality contamination of the culture by Rickettsia typhi ( 213 , 327 , 328 ). Thus, the culture of R. felis has been credited to Raoult et al. ( 40 ), who cultivated this bacterium with success in an amphibian cell line derived from Xenopus laevis (XTC2) after 6 days of incubation at 28°C. The bacterium also grows slowly on Vero cells at 28°C and 32°C ( 40 , 329 ). The growth of R. felis in mammalians cell lines is inconstant but was improved by the addition of tryptose phosphate broth in culture media, which seems to be a critical component based on its involvement in the electron transport chain of the Krebs cycle ( 330 ). More recently, growth of R. felis in Drosophila melanogaster S2 cells was reported, with a cell infection rate of 100% after 19 days of incubation at 25°C ( 331 ). Other arthropod-borne bacteria, such as Diplorickettsia massiliensis ( 332 ), Wolbachia sp., and Bartonella bacilliformis ( 40 ), as well as other bacteria such as Piscirickettsia salmonis ( 333 ) and M. ulcerans ( 334 ) can also be cultivated more effectively by using this cell line.

Approximately 40 different tick cell lines are available, primarily from ixodid rather than argasid tick species ( 335 ). These cells have the advantage of partially reproducing the natural environment of tick-borne pathogens. Most tick cells grow at incubation temperatures of between 28 and 34°C, and acidic or alkaline pH conditions vary according to the cell line. Cells from ticks divide slowly, require up to 1 month between passages, and are generally not strongly adherent, generating partial monolayers or a suspension culture. Tick cell lines have been used successfully for the growth of A. phagocytophilum , Anaplasma marginale , Ehrlichia canis , Ehrlichia ruminantium , Borrelia spp., Rickettsia spp., Bartonella spp., and C. burnetii ( 335 , – 340 ) as well as for the isolation of unknown bacteria, such as a new Anaplasma sp. ( 341 ), Borrelia lonestari ( 342 ), and Rickettsia monacensis ( 343 ). Mosquito cell lines are permissive to multiple arthropod-borne infections and offer the advantage of being easier to culture than tick cell lines. Mosquito cell lines can be passaged in 1 week and grown at room temperature under an ambient atmosphere and have the potential to develop high bacterial titers, and infection is stable in the cells for >40 passages ( 339 ). In particular, Anopheles gambiae (Sua5B) and Aedes albopictus (Aa23 or C6/36) cell lines have been used to grow R. felis , R. montanensis , R. peacockii , and R. typhi as well as several strains of the bacterial symbiont Wolbachia ( 37 , 339 , 344 , – 349 ).

Few assays have been performed to grow fish-pathogenic bacteria in a fish cell line at temperatures between 21°C and 30°C. Mycobacterium marinum was cultivated successfully in a carp monocyte cell line at 28°C ( 350 ), and Piscirickettsia salmonis was cultivated in a Chinook salmon cell line ( 351 ) ( Table 4 ).

The most susceptible cells line used for the culture of intracellular bacteria a

Specific devices (shell vial).

The shell vial assay is currently the method used in specialized laboratories to isolate fastidious bacteria from clinical samples ( Fig. 4 ). This technique was adapted to bacterial culture in 1989 ( 352 ) from a centrifugation-cell microculture system, which was described in 1976 and used for virus isolation ( 353 ). The principles of this technique are based on the inoculation of clinical specimens by low-speed centrifugation on confluent cell monolayers seeded onto 1-cm 2 -round coverslips and incubated with 1 ml of culture medium in a shell vial. The centrifugation step is critical because this step enhances the attachment and penetration of the bacteria in cells ( 354 ). The sensitivity of this cell culture technique is increased by the small surface area of the coverslip, which contains cells that enhance the ratio of the number of intracellular bacteria to the number of cells and that allow more efficient recovery. Moreover, the cell type, length, atmosphere, and incubation temperature can be optimized according to the suspected pathogen. Different protocols can be used for the specific isolation of Rickettsia spp., Bartonella spp., C. burnetii , or Tropheryma whipplei , and one protocol, which is called “JNSP,” is based on the French abbreviation of the sentence “ Je ne sais pas ,” which means “I do not know” what I am growing, for unspecific research into other intracellular bacteria ( Fig. 4 ). HEL fibroblasts are used because of their high level of susceptibility to intracellular bacterial infection and their ease of maintenance. To avoid bacterial contamination, antibiotics with no activity against the bacteria of interest can be added. The growth of bacteria is detected by observation of cytopathic effect; acridine orange, Giemsa, and/or Gimenez staining; or immunofluorescence revelation using the patient's serum or sera from immune animals as the primary antibody ( 197 ). Definitive identification of the bacteria is performed by PCR. This technique is used routinely in our laboratory and permits the successful isolation of spotted fever group Rickettsia ( 355 ), Rickettsia prowazekii ( 356 ), C. burnetii ( 357 ), C. trachomatis ( 358 ), and fastidious bacteria such as Bartonella sp. ( 48 , 359 ), F. tularensis ( 360 ), Mycobacterium sp. ( 359 , 361 ), L. pneumophila ( 362 ), Brucella melitensis ( 363 ), Actinomyces sp. ( 197 ), Streptobacillus moniliformis ( 364 ), and Nocardia sp. ( 197 ). The shell vial assay exhibits a low rate of success, but this success can be extremely valuable ( 197 ). This technique facilitated the establishment of T. whipplei from a cardiac valve of a patient with Whipple's disease endocarditis in the context of JNSP protocols ( 309 ). In some cases, this technique is the only technique that allows microbiological diagnosis, and the shell vial system was also used for testing the antibiotic susceptibilities of intracellular bacteria ( 365 , 366 ).

Shell vial system protocols used for specific isolation of Bartonella , Coxiella burnetii , rickettsiae, or T. whipplei and for unspecific research into other intracellular bacteria according to JNSP protocols.

Axenic Culture Media for Intracellular Bacteria

Knowledge of pathogen physiology and the host cell-pathogen interaction and the use of new tools, such as genomic and transcriptomic analyses, have enabled the study of metabolic pathways, enhancing the success of axenic cultivation ( 198 , 367 , 368 ). The establishment of axenic culture media could have significant effects on the study of their pathogenicity, virulence, and antibiotic susceptibility and on the design of new diagnostic tools ( 140 ).

Coxiella burnetii .

Coxiella burnetii has been described as being cultivable in an acellular chemically defined medium ( 11 , 369 , 370 ), a medium designed after a comprehensive analysis of the nutrient consumption of C. burnetii . First, genomic analysis identified three critical components for this complex Coxiella medium (CCM) to sustain the metabolic activity of the bacteria as well as 3 complex nutrient sources (Neopeptone, fetal bovine serum, and RPMI cell culture medium) and a high concentration of chloride and citrate buffer, mimicking the acidic environment of phagosomes ( 370 ). A transcriptomic analysis resulted in the conception of ACCM (acidified citrate cysteine medium) enriched with cysteine and Casamino Acids that support the growth of C. burnetii by >3 log 10 units after 6 days of incubation in a microaerophilic atmosphere ( 11 , 371 ). This medium was then optimized through the replacement of fetal bovine serum by methyl-β-cyclodextrin and was named ACCM-2; ACCM-2 sustained 4- to 5-log 10 growth of C. burnetii over 7 days with the addition of moderate shaking ( 372 ). Moreover, colonies were observed on ACCM-2 agarose ( 372 ). More recently, Singh et al. showed the exponential growth of C. burnetii in an empirical medium containing eukaryotic cell extracts ( 120 ). With the medium being refreshed every 2 days, low oxygen tension and the presence of small hydrophilic molecules and short peptides were critical for its growth. However, this medium harbors a neutral pH, suggesting that the growth of C. burnetii does not require acid activation of its metabolic pathways. The antigenicity and the virulent form (phase I) of C. burnetii were conserved, revealing the potential for this culture medium to be used for antigen and vaccine production. Furthermore, this medium permitted the successful isolation of C. burnetii from pathological samples.

Tropheryma whipplei .

Culture of T. whipplei permitted the obtaining of the complete genomes of two different strains of T. whipplei ( 7 , 313 ). Genomic analysis revealed a reduced genome size (0.9 Mb) and a specific metabolic deficiency. The genes coding for metabolic pathways for nine amino acids were entirely missing, and the pathways of glutamate and phenylalanine synthesis were nonfunctional ( 13 ). An axenic medium compensating for these deficiencies was designed based on these genomic analyses ( 13 , 368 ). This medium consisted of cell culture medium that provided the missing amino acids, supplemented with 10% fetal calf serum, 1% glutamine, and 1% human nonessential amino acids ( 13 ). This approach permitted the successful growth of T. whipplei from a great variety of clinical samples as well as antibiotic susceptibility testing ( 4 , 373 ). The development of an axenic medium permitted the culturing of isolates from sterile and nonsterile sources, such as saliva and stool samples, after a decontamination procedure with glutaraldehyde ( 312 ) or with filtration ( 374 , 375 ).

Chlamydia species.

C. trachomatis , similar to other members of the Chlamydiaceae family, presents a biphasic developmental cycle, with important consequences for its metabolic activity and axenic growth. During their biphasic intracellular development cycle, the surviving infectious elementary bodies (EBs) extracellularly differentiate into reticulate bodies (RBs) after entry into the cytoplasm of the host cells, which multiply by binary fission. Two recent studies demonstrated the ability of EBs to conduct metabolic activity outside the cytoplasm of host cells ( 198 , 376 ). The development of a host cell-free medium was based on analysis of the C. trachomatis genome. A reduced genome (1.04 Mb) and the several missing enzymes and entire metabolic pathways suggested that C. trachomatis uses nutrients from host cells for its growth ( 377 ). The axenic medium supporting the metabolic activity of C. trachomatis consisted of a novel intracellular phosphate buffer and an ion concentration mimicking the composition of the eukaryotic cytoplasm ( 14 ). Incubation in a microaerophilic atmosphere and an energy source were critical for the metabolic activity of C. trachomatis . EBs have been historically described as metabolically dormant; however, in cell-free media, high levels of metabolic and biosynthetic activity were observed for both EBs and RBs, although the EBs primarily used glucose-6-phosphate, and the RBs used ATP as an energy source. This axenic medium, with further modifications, promises to support the replication of C. trachomatis and the study of the metabolism and physiology of Chlamydiae .

Empirical broad-spectrum medium.

Singh et al. developed an axenic liquid medium based on an extract of eukaryotic cells, allowing the growth of a broad spectrum of bacteria. The eukaryotic cell extract-based universal empirical medium, which was tested for culturing of C. burnetii as a proof of concept, supported the growth of other fastidious bacteria and putative bacterial bioterrorism agents ( 120 ). Rapid growth (<72 h) was observed for T. whipplei , Yersinia pestis , Vibrio cholerae , Bacillus anthracis , Shigella dysenteriae , Brucella melitensis , Bordetella pertussis , and Bartonella henselae. F. tularensis and M. bovis were able to grow on this medium in 20 and 8 days, respectively, and Campylobacter spp. were able to grow in 5 days under microaerophilic conditions. In addition, this medium was the first to permit the growth of two spirochetes belonging to different genera, B. crocidurae and L. interrogans , with delays of 14 and 4 days, respectively. However, two limitations of this medium are its inability to sustain the growth of other intracellular bacteria, including Rickettsia and Legionella species, and its variability, which is caused by the use of uncharacterized biological products. The primary advantage of this empirical medium is its versatility; thus, this medium can be used for many applications, including the isolation and culture of unknown fastidious bacteria from clinical samples, which encourages its use as a replacement for cell-based culture systems. Moreover, this medium offers promising perspectives for culturing other intracellular fastidious bacteria by testing variations in temperature, atmosphere of incubation, and supplementation with nutrients and growth factors required by the bacteria.

Future challenges.

We have reviewed older and more current approaches used to cultivate intracellular bacteria. No single approach exists. Older techniques, such as animal inoculation, are still used to propagate some bacteria and to isolate new bacterial species, such as “ Candidatus Neoehrlichia mikurensis.” Cell culture permits the growth of a large number of intracellular bacteria; however, in some cases, cell culture must be more specifically adapted to particular species, mimicking their natural environment to optimize their growth. Future prospects will be based largely on the development of axenic media, facilitating genetic manipulation, and on an understanding of microbial ecology and the pathogenicity of fastidious bacteria. Axenic growth of the historically “uncultivable” T. pallidum and M. leprae has remained a large challenge for a century. The success of axenic growth of C. burnetii and T. whipplei , as well as the design of axenic media for the growth of Rickettsia , Anaplasma , Ehrlichia , Orientia , and Chlamydia , should accelerate soon.

Our laboratory (URMITE, Marseille, France), which was created ex nihilo in 1984 and which initially specialized in intracellular bacterial culture, has now extended its experience into many fields of clinical microbiology, tirelessly demonstrating the central role of pure culture in understanding infectious diseases. The shell vial procedure and intracellular culture have allowed us to culture 18 different species of Rickettsia and have facilitated participation in the description of 7 new bacterial species or subspecies ( 195 , 378 , – 383 ) and in the first culture of 4 new rickettsial species ( 40 , 384 , – 386 ). Interestingly, Rickettsia felis is now associated with acute febrile illness ( 387 , 388 ) and with a vesicular fever called “yaaf” in Africa ( 389 ). Currently, we have cultured 41 different T. whipplei strains from diverse tissues and fluid ( 310 , 312 , 374 , 375 , 390 , 391 ), playing a key role in the understanding of the clinical manifestations caused by this bacterium ( 194 ). Coculture with amoebae, which was performed in our laboratory, has allowed us to identify 139 different bacterial species from both clinical and environmental samples, including 10 new bacterial species ( 224 , 235 , 239 , 251 , 261 , 392 , – 396 ). In our laboratory, the great qualities of several “cultivators,” technicians, or researchers were identified and recognized in the names of bacterial species to honor these qualities (i.e., Rickettsia raoultii , Legionella drancourtii , Mycobacterium barrassiae , Afipia birgiae , Bosea vestrisii , and Bosea eneae , etc. [ 251 , 384 , 392 , 396 , 397 ]), as the names of other species acknowledge several “cultivators,” such as Finegoldia magna , Murdochiella asaccharolytica , or Gordonibacterium pamelaeae ( 398 , 399 ).

Despite the spectacular advances in microbial culture in recent years, there are still unsatisfactory results remaining. A large part of anaerobic bacteria detected by molecular methods remain as yet uncultured. The optimization of transport time and the use of antioxidants will allow a dramatic increase of this repertoire. The development of axenic culture of spirochetes such as T. pallidum or M. leprae remains an important challenge in clinical microbiology.

Some bacteria have not yet been cultivated due only to the lack of investigators; Rickettsia raoultii was cultivated for the first time by Oleg Mediannikov, whereas we failed to culture this strain in the same laboratory for 8 years ( 384 ). The patience and energy required for the primary culture of a bacterium are often considerable. Culture is a complex and difficult art, in which the individual qualities of the scientist are not comparable with the individual qualities required by researchers using a more biochemically defined method.

ACKNOWLEDGMENTS

We thank all the technicians and researchers working in our laboratory implicated in bacterial culture.

Michel Drancourt and Didier Raoult are coinventors of pending patents on the culture and subculture of M. tuberculosis , and Didier Raoult is coinventor of a pending patent on the culture of anaerobes and Vero cell extract medium. All these patents are owned by Aix Marseille University.

Biographies

Jean-Christophe Lagier , M.D., Ph.D., who specializes in infectious diseases, has been an associate professor at the Faculty of Medicine of Marseille, Aix Marseille University, since 2013. His clinical medical activity occurs at the University Hospital Institute Mediterranée Infection of Marseille. His research interests include Tropheryma whipplei infections and gut microbiota diversity, which he has specifically explored by culture on the team of D. Raoult since 2007. He coordinates with S. Khelaifia, the culturomics team directed by Prof. D. Raoult. By October 2014, Dr. Lagier had coauthored more than 85 publications in the international literature.

Sophie Edouard , Pharm.D., Ph.D., is a microbiologist specializing in diagnosing infectious diseases, specifically infections caused by intracellular bacteria, at Aix Marseille University, France. She obtained the title of doctor of pharmacy in 2011. Subsequently, she obtained her Ph.D. at the Faculty of Medicine of Marseille, Aix Marseille University, France, under the direction of Didier Raoult in 2013. She is now in charge of diagnosing and managing infection with fastidious bacteria at the infectious diseases department of the University Hospital of Marseille and in the French reference center for the diagnosis of rickettsioses, bartonelloses, and Q fever. She continues her research activity with the team of Didier Raoult. Her research interests focus on the diagnosis of and culture techniques for fastidious and vector-borne bacteria, including C. burnetii , Bartonella spp., Rickettsia spp., and Anaplasma spp.

Isabelle Pagnier , Pharm.D., Ph.D., is a French pharmacist and associate professor of microbiology at the Faculty of Pharmacy, and her research activity occurs at the Faculty of Medicine, Aix Marseille University. She completed her Ph.D. under the direction of Prof. Bernard La Scola. Her research interests focus on amoeba-associated microorganisms, primarily giant viruses such as Mimiviridae and marseilleviruses. Since 2013, she has been in charge of the scientific management of the electron microscopy platform of the URMITE laboratory, supervising structural and morphological studies of diverse bacteria, viruses, and microorganisms associated with protozoa. She has published more than 30 research articles regarding protozoon-associated microorganisms.

Oleg Mediannikov , M.D., Ph.D., graduated from the Medical University in Khabarovsk, Russia, in 1998 and specializes in infectious diseases. He obtained his Ph.D. in 2004, identifying the role of Rickettsia . He joined Didier Raoult's team in France, focusing on vector-borne diseases, including anaplasmosis, spotted fevers, borrelioses, and their vectors. Since 2011, he has led a research group within the IRD component in Senegal, where he continues his studies of the origins of acute febrile diseases in Africa, particularly emerging vector-borne diseases (not only relapsing fever, spotted fevers, and bartonelloses but also filarioses and malaria). He is an expert on the isolation of fastidious bacteria, particularly intracellular ones.

Michel Drancourt , M.D., Ph.D., is a professor of medical microbiology at Aix Marseille University at Marseilles Medical School. He graduated in Marseille and visited the CDC in Atlanta, GA, as a postdoc. He developed expertise in paleomicrobiology and fastidious organisms, including mycobacteria, archaea, and planctomycetes.

Didier Raoult , M.D., Ph.D., who specializes in infectious diseases, is a professor of microbiology at the Faculty of Medicine of Marseille, Aix Marseille University. In 1984, he created ex nihilo his research laboratory, the Rickettsia Unit. This unit has now become the Research Unit in Infectious and Tropical Emergent Diseases (URMITE), collaborating with the CNRS (National Center for the Scientific Research), the IRD (Institute of Research for Development), and INSERM (National Institute of Health and Medical Research). In 2011, he became the director of the University Hospital Institute Mediterranée Infection, which is a 600-person medical institute focused on infectious diseases. This facility includes the largest diagnostic and research microbiology laboratory in France. As of 2014, Prof. Raoult has published more than 1,900 indexed publications. In the last 30 years, he has cultured approximately 16% of the bacteria isolated for the first time in humans, including T. whipplei , in his laboratory and developed a culturomics team in 2011.

Cultural Relativity and Acceptance of Embryonic Stem Cell Research

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There is a debate about the ethical implications of using human embryos in stem cell research, which can be influenced by cultural, moral, and social values. This paper argues for an adaptable framework to accommodate diverse cultural and religious perspectives. By using an adaptive ethics model, research protections can reflect various populations and foster growth in stem cell research possibilities.

INTRODUCTION

Stem cell research combines biology, medicine, and technology, promising to alter health care and the understanding of human development. Yet, ethical contention exists because of individuals’ perceptions of using human embryos based on their various cultural, moral, and social values. While these disagreements concerning policy, use, and general acceptance have prompted the development of an international ethics policy, such a uniform approach can overlook the nuanced ethical landscapes between cultures. With diverse viewpoints in public health, a single global policy, especially one reflecting Western ethics or the ethics prevalent in high-income countries, is impractical. This paper argues for a culturally sensitive, adaptable framework for the use of embryonic stem cells. Stem cell policy should accommodate varying ethical viewpoints and promote an effective global dialogue. With an extension of an ethics model that can adapt to various cultures, we recommend localized guidelines that reflect the moral views of the people those guidelines serve.

Stem cells, characterized by their unique ability to differentiate into various cell types, enable the repair or replacement of damaged tissues. Two primary types of stem cells are somatic stem cells (adult stem cells) and embryonic stem cells. Adult stem cells exist in developed tissues and maintain the body’s repair processes. [1] Embryonic stem cells (ESC) are remarkably pluripotent or versatile, making them valuable in research. [2] However, the use of ESCs has sparked ethics debates. Considering the potential of embryonic stem cells, research guidelines are essential. The International Society for Stem Cell Research (ISSCR) provides international stem cell research guidelines. They call for “public conversations touching on the scientific significance as well as the societal and ethical issues raised by ESC research.” [3] The ISSCR also publishes updates about culturing human embryos 14 days post fertilization, suggesting local policies and regulations should continue to evolve as ESC research develops. [4]  Like the ISSCR, which calls for local law and policy to adapt to developing stem cell research given cultural acceptance, this paper highlights the importance of local social factors such as religion and culture.

I.     Global Cultural Perspective of Embryonic Stem Cells

Views on ESCs vary throughout the world. Some countries readily embrace stem cell research and therapies, while others have stricter regulations due to ethical concerns surrounding embryonic stem cells and when an embryo becomes entitled to moral consideration. The philosophical issue of when the “someone” begins to be a human after fertilization, in the morally relevant sense, [5] impacts when an embryo becomes not just worthy of protection but morally entitled to it. The process of creating embryonic stem cell lines involves the destruction of the embryos for research. [6] Consequently, global engagement in ESC research depends on social-cultural acceptability.

a.     US and Rights-Based Cultures

In the United States, attitudes toward stem cell therapies are diverse. The ethics and social approaches, which value individualism, [7] trigger debates regarding the destruction of human embryos, creating a complex regulatory environment. For example, the 1996 Dickey-Wicker Amendment prohibited federal funding for the creation of embryos for research and the destruction of embryos for “more than allowed for research on fetuses in utero.” [8] Following suit, in 2001, the Bush Administration heavily restricted stem cell lines for research. However, the Stem Cell Research Enhancement Act of 2005 was proposed to help develop ESC research but was ultimately vetoed. [9] Under the Obama administration, in 2009, an executive order lifted restrictions allowing for more development in this field. [10] The flux of research capacity and funding parallels the different cultural perceptions of human dignity of the embryo and how it is socially presented within the country’s research culture. [11]

b.     Ubuntu and Collective Cultures

African bioethics differs from Western individualism because of the different traditions and values. African traditions, as described by individuals from South Africa and supported by some studies in other African countries, including Ghana and Kenya, follow the African moral philosophies of Ubuntu or Botho and Ukama , which “advocates for a form of wholeness that comes through one’s relationship and connectedness with other people in the society,” [12] making autonomy a socially collective concept. In this context, for the community to act autonomously, individuals would come together to decide what is best for the collective. Thus, stem cell research would require examining the value of the research to society as a whole and the use of the embryos as a collective societal resource. If society views the source as part of the collective whole, and opposes using stem cells, compromising the cultural values to pursue research may cause social detachment and stunt research growth. [13] Based on local culture and moral philosophy, the permissibility of stem cell research depends on how embryo, stem cell, and cell line therapies relate to the community as a whole. Ubuntu is the expression of humanness, with the person’s identity drawn from the “’I am because we are’” value. [14] The decision in a collectivistic culture becomes one born of cultural context, and individual decisions give deference to others in the society.

Consent differs in cultures where thought and moral philosophy are based on a collective paradigm. So, applying Western bioethical concepts is unrealistic. For one, Africa is a diverse continent with many countries with different belief systems, access to health care, and reliance on traditional or Western medicines. Where traditional medicine is the primary treatment, the “’restrictive focus on biomedically-related bioethics’” [is] problematic in African contexts because it neglects bioethical issues raised by traditional systems.” [15] No single approach applies in all areas or contexts. Rather than evaluating the permissibility of ESC research according to Western concepts such as the four principles approach, different ethics approaches should prevail.

Another consideration is the socio-economic standing of countries. In parts of South Africa, researchers have not focused heavily on contributing to the stem cell discourse, either because it is not considered health care or a health science priority or because resources are unavailable. [16] Each country’s priorities differ given different social, political, and economic factors. In South Africa, for instance, areas such as maternal mortality, non-communicable diseases, telemedicine, and the strength of health systems need improvement and require more focus. [17] Stem cell research could benefit the population, but it also could divert resources from basic medical care. Researchers in South Africa adhere to the National Health Act and Medicines Control Act in South Africa and international guidelines; however, the Act is not strictly enforced, and there is no clear legislation for research conduct or ethical guidelines. [18]

Some parts of Africa condemn stem cell research. For example, 98.2 percent of the Tunisian population is Muslim. [19] Tunisia does not permit stem cell research because of moral conflict with a Fatwa. Religion heavily saturates the regulation and direction of research. [20] Stem cell use became permissible for reproductive purposes only recently, with tight restrictions preventing cells from being used in any research other than procedures concerning ART/IVF.  Their use is conditioned on consent, and available only to married couples. [21] The community's receptiveness to stem cell research depends on including communitarian African ethics.

c.     Asia

Some Asian countries also have a collective model of ethics and decision making. [22] In China, the ethics model promotes a sincere respect for life or human dignity, [23] based on protective medicine. This model, influenced by Traditional Chinese Medicine (TCM), [24] recognizes Qi as the vital energy delivered via the meridians of the body; it connects illness to body systems, the body’s entire constitution, and the universe for a holistic bond of nature, health, and quality of life. [25] Following a protective ethics model, and traditional customs of wholeness, investment in stem cell research is heavily desired for its applications in regenerative therapies, disease modeling, and protective medicines. In a survey of medical students and healthcare practitioners, 30.8 percent considered stem cell research morally unacceptable while 63.5 percent accepted medical research using human embryonic stem cells. Of these individuals, 89.9 percent supported increased funding for stem cell research. [26] The scientific community might not reflect the overall population. From 1997 to 2019, China spent a total of $576 million (USD) on stem cell research at 8,050 stem cell programs, increased published presence from 0.6 percent to 14.01 percent of total global stem cell publications as of 2014, and made significant strides in cell-based therapies for various medical conditions. [27] However, while China has made substantial investments in stem cell research and achieved notable progress in clinical applications, concerns linger regarding ethical oversight and transparency. [28] For example, the China Biosecurity Law, promoted by the National Health Commission and China Hospital Association, attempted to mitigate risks by introducing an institutional review board (IRB) in the regulatory bodies. 5800 IRBs registered with the Chinese Clinical Trial Registry since 2021. [29] However, issues still need to be addressed in implementing effective IRB review and approval procedures.

The substantial government funding and focus on scientific advancement have sometimes overshadowed considerations of regional cultures, ethnic minorities, and individual perspectives, particularly evident during the one-child policy era. As government policy adapts to promote public stability, such as the change from the one-child to the two-child policy, [30] research ethics should also adapt to ensure respect for the values of its represented peoples.

Japan is also relatively supportive of stem cell research and therapies. Japan has a more transparent regulatory framework, allowing for faster approval of regenerative medicine products, which has led to several advanced clinical trials and therapies. [31] South Korea is also actively engaged in stem cell research and has a history of breakthroughs in cloning and embryonic stem cells. [32] However, the field is controversial, and there are issues of scientific integrity. For example, the Korean FDA fast-tracked products for approval, [33] and in another instance, the oocyte source was unclear and possibly violated ethical standards. [34] Trust is important in research, as it builds collaborative foundations between colleagues, trial participant comfort, open-mindedness for complicated and sensitive discussions, and supports regulatory procedures for stakeholders. There is a need to respect the culture’s interest, engagement, and for research and clinical trials to be transparent and have ethical oversight to promote global research discourse and trust.

d.     Middle East

Countries in the Middle East have varying degrees of acceptance of or restrictions to policies related to using embryonic stem cells due to cultural and religious influences. Saudi Arabia has made significant contributions to stem cell research, and conducts research based on international guidelines for ethical conduct and under strict adherence to guidelines in accordance with Islamic principles. Specifically, the Saudi government and people require ESC research to adhere to Sharia law. In addition to umbilical and placental stem cells, [35] Saudi Arabia permits the use of embryonic stem cells as long as they come from miscarriages, therapeutic abortions permissible by Sharia law, or are left over from in vitro fertilization and donated to research. [36] Laws and ethical guidelines for stem cell research allow the development of research institutions such as the King Abdullah International Medical Research Center, which has a cord blood bank and a stem cell registry with nearly 10,000 donors. [37] Such volume and acceptance are due to the ethical ‘permissibility’ of the donor sources, which do not conflict with religious pillars. However, some researchers err on the side of caution, choosing not to use embryos or fetal tissue as they feel it is unethical to do so. [38]

Jordan has a positive research ethics culture. [39] However, there is a significant issue of lack of trust in researchers, with 45.23 percent (38.66 percent agreeing and 6.57 percent strongly agreeing) of Jordanians holding a low level of trust in researchers, compared to 81.34 percent of Jordanians agreeing that they feel safe to participate in a research trial. [40] Safety testifies to the feeling of confidence that adequate measures are in place to protect participants from harm, whereas trust in researchers could represent the confidence in researchers to act in the participants’ best interests, adhere to ethical guidelines, provide accurate information, and respect participants’ rights and dignity. One method to improve trust would be to address communication issues relevant to ESC. Legislation surrounding stem cell research has adopted specific language, especially concerning clarification “between ‘stem cells’ and ‘embryonic stem cells’” in translation. [41] Furthermore, legislation “mandates the creation of a national committee… laying out specific regulations for stem-cell banking in accordance with international standards.” [42] This broad regulation opens the door for future global engagement and maintains transparency. However, these regulations may also constrain the influence of research direction, pace, and accessibility of research outcomes.

e.     Europe

In the European Union (EU), ethics is also principle-based, but the principles of autonomy, dignity, integrity, and vulnerability are interconnected. [43] As such, the opportunity for cohesion and concessions between individuals’ thoughts and ideals allows for a more adaptable ethics model due to the flexible principles that relate to the human experience The EU has put forth a framework in its Convention for the Protection of Human Rights and Dignity of the Human Being allowing member states to take different approaches. Each European state applies these principles to its specific conventions, leading to or reflecting different acceptance levels of stem cell research. [44]

For example, in Germany, Lebenzusammenhang , or the coherence of life, references integrity in the unity of human culture. Namely, the personal sphere “should not be subject to external intervention.” [45]  Stem cell interventions could affect this concept of bodily completeness, leading to heavy restrictions. Under the Grundgesetz, human dignity and the right to life with physical integrity are paramount. [46] The Embryo Protection Act of 1991 made producing cell lines illegal. Cell lines can be imported if approved by the Central Ethics Commission for Stem Cell Research only if they were derived before May 2007. [47] Stem cell research respects the integrity of life for the embryo with heavy specifications and intense oversight. This is vastly different in Finland, where the regulatory bodies find research more permissible in IVF excess, but only up to 14 days after fertilization. [48] Spain’s approach differs still, with a comprehensive regulatory framework. [49] Thus, research regulation can be culture-specific due to variations in applied principles. Diverse cultures call for various approaches to ethical permissibility. [50] Only an adaptive-deliberative model can address the cultural constructions of self and achieve positive, culturally sensitive stem cell research practices. [51]

II.     Religious Perspectives on ESC

Embryonic stem cell sources are the main consideration within religious contexts. While individuals may not regard their own religious texts as authoritative or factual, religion can shape their foundations or perspectives.

The Qur'an states:

“And indeed We created man from a quintessence of clay. Then We placed within him a small quantity of nutfa (sperm to fertilize) in a safe place. Then We have fashioned the nutfa into an ‘alaqa (clinging clot or cell cluster), then We developed the ‘alaqa into mudgha (a lump of flesh), and We made mudgha into bones, and clothed the bones with flesh, then We brought it into being as a new creation. So Blessed is Allah, the Best of Creators.” [52]

Many scholars of Islam estimate the time of soul installment, marked by the angel breathing in the soul to bring the individual into creation, as 120 days from conception. [53] Personhood begins at this point, and the value of life would prohibit research or experimentation that could harm the individual. If the fetus is more than 120 days old, the time ensoulment is interpreted to occur according to Islamic law, abortion is no longer permissible. [54] There are a few opposing opinions about early embryos in Islamic traditions. According to some Islamic theologians, there is no ensoulment of the early embryo, which is the source of stem cells for ESC research. [55]

In Buddhism, the stance on stem cell research is not settled. The main tenets, the prohibition against harming or destroying others (ahimsa) and the pursuit of knowledge (prajña) and compassion (karuna), leave Buddhist scholars and communities divided. [56] Some scholars argue stem cell research is in accordance with the Buddhist tenet of seeking knowledge and ending human suffering. Others feel it violates the principle of not harming others. Finding the balance between these two points relies on the karmic burden of Buddhist morality. In trying to prevent ahimsa towards the embryo, Buddhist scholars suggest that to comply with Buddhist tenets, research cannot be done as the embryo has personhood at the moment of conception and would reincarnate immediately, harming the individual's ability to build their karmic burden. [57] On the other hand, the Bodhisattvas, those considered to be on the path to enlightenment or Nirvana, have given organs and flesh to others to help alleviate grieving and to benefit all. [58] Acceptance varies on applied beliefs and interpretations.

Catholicism does not support embryonic stem cell research, as it entails creation or destruction of human embryos. This destruction conflicts with the belief in the sanctity of life. For example, in the Old Testament, Genesis describes humanity as being created in God’s image and multiplying on the Earth, referencing the sacred rights to human conception and the purpose of development and life. In the Ten Commandments, the tenet that one should not kill has numerous interpretations where killing could mean murder or shedding of the sanctity of life, demonstrating the high value of human personhood. In other books, the theological conception of when life begins is interpreted as in utero, [59] highlighting the inviolability of life and its formation in vivo to make a religious point for accepting such research as relatively limited, if at all. [60] The Vatican has released ethical directives to help apply a theological basis to modern-day conflicts. The Magisterium of the Church states that “unless there is a moral certainty of not causing harm,” experimentation on fetuses, fertilized cells, stem cells, or embryos constitutes a crime. [61] Such procedures would not respect the human person who exists at these stages, according to Catholicism. Damages to the embryo are considered gravely immoral and illicit. [62] Although the Catholic Church officially opposes abortion, surveys demonstrate that many Catholic people hold pro-choice views, whether due to the context of conception, stage of pregnancy, threat to the mother’s life, or for other reasons, demonstrating that practicing members can also accept some but not all tenets. [63]

Some major Jewish denominations, such as the Reform, Conservative, and Reconstructionist movements, are open to supporting ESC use or research as long as it is for saving a life. [64] Within Judaism, the Talmud, or study, gives personhood to the child at birth and emphasizes that life does not begin at conception: [65]

“If she is found pregnant, until the fortieth day it is mere fluid,” [66]

Whereas most religions prioritize the status of human embryos, the Halakah (Jewish religious law) states that to save one life, most other religious laws can be ignored because it is in pursuit of preservation. [67] Stem cell research is accepted due to application of these religious laws.

We recognize that all religions contain subsets and sects. The variety of environmental and cultural differences within religious groups requires further analysis to respect the flexibility of religious thoughts and practices. We make no presumptions that all cultures require notions of autonomy or morality as under the common morality theory , which asserts a set of universal moral norms that all individuals share provides moral reasoning and guides ethical decisions. [68] We only wish to show that the interaction with morality varies between cultures and countries.

III.     A Flexible Ethical Approach

The plurality of different moral approaches described above demonstrates that there can be no universally acceptable uniform law for ESC on a global scale. Instead of developing one standard, flexible ethical applications must be continued. We recommend local guidelines that incorporate important cultural and ethical priorities.

While the Declaration of Helsinki is more relevant to people in clinical trials receiving ESC products, in keeping with the tradition of protections for research subjects, consent of the donor is an ethical requirement for ESC donation in many jurisdictions including the US, Canada, and Europe. [69] The Declaration of Helsinki provides a reference point for regulatory standards and could potentially be used as a universal baseline for obtaining consent prior to gamete or embryo donation.

For instance, in Columbia University’s egg donor program for stem cell research, donors followed standard screening protocols and “underwent counseling sessions that included information as to the purpose of oocyte donation for research, what the oocytes would be used for, the risks and benefits of donation, and process of oocyte stimulation” to ensure transparency for consent. [70] The program helped advance stem cell research and provided clear and safe research methods with paid participants. Though paid participation or covering costs of incidental expenses may not be socially acceptable in every culture or context, [71] and creating embryos for ESC research is illegal in many jurisdictions, Columbia’s program was effective because of the clear and honest communications with donors, IRBs, and related stakeholders.  This example demonstrates that cultural acceptance of scientific research and of the idea that an egg or embryo does not have personhood is likely behind societal acceptance of donating eggs for ESC research. As noted, many countries do not permit the creation of embryos for research.

Proper communication and education regarding the process and purpose of stem cell research may bolster comprehension and garner more acceptance. “Given the sensitive subject material, a complete consent process can support voluntary participation through trust, understanding, and ethical norms from the cultures and morals participants value. This can be hard for researchers entering countries of different socioeconomic stability, with different languages and different societal values. [72]

An adequate moral foundation in medical ethics is derived from the cultural and religious basis that informs knowledge and actions. [73] Understanding local cultural and religious values and their impact on research could help researchers develop humility and promote inclusion.

IV.     Concerns

Some may argue that if researchers all adhere to one ethics standard, protection will be satisfied across all borders, and the global public will trust researchers. However, defining what needs to be protected and how to define such research standards is very specific to the people to which standards are applied. We suggest that applying one uniform guide cannot accurately protect each individual because we all possess our own perceptions and interpretations of social values. [74] Therefore, the issue of not adjusting to the moral pluralism between peoples in applying one standard of ethics can be resolved by building out ethics models that can be adapted to different cultures and religions.

Other concerns include medical tourism, which may promote health inequities. [75] Some countries may develop and approve products derived from ESC research before others, compromising research ethics or drug approval processes. There are also concerns about the sale of unauthorized stem cell treatments, for example, those without FDA approval in the United States. Countries with robust research infrastructures may be tempted to attract medical tourists, and some customers will have false hopes based on aggressive publicity of unproven treatments. [76]

For example, in China, stem cell clinics can market to foreign clients who are not protected under the regulatory regimes. Companies employ a marketing strategy of “ethically friendly” therapies. Specifically, in the case of Beike, China’s leading stem cell tourism company and sprouting network, ethical oversight of administrators or health bureaus at one site has “the unintended consequence of shifting questionable activities to another node in Beike's diffuse network.” [77] In contrast, Jordan is aware of stem cell research’s potential abuse and its own status as a “health-care hub.” Jordan’s expanded regulations include preserving the interests of individuals in clinical trials and banning private companies from ESC research to preserve transparency and the integrity of research practices. [78]

The social priorities of the community are also a concern. The ISSCR explicitly states that guidelines “should be periodically revised to accommodate scientific advances, new challenges, and evolving social priorities.” [79] The adaptable ethics model extends this consideration further by addressing whether research is warranted given the varying degrees of socioeconomic conditions, political stability, and healthcare accessibilities and limitations. An ethical approach would require discussion about resource allocation and appropriate distribution of funds. [80]

While some religions emphasize the sanctity of life from conception, which may lead to public opposition to ESC research, others encourage ESC research due to its potential for healing and alleviating human pain. Many countries have special regulations that balance local views on embryonic personhood, the benefits of research as individual or societal goods, and the protection of human research subjects. To foster understanding and constructive dialogue, global policy frameworks should prioritize the protection of universal human rights, transparency, and informed consent. In addition to these foundational global policies, we recommend tailoring local guidelines to reflect the diverse cultural and religious perspectives of the populations they govern. Ethics models should be adapted to local populations to effectively establish research protections, growth, and possibilities of stem cell research.

For example, in countries with strong beliefs in the moral sanctity of embryos or heavy religious restrictions, an adaptive model can allow for discussion instead of immediate rejection. In countries with limited individual rights and voice in science policy, an adaptive model ensures cultural, moral, and religious views are taken into consideration, thereby building social inclusion. While this ethical consideration by the government may not give a complete voice to every individual, it will help balance policies and maintain the diverse perspectives of those it affects. Embracing an adaptive ethics model of ESC research promotes open-minded dialogue and respect for the importance of human belief and tradition. By actively engaging with cultural and religious values, researchers can better handle disagreements and promote ethical research practices that benefit each society.

This brief exploration of the religious and cultural differences that impact ESC research reveals the nuances of relative ethics and highlights a need for local policymakers to apply a more intense adaptive model.

[1] Poliwoda, S., Noor, N., Downs, E., Schaaf, A., Cantwell, A., Ganti, L., Kaye, A. D., Mosel, L. I., Carroll, C. B., Viswanath, O., & Urits, I. (2022). Stem cells: a comprehensive review of origins and emerging clinical roles in medical practice.  Orthopedic reviews ,  14 (3), 37498. https://doi.org/10.52965/001c.37498

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[3] International Society for Stem Cell Research. (2023). Laboratory-based human embryonic stem cell research, embryo research, and related research activities . International Society for Stem Cell Research. https://www.isscr.org/guidelines/blog-post-title-one-ed2td-6fcdk ; Kimmelman, J., Hyun, I., Benvenisty, N.  et al.  Policy: Global standards for stem-cell research.  Nature   533 , 311–313 (2016). https://doi.org/10.1038/533311a

[4] International Society for Stem Cell Research. (2023). Laboratory-based human embryonic stem cell research, embryo research, and related research activities . International Society for Stem Cell Research. https://www.isscr.org/guidelines/blog-post-title-one-ed2td-6fcdk

[5] Concerning the moral philosophies of stem cell research, our paper does not posit a personal moral stance nor delve into the “when” of human life begins. To read further about the philosophical debate, consider the following sources:

Sandel M. J. (2004). Embryo ethics--the moral logic of stem-cell research.  The New England journal of medicine ,  351 (3), 207–209. https://doi.org/10.1056/NEJMp048145 ; George, R. P., & Lee, P. (2020, September 26). Acorns and Embryos . The New Atlantis. https://www.thenewatlantis.com/publications/acorns-and-embryos ; Sagan, A., & Singer, P. (2007). The moral status of stem cells. Metaphilosophy , 38 (2/3), 264–284. http://www.jstor.org/stable/24439776 ; McHugh P. R. (2004). Zygote and "clonote"--the ethical use of embryonic stem cells.  The New England journal of medicine ,  351 (3), 209–211. https://doi.org/10.1056/NEJMp048147 ; Kurjak, A., & Tripalo, A. (2004). The facts and doubts about beginning of the human life and personality.  Bosnian journal of basic medical sciences ,  4 (1), 5–14. https://doi.org/10.17305/bjbms.2004.3453

[6] Vazin, T., & Freed, W. J. (2010). Human embryonic stem cells: derivation, culture, and differentiation: a review.  Restorative neurology and neuroscience ,  28 (4), 589–603. https://doi.org/10.3233/RNN-2010-0543

[7] Socially, at its core, the Western approach to ethics is widely principle-based, autonomy being one of the key factors to ensure a fundamental respect for persons within research. For information regarding autonomy in research, see: Department of Health, Education, and Welfare, & National Commission for the Protection of Human Subjects of Biomedical and Behavioral Research (1978). The Belmont Report. Ethical principles and guidelines for the protection of human subjects of research.; For a more in-depth review of autonomy within the US, see: Beauchamp, T. L., & Childress, J. F. (1994). Principles of Biomedical Ethics . Oxford University Press.

[8] Sherley v. Sebelius , 644 F.3d 388 (D.C. Cir. 2011), citing 45 C.F.R. 46.204(b) and [42 U.S.C. § 289g(b)]. https://www.cadc.uscourts.gov/internet/opinions.nsf/6c690438a9b43dd685257a64004ebf99/$file/11-5241-1391178.pdf

[9] Stem Cell Research Enhancement Act of 2005, H. R. 810, 109 th Cong. (2001). https://www.govtrack.us/congress/bills/109/hr810/text ; Bush, G. W. (2006, July 19). Message to the House of Representatives . National Archives and Records Administration. https://georgewbush-whitehouse.archives.gov/news/releases/2006/07/20060719-5.html

[10] National Archives and Records Administration. (2009, March 9). Executive order 13505 -- removing barriers to responsible scientific research involving human stem cells . National Archives and Records Administration. https://obamawhitehouse.archives.gov/the-press-office/removing-barriers-responsible-scientific-research-involving-human-stem-cells

[11] Hurlbut, W. B. (2006). Science, Religion, and the Politics of Stem Cells.  Social Research ,  73 (3), 819–834. http://www.jstor.org/stable/40971854

[12] Akpa-Inyang, Francis & Chima, Sylvester. (2021). South African traditional values and beliefs regarding informed consent and limitations of the principle of respect for autonomy in African communities: a cross-cultural qualitative study. BMC Medical Ethics . 22. 10.1186/s12910-021-00678-4.

[13] Source for further reading: Tangwa G. B. (2007). Moral status of embryonic stem cells: perspective of an African villager. Bioethics , 21(8), 449–457. https://doi.org/10.1111/j.1467-8519.2007.00582.x , see also Mnisi, F. M. (2020). An African analysis based on ethics of Ubuntu - are human embryonic stem cell patents morally justifiable? African Insight , 49 (4).

[14] Jecker, N. S., & Atuire, C. (2021). Bioethics in Africa: A contextually enlightened analysis of three cases. Developing World Bioethics , 22 (2), 112–122. https://doi.org/10.1111/dewb.12324

[15] Jecker, N. S., & Atuire, C. (2021). Bioethics in Africa: A contextually enlightened analysis of three cases. Developing World Bioethics, 22(2), 112–122. https://doi.org/10.1111/dewb.12324

[16] Jackson, C.S., Pepper, M.S. Opportunities and barriers to establishing a cell therapy programme in South Africa.  Stem Cell Res Ther   4 , 54 (2013). https://doi.org/10.1186/scrt204 ; Pew Research Center. (2014, May 1). Public health a major priority in African nations . Pew Research Center’s Global Attitudes Project. https://www.pewresearch.org/global/2014/05/01/public-health-a-major-priority-in-african-nations/

[17] Department of Health Republic of South Africa. (2021). Health Research Priorities (revised) for South Africa 2021-2024 . National Health Research Strategy. https://www.health.gov.za/wp-content/uploads/2022/05/National-Health-Research-Priorities-2021-2024.pdf

[18] Oosthuizen, H. (2013). Legal and Ethical Issues in Stem Cell Research in South Africa. In: Beran, R. (eds) Legal and Forensic Medicine. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-32338-6_80 , see also: Gaobotse G (2018) Stem Cell Research in Africa: Legislation and Challenges. J Regen Med 7:1. doi: 10.4172/2325-9620.1000142

[19] United States Bureau of Citizenship and Immigration Services. (1998). Tunisia: Information on the status of Christian conversions in Tunisia . UNHCR Web Archive. https://webarchive.archive.unhcr.org/20230522142618/https://www.refworld.org/docid/3df0be9a2.html

[20] Gaobotse, G. (2018) Stem Cell Research in Africa: Legislation and Challenges. J Regen Med 7:1. doi: 10.4172/2325-9620.1000142

[21] Kooli, C. Review of assisted reproduction techniques, laws, and regulations in Muslim countries.  Middle East Fertil Soc J   24 , 8 (2020). https://doi.org/10.1186/s43043-019-0011-0 ; Gaobotse, G. (2018) Stem Cell Research in Africa: Legislation and Challenges. J Regen Med 7:1. doi: 10.4172/2325-9620.1000142

[22] Pang M. C. (1999). Protective truthfulness: the Chinese way of safeguarding patients in informed treatment decisions. Journal of medical ethics , 25(3), 247–253. https://doi.org/10.1136/jme.25.3.247

[23] Wang, L., Wang, F., & Zhang, W. (2021). Bioethics in China’s biosecurity law: Forms, effects, and unsettled issues. Journal of law and the biosciences , 8(1).  https://doi.org/10.1093/jlb/lsab019 https://academic.oup.com/jlb/article/8/1/lsab019/6299199

[24] Wang, Y., Xue, Y., & Guo, H. D. (2022). Intervention effects of traditional Chinese medicine on stem cell therapy of myocardial infarction.  Frontiers in pharmacology ,  13 , 1013740. https://doi.org/10.3389/fphar.2022.1013740

[25] Li, X.-T., & Zhao, J. (2012). Chapter 4: An Approach to the Nature of Qi in TCM- Qi and Bioenergy. In Recent Advances in Theories and Practice of Chinese Medicine (p. 79). InTech.

[26] Luo, D., Xu, Z., Wang, Z., & Ran, W. (2021). China's Stem Cell Research and Knowledge Levels of Medical Practitioners and Students.  Stem cells international ,  2021 , 6667743. https://doi.org/10.1155/2021/6667743

[27] Luo, D., Xu, Z., Wang, Z., & Ran, W. (2021). China's Stem Cell Research and Knowledge Levels of Medical Practitioners and Students.  Stem cells international ,  2021 , 6667743. https://doi.org/10.1155/2021/6667743

[28] Zhang, J. Y. (2017). Lost in translation? accountability and governance of Clinical Stem Cell Research in China. Regenerative Medicine , 12 (6), 647–656. https://doi.org/10.2217/rme-2017-0035

[29] Wang, L., Wang, F., & Zhang, W. (2021). Bioethics in China’s biosecurity law: Forms, effects, and unsettled issues. Journal of law and the biosciences , 8(1).  https://doi.org/10.1093/jlb/lsab019 https://academic.oup.com/jlb/article/8/1/lsab019/6299199

[30] Chen, H., Wei, T., Wang, H.  et al.  Association of China’s two-child policy with changes in number of births and birth defects rate, 2008–2017.  BMC Public Health   22 , 434 (2022). https://doi.org/10.1186/s12889-022-12839-0

[31] Azuma, K. Regulatory Landscape of Regenerative Medicine in Japan.  Curr Stem Cell Rep   1 , 118–128 (2015). https://doi.org/10.1007/s40778-015-0012-6

[32] Harris, R. (2005, May 19). Researchers Report Advance in Stem Cell Production . NPR. https://www.npr.org/2005/05/19/4658967/researchers-report-advance-in-stem-cell-production

[33] Park, S. (2012). South Korea steps up stem-cell work.  Nature . https://doi.org/10.1038/nature.2012.10565

[34] Resnik, D. B., Shamoo, A. E., & Krimsky, S. (2006). Fraudulent human embryonic stem cell research in South Korea: lessons learned.  Accountability in research ,  13 (1), 101–109. https://doi.org/10.1080/08989620600634193 .

[35] Alahmad, G., Aljohani, S., & Najjar, M. F. (2020). Ethical challenges regarding the use of stem cells: interviews with researchers from Saudi Arabia. BMC medical ethics, 21(1), 35. https://doi.org/10.1186/s12910-020-00482-6

[36] Association for the Advancement of Blood and Biotherapies.  https://www.aabb.org/regulatory-and-advocacy/regulatory-affairs/regulatory-for-cellular-therapies/international-competent-authorities/saudi-arabia

[37] Alahmad, G., Aljohani, S., & Najjar, M. F. (2020). Ethical challenges regarding the use of stem cells: Interviews with researchers from Saudi Arabia.  BMC medical ethics ,  21 (1), 35. https://doi.org/10.1186/s12910-020-00482-6

[38] Alahmad, G., Aljohani, S., & Najjar, M. F. (2020). Ethical challenges regarding the use of stem cells: Interviews with researchers from Saudi Arabia. BMC medical ethics , 21(1), 35. https://doi.org/10.1186/s12910-020-00482-6

Culturally, autonomy practices follow a relational autonomy approach based on a paternalistic deontological health care model. The adherence to strict international research policies and religious pillars within the regulatory environment is a great foundation for research ethics. However, there is a need to develop locally targeted ethics approaches for research (as called for in Alahmad, G., Aljohani, S., & Najjar, M. F. (2020). Ethical challenges regarding the use of stem cells: interviews with researchers from Saudi Arabia. BMC medical ethics, 21(1), 35. https://doi.org/10.1186/s12910-020-00482-6), this decision-making approach may help advise a research decision model. For more on the clinical cultural autonomy approaches, see: Alabdullah, Y. Y., Alzaid, E., Alsaad, S., Alamri, T., Alolayan, S. W., Bah, S., & Aljoudi, A. S. (2022). Autonomy and paternalism in Shared decision‐making in a Saudi Arabian tertiary hospital: A cross‐sectional study. Developing World Bioethics , 23 (3), 260–268. https://doi.org/10.1111/dewb.12355 ; Bukhari, A. A. (2017). Universal Principles of Bioethics and Patient Rights in Saudi Arabia (Doctoral dissertation, Duquesne University). https://dsc.duq.edu/etd/124; Ladha, S., Nakshawani, S. A., Alzaidy, A., & Tarab, B. (2023, October 26). Islam and Bioethics: What We All Need to Know . Columbia University School of Professional Studies. https://sps.columbia.edu/events/islam-and-bioethics-what-we-all-need-know

[39] Ababneh, M. A., Al-Azzam, S. I., Alzoubi, K., Rababa’h, A., & Al Demour, S. (2021). Understanding and attitudes of the Jordanian public about clinical research ethics.  Research Ethics ,  17 (2), 228-241.  https://doi.org/10.1177/1747016120966779

[40] Ababneh, M. A., Al-Azzam, S. I., Alzoubi, K., Rababa’h, A., & Al Demour, S. (2021). Understanding and attitudes of the Jordanian public about clinical research ethics.  Research Ethics ,  17 (2), 228-241.  https://doi.org/10.1177/1747016120966779

[41] Dajani, R. (2014). Jordan’s stem-cell law can guide the Middle East.  Nature  510, 189. https://doi.org/10.1038/510189a

[42] Dajani, R. (2014). Jordan’s stem-cell law can guide the Middle East.  Nature  510, 189. https://doi.org/10.1038/510189a

[43] The EU’s definition of autonomy relates to the capacity for creating ideas, moral insight, decisions, and actions without constraint, personal responsibility, and informed consent. However, the EU views autonomy as not completely able to protect individuals and depends on other principles, such as dignity, which “expresses the intrinsic worth and fundamental equality of all human beings.” Rendtorff, J.D., Kemp, P. (2019). Four Ethical Principles in European Bioethics and Biolaw: Autonomy, Dignity, Integrity and Vulnerability. In: Valdés, E., Lecaros, J. (eds) Biolaw and Policy in the Twenty-First Century. International Library of Ethics, Law, and the New Medicine, vol 78. Springer, Cham. https://doi.org/10.1007/978-3-030-05903-3_3

[44] Council of Europe. Convention for the protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine (ETS No. 164) https://www.coe.int/en/web/conventions/full-list?module=treaty-detail&treatynum=164 (forbidding the creation of embryos for research purposes only, and suggests embryos in vitro have protections.); Also see Drabiak-Syed B. K. (2013). New President, New Human Embryonic Stem Cell Research Policy: Comparative International Perspectives and Embryonic Stem Cell Research Laws in France.  Biotechnology Law Report ,  32 (6), 349–356. https://doi.org/10.1089/blr.2013.9865

[45] Rendtorff, J.D., Kemp, P. (2019). Four Ethical Principles in European Bioethics and Biolaw: Autonomy, Dignity, Integrity and Vulnerability. In: Valdés, E., Lecaros, J. (eds) Biolaw and Policy in the Twenty-First Century. International Library of Ethics, Law, and the New Medicine, vol 78. Springer, Cham. https://doi.org/10.1007/978-3-030-05903-3_3

[46] Tomuschat, C., Currie, D. P., Kommers, D. P., & Kerr, R. (Trans.). (1949, May 23). Basic law for the Federal Republic of Germany. https://www.btg-bestellservice.de/pdf/80201000.pdf

[47] Regulation of Stem Cell Research in Germany . Eurostemcell. (2017, April 26). https://www.eurostemcell.org/regulation-stem-cell-research-germany

[48] Regulation of Stem Cell Research in Finland . Eurostemcell. (2017, April 26). https://www.eurostemcell.org/regulation-stem-cell-research-finland

[49] Regulation of Stem Cell Research in Spain . Eurostemcell. (2017, April 26). https://www.eurostemcell.org/regulation-stem-cell-research-spain

[50] Some sources to consider regarding ethics models or regulatory oversights of other cultures not covered:

Kara MA. Applicability of the principle of respect for autonomy: the perspective of Turkey. J Med Ethics. 2007 Nov;33(11):627-30. doi: 10.1136/jme.2006.017400. PMID: 17971462; PMCID: PMC2598110.

Ugarte, O. N., & Acioly, M. A. (2014). The principle of autonomy in Brazil: one needs to discuss it ...  Revista do Colegio Brasileiro de Cirurgioes ,  41 (5), 374–377. https://doi.org/10.1590/0100-69912014005013

Bharadwaj, A., & Glasner, P. E. (2012). Local cells, global science: The rise of embryonic stem cell research in India . Routledge.

For further research on specific European countries regarding ethical and regulatory framework, we recommend this database: Regulation of Stem Cell Research in Europe . Eurostemcell. (2017, April 26). https://www.eurostemcell.org/regulation-stem-cell-research-europe   

[51] Klitzman, R. (2006). Complications of culture in obtaining informed consent. The American Journal of Bioethics, 6(1), 20–21. https://doi.org/10.1080/15265160500394671 see also: Ekmekci, P. E., & Arda, B. (2017). Interculturalism and Informed Consent: Respecting Cultural Differences without Breaching Human Rights.  Cultura (Iasi, Romania) ,  14 (2), 159–172.; For why trust is important in research, see also: Gray, B., Hilder, J., Macdonald, L., Tester, R., Dowell, A., & Stubbe, M. (2017). Are research ethics guidelines culturally competent?  Research Ethics ,  13 (1), 23-41.  https://doi.org/10.1177/1747016116650235

[52] The Qur'an  (M. Khattab, Trans.). (1965). Al-Mu’minun, 23: 12-14. https://quran.com/23

[53] Lenfest, Y. (2017, December 8). Islam and the beginning of human life . Bill of Health. https://blog.petrieflom.law.harvard.edu/2017/12/08/islam-and-the-beginning-of-human-life/

[54] Aksoy, S. (2005). Making regulations and drawing up legislation in Islamic countries under conditions of uncertainty, with special reference to embryonic stem cell research. Journal of Medical Ethics , 31: 399-403.; see also: Mahmoud, Azza. "Islamic Bioethics: National Regulations and Guidelines of Human Stem Cell Research in the Muslim World." Master's thesis, Chapman University, 2022. https://doi.org/10.36837/ chapman.000386

[55] Rashid, R. (2022). When does Ensoulment occur in the Human Foetus. Journal of the British Islamic Medical Association , 12 (4). ISSN 2634 8071. https://www.jbima.com/wp-content/uploads/2023/01/2-Ethics-3_-Ensoulment_Rafaqat.pdf.

[56] Sivaraman, M. & Noor, S. (2017). Ethics of embryonic stem cell research according to Buddhist, Hindu, Catholic, and Islamic religions: perspective from Malaysia. Asian Biomedicine,8(1) 43-52.  https://doi.org/10.5372/1905-7415.0801.260

[57] Jafari, M., Elahi, F., Ozyurt, S. & Wrigley, T. (2007). 4. Religious Perspectives on Embryonic Stem Cell Research. In K. Monroe, R. Miller & J. Tobis (Ed.),  Fundamentals of the Stem Cell Debate: The Scientific, Religious, Ethical, and Political Issues  (pp. 79-94). Berkeley: University of California Press.  https://escholarship.org/content/qt9rj0k7s3/qt9rj0k7s3_noSplash_f9aca2e02c3777c7fb76ea768ba458f0.pdf https://doi.org/10.1525/9780520940994-005

[58] Lecso, P. A. (1991). The Bodhisattva Ideal and Organ Transplantation.  Journal of Religion and Health ,  30 (1), 35–41. http://www.jstor.org/stable/27510629 ; Bodhisattva, S. (n.d.). The Key of Becoming a Bodhisattva . A Guide to the Bodhisattva Way of Life. http://www.buddhism.org/Sutras/2/BodhisattvaWay.htm

[59] There is no explicit religious reference to when life begins or how to conduct research that interacts with the concept of life. However, these are relevant verses pertaining to how the fetus is viewed. (( King James Bible . (1999). Oxford University Press. (original work published 1769))

Jerimiah 1: 5 “Before I formed thee in the belly I knew thee; and before thou camest forth out of the womb I sanctified thee…”

In prophet Jerimiah’s insight, God set him apart as a person known before childbirth, a theme carried within the Psalm of David.

Psalm 139: 13-14 “…Thou hast covered me in my mother's womb. I will praise thee; for I am fearfully and wonderfully made…”

These verses demonstrate David’s respect for God as an entity that would know of all man’s thoughts and doings even before birth.

[60] It should be noted that abortion is not supported as well.

[61] The Vatican. (1987, February 22). Instruction on Respect for Human Life in Its Origin and on the Dignity of Procreation Replies to Certain Questions of the Day . Congregation For the Doctrine of the Faith. https://www.vatican.va/roman_curia/congregations/cfaith/documents/rc_con_cfaith_doc_19870222_respect-for-human-life_en.html

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Mifrah Hayath

SM Candidate Harvard Medical School, MS Biotechnology Johns Hopkins University

Olivia Bowers

MS Bioethics Columbia University (Disclosure: affiliated with Voices in Bioethics)

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