Although there are a large number of reasons that a soil bacterium may not be easily cultured in the lab, most of these can be reduced to the difficulty of replicating the very precise environment conditions certain microbes require for growth. Early culturing efforts focused on microbes that were easily grown under standard conditions, largely due to adaptation to the incubator-like conditions of an animal body. By contrast, free-living environmental microbes can have a broad array of environmental requirements.

One example that I find interesting is the discovery that many organisms cannot survive in the high-nutrient conditions favored by standard cultural practices (e.g. the linked paper). The development of low-nutrient media has greatly increased the number of organisms that have been successfully cultured.

Many soil organisms are incapable of surviving under high-oxygen conditions. These obligate anaerobes would naturally not survive standard culturing approaches, but there have been some successes with growing them in anaerobic chambers.

A more pernicious problem is that many soil microorganisms have adapted to their low resource environment by growing very slowly. A standard culturing assay would not detect these organisms, as they would have very few replications in the time it would take a faster organism to completely overgrow the plate. Most to nearly all bacteria in a soil are dormant at a given point in time, so for some of them it may be necessary to find the right conditions to "wake" them.

You might note that these examples all have workarounds. In general, I would say that the statement that "99% of bacterial strains are unculturable" is quite outdated at this point. The challenge lies in identifying the necessary conditions for promoting their growth in a laboratory setting. Given sufficient time and interest, I think a surprisingly large number of soil bacteria could be grown in isolation. That said, the diversity of bacteria in the soil is truly mind-bogling, and culturing most of those might take months or years of trial and error. Culturing a large percentage of soil bacteria is completely unfeasible at this time.Moreover, culture-independent methods (e.g. metagenomic sequencing) are improving rapidly, and have perhaps removed some of the impetus for working toward anything like comprehensive culturing of soil bacteria.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC321273/

Although there are a large number of reasons that a soil bacterium may not be easily cultured in the lab, most of these can be reduced to the difficulty of replicating the very precise environment conditions certain microbes require for growth. Early culturing efforts focused on microbes that were easily grown under standard conditions, largely due to adaptation to the incubator-like conditions of an animal body. By contrast, free-living environmental microbes can have a broad array of environmental requirements.

One example that I find interesting is the discovery that many organisms cannot survive in the high-nutrient conditions favored by standard cultural practices (e.g. the linked paper). The development of low-nutrient media has greatly increased the number of organisms that have been successfully cultured.

Many soil organisms are incapable of surviving under high-oxygen conditions. These obligate anaerobes would naturally not survive standard culturing approaches, but there have been some successes with growing them in anaerobic chambers.

A more pernicious problem is that many soil microorganisms have adapted to their low resource environment by growing very slowly. A standard culturing assay would not detect these organisms, as they would have very few replications in the time it would take a faster organism to completely overgrow the plate. Most to nearly all bacteria in a soil are dormant at a given point in time, so for some of them it may be necessary to find the right conditions to "wake" them.

You might note that these examples all have workarounds. In general, I would say that the statement that "99% of bacterial strains are unculturable" is quite outdated at this point. The challenge lies in identifying the necessary conditions for promoting their growth in a laboratory setting. Given sufficient time and interest, I think a surprisingly large number of soil bacteria could be grown in isolation. That said, the diversity of bacteria in the soil is truly mind-bogling, and culturing most of those might take months or years of trial and error. Culturing a large percentage of soil bacteria is completely unfeasible at this time.Moreover, culture-independent methods (e.g. metagenomic sequencing) are improving rapidly, and have perhaps removed some of the impetus for working toward anything like comprehensive culturing of soil bacteria.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC321273/

Dear David,

I really appreciate very much your very informative answer. I am a pharma synthetic chemist and I was not aware of much of what you write about. Thank you.

One note I have to make is that the Lewis and Epstein team came up with a device called iChip, which is supposed to increase the percentage of culturable strains to about 50%... and that would be great.

Dear Jacek,

I should like to give my considerations on this subject. It would have been risky to answer these very interesting questions by a simple "99% of soil bacterial strains cannot be culture-grown in the lab". This value reflects only the surprise of the researchers, who use modern molecular genetic methods. Moreover, this indicator is not constant and varies in a wide range of values. The existence of the nonculturable bacteria was predicted by S.N. Vinogradskii (Winogradsky) in late twenties of the last century based on direct microscopic examinations of the soil. Long before he showed tremendous functionality of natural microbial communities. "In 1896, while actively engaged in his elective culture research, he delivered a popular lecture at the IEM ‘‘On the Role of Microbes in the General Cycle of Life.’’ Sounding the same note that reverberated in his scientific nvestigations, he described the cycle of life as ‘‘a single huge organism.’’ The body of this entity was composed of the entire planet’s organic material; it borrowed what it needed from nature’s inorganic reserves, used it purposively and, finally, returned it to the dead, inorganic realm of nature. In this vision of the cycle of life, he delegated the essential role to microorganisms. Only through their agency did matter circulate between nature’s two realms. Microbes regulated this circulation by the endlessly varied reagents in their

physiological processes that constantly decompose and synthesize organic compounds. " (LLOYD T. ACKERT JR., The ‘‘Cycle of Life’’ in Ecology: Sergei Vinogradskii’s Soil

Microbiology, 1885–1940// Journal of the History of Biology (2007) 40:109–145).

Some examples show the excellence and exceptional potential in natural microbial communities. "These and other examples demonstrate that the term “antibiotics” (against life), introduced by S. Waksman in 1942, seems to be rather limited at present. At the same time, practical application of this particular mechanism of regulation of microbial communities (antibiosis) has resulted in outstanding revolutionary results in medicine and related fields. It is

possible to say that delocalization of a single event in the soil’s microzone (antibiosis) occurred with subsequent use of the mechanism on the scale of the global planet. However, an increase in microorganism resistance to antibiotics is now manifested at the same scale; according to some pessimistic forecasts, it means a return to the preantibiotic era. Science has to return again to the level of the soil’s microzone in order to implement the particular character of the killer effect of antibiotics and huge functional potential of natural microbial communities, in a structure in which uncultivated forms account for the main portion". (Kozhevin, Vinogradova, Bulgakova // Natural Functions of Antibiotics as “Informbiotics // Moscow University Soil Science Bulletin, 2014, Vol. 69, No. 3, pp. 93–98

We have shown that it is possible to obtain complex microbial fertilizers for different applications based on microbial communities without growing microorganisms on nutrient media. Management of the natural microbial community may be carried out using the methods of mathematical experimental design in the course of microbial successions (Andreeva , Kozhevin Optimization of Natural Communities of Soil Microorganisms as a Way to Create Microbial Fertilizers // Moscow University Soil Science Bulletin, 2014, Vol. 69, No. 4, pp. 184–187)

And finally I will give my old analysis of this phenomenon (Kozhevin P.A. Microbial Populations in Nature.MSU, Moscow (in Russian), 1989.

Direct microscopy and the plating method carry different information which can be presented in the form of the equa­tion M1 +M0 = P1 + P2 + P3 + P4 + P5 ..., where M1 is the bacterial count obtained by the direct method; M0 is the number of particles which can be counted as cells in microscopy; P1 is the quantity of bac­teria that can be counted on the medium used; P2 is the quantity of bacteria having different food or other requirements and cannot be estimated on a given medium; P3 is the quantity of bacteria having a low total growth rate. They do not appear on the plate because their growth is suppressed by the fast growing cultures; P4 is the quantity of bacteria which can grow on a given medium but cannot be estimated because of stresses. One of the examples of stress could be the mortality of the bacteria transferred from a medium with low level of nutrients to a rich medium ("mortality enhanced by the substrate"); P5 are dead cells that are visible under microscopy but do not give colonies on the nutrient medium. The ratio between these methods (coefficient Kozhevin, К ) can be represented by the following formula: K= (M1 +M0)/ P1 .The coefficient К will increase if there is an increase in the components of P2 (increase in variability, biodiversity), P3 (development of the slow-growing species), P4 (cells growing with the minimum quantity of the substrate), and P5 (the fraction of inviable cells). This is a characteristic property of the "climax" ecosystems. Consequently, a higher value of К should reflect the late stages of microbial succession, i.e., the "ma­tured" ecosystems.

A low value of К indicates an increase in the role of the fast growing microorganisms that vigorously consume the nutrient substrate, which is characteristic of the young ecosystems in the initial stages of succes­sion. A regular change in the value of К in the process of the experiment fully confirms that this coefficient is an indicator of different stages of microbial succession. In reality, on the 7th day after addition of glucose, the value of К declined sharply so that the population indexes according to the data of microscopy and plating differ by not more than 11-12 times. The value of К gradually increases in the later stages of succession, and the in­crease in the value of К is more pronounced for the lower horizon.

Thus, the ratio of bacterial counts from direct microscopy to those obtained by the plating method (coeffi­cient K) is a valuable quantitative index on the basis of which a general idea about the stage of microbial suc­cession in the soil can be obtained and the microbial system as a whole characterized.

Dear Dr. Kozhevin,

Thank you for sharing some of the gems of microbiological thought you yourself contributed to. Very interesting. I just have one comment regarding the following fragment taken from your post above: "Science has to return again to the level of the soil’s microzone in order to implement the particular character of the killer effect of antibiotics and huge functional potential of natural microbial communities, in a structure in which uncultivated forms account for the main portion". I would say, parallel to that and for practical reasons, we have to continue efforts in the area of de novo design of antibacterials based on the known molecular structures of bacterial targets. But that is just the impatient synthetic chemist speaking here...

Thank you again very, very much.

Jacek Martynow

From the statistics of this particular problem it is evident that this is an area of continued great interest. No wonder, the resistant strains are increasingly prevalent, causing a lot of harm and suffering...

I am continually very much interested in your thoughts about the present topic.

Dear Jacek,

your question once again highlighted the fundamental problem. To characterize its significance, one can use the simplest classification of scientific works.

Several categories of scientific works can be distinguished. 1. The upper class - staying in the archives from the moment of their birth. 2. Used sometimes and indirectly associated with an effective solution (scrap). 3. Widely used and quoted "pop" ("scientometric" indicators), which often has nothing but fashion, hinders the development of true science, and then falls not even in scrap, but in the toilet. For my part, I'll venture to add another category - Superclass. This is the information that the researcher, on the basis of his principles or external constraints, completely closes for others. From the history of science we know about such cases. This is primarily about morality in cases where the researcher analyzes undesirable risks. This is obvious even in ordinary life. For example, if you find an incredibly beautiful gemstone, then it's hardly reasonable to carry it with a joyful dance for a gang of avaricious villains.

Your question relates to scientific research at higher levels. Therefore, one can hardly expect an unambiguous answer with an effective solution.

For me, this problem is of system interest. The natural microbial community is a very complex system with pronounced teleonomy. This system responds in this case with antibiotic resistance in response not only to the mass use of antibiotics, but also to anthropogenic pollution of the environment (for example, heavy metals). In such cases, in the lateral transfer of genes, co-selection is observed. It can be said that the microbial system strikes back. The main target is a Homo sapiens who pollutes nature. I believe that the system approach should be combined with the search for reduction solutions. In particular, it is possible to ensure the synthesis of a complex of antibiotics by microorganisms directly in a natural habitat. In this case there is no problem of non-cultivable microorganisms. The result of the life activity of the microbial community under certain conditions can be a complex of antibiotics, including new and unknown compounds. However, such a decision in principle is not new. Suffice it to say that before the era of antibiotics for the treatment of purulent wounds, an ointment was used on the basis of non-sterile (!) soil after special treatment.

Dear Dr. Kozhevin,

I read your post with great interest, thank you very much.

Professor Kozhevin,

Russian researchers always surprise me with great mathematical works and deep philosophical thinking. But, What do you think about the Black Queen hypotheses as the main reason for non-cultivable microorganisms?

Article Morris JJ, Lenski RE, Zinser ER.. The Black Queen Hypothesis...

This is a nice, very thoughtful and interesting discussion, spanning beyond traditionally divided science disciplines - something rarely seen nowadays. I really appreciate the input of scientists sharing their thoughts in this thread.

Dear Vladimir,

Thank you for your positive thoughts.

Perhaps solving many of the new problems we humans are facing does require reaching far beyond our fields? Anyways, we always need to be curious...

Dear Raúl Montañez Martínez! I thank you for your kind words about the Russian scientific school. A feature of this scientific school is a systematic view of nature as a surprisingly complex object with its goal-setting. Unfortunately, science is now going through difficult times. I do not consider myself a venerable scholar, but I studied and worked, for example, with such an outstanding scientist as GA. Zavarzin. Russian scientific school has always respected the works of leading foreign researchers, books and articles of which have always been presented in the lists of cited literature. I still remember the definition of the natural habitat of microorganisms (Thomas Brock), the equifinal description of successions (Clements), the analysis of the dynamics of microbial populations and comments to my address (M. Alexander, T. Gray). Maybe I'm wrong, but most of the current publications in the world science are not so interesting (reductionism, postmodernism, priority of genotypic methods with the accumulation of redundant and often meaningless information). Therefore, I am grateful to you for a very interesting article. It remains only to understand whether there is an evolution in the traditional sense (Darvin) in the case of the microbial system, which for billions of years has surprisingly preserved itself and ensured the stability of the biosphere.

Until now, the main focus is on negative mechanisms (competition, antagonism, predation, parasitism), which are included in the hypothesis of the Red Queen. However, only negative mechanisms do not give a sustainable community. For a sustainable community, negative mechanisms must be compensated by mechanisms of regulation of the opposite sign. These include the mechanisms of regulation described in the article as the hypothesis of the Black Queen. Therefore, the metaphor is directly related to the problem of uncultivated populations. Here everything depends on the scale of the consideration of the problem. For example, depending on the concentration, the effect of the antibiotic on the target can be not only negative but also positive (hormesis).

This can be shown in the figure with the dynamics of population A and population B. To characterize iinteractions, we take the simplest tetrachoric coefficient (correlation).Here r0 affine association. It is clear that with the growth of the coefficient r the role of complex trophic and signaling mechanisms of regulation increases. Therefore, the problem of uncultivated microorganisms is obvious for populations from mature microbial systems (K-strategy). The problem of uncultivated populations is usually a laboratory problem. In fact, these populations often perfectly exist in a dynamic regime in nature (compensation of natural death by reproduction processes). These populations always live in an ecological system that ensures the possibility of the existence of all its elements (cultivated and uncultivated in the laboratory). Thus, it is necessary to follow the logic of analyzing complex systems from "top to bottom". It is obvious that the key object in the ecology of microorganisms should be a microbial community.

This is a fundamental question; of the answer depends advances in science. Petr Kazhevin gave the right answer and I quote a part of it : "The existence of the nonculturable bacteria was predicted by S.N. Vinogradskii (Winogradsky) in late twenties of the last century based on direct microscopic examinations of the soil. Long before he showed tremendous functionality of natural microbial communities. "In 1896, while actively engaged in his elective culture research, he delivered a popular lecture at the IEM ‘‘On the Role of Microbes in the General Cycle of Life.’’ Sounding the same note that reverberated in his scientific investigations, he described the cycle of life as ‘‘a single huge organism.’’ " This is the key. If we start considering nature as a single organism in which we have our existence, we will find new answers because our scientific problem resides in the separation of what we study from its natural piezoelectric environment. This following article can also shed light on this:https://www.nature.com/articles/s41598-017-12063-6.

Dear Jacek, Indeed, the interest in the discussion that you initiated was maintained for several years. Finally, an article appeared in which the authors (using molecular tools!) abandon the paradigm about 99% of "unculturable" microorganisms https://www.researchgate.net/publication/332228979_High_proportions_of_bacteria_are_culturable_across_major_biomes

Thank you very much.