The Great Plate Count Anomaly

Forty years on

I am veering away from the line I have been developing over the past few weeks to honor a mentor and friend. I learned last weekend that my PhD mentor, Jim Staley, passed away. Jim asked me to help write a review article, “Measurement of in situ activities of nonphotosynthetic microorganisms in aquatic and terrestrial habitats” that was published in 1985. In it he cited observations made by others as well as his own data that “as a general rule we have found that the maximum recovery of heterotrophic bacteria is 1% of the total direct count using … viable enumeration methods from a variety of oligotrophic to mesotrophic aquatic habitats, whereas higher recoveries (approaching 80-90%) can be achieved from eutrophic habitats” This he called the Great Plate Count Anomaly.

A number of molecular microbial ecologists told me that in the early days of applying direct nucleic acid extraction and analysis to environmental samples, they always cited this paper in their grant proposals as a justification to not worry about cultivating bacteria from the habitat. Several thoughts occur to me. First, note that Jim points out that there are habitats where recovery is not that poor. Second, over the past 40 years there have been important improvements in cultivation theory and practice (and given the very high cost of sequencing in those early days, I wonder want an equivalent amount of money spent on grad student fellowships devoted to improving cultivability might have yielded¹). Third, it strikes me that the most important issue is why there is this enormous difference between direct and viable counts. I would like to pursue this last point here, as a means of honoring Jim’s legacy.

I would be remiss to not point out the excellent work that Slava Epstein and his colleagues at Northeastern University have done. In 2009 he edited a book Uncultivated Microorganisms (Springer-Verlag Berlin Heidelberg) that covered a broad variety of these issues. My discussion here will necessarily be brief -- his book remains a valuable resource as I’m not sure that there have been enormous breakthroughs on this topic in the last 15 years.¹ Kapinusava et al. (2023) recently reviewed a number of factors related to culturability.

Why the Great Plate Count Anomaly?

Several explanations have been put forward.

1.     Many of the cells are dead / dormant / “Viable But Non-Culturable”

2.     The culture medium is selecting against most of the microbes

3.     They are impossible to grow in culture

How many cells are active/alive?

This actually was a central element of the review article Jim and I wrote in 1985. Historically, tetrazolium dye reduction and deposition has been used to identify active cells microscopically. However, that method is far from perfect. The ratio of high- to low-nucleic acid containing cells has been used to infer active populations. More recently, there have been a small number of papers that applied Nano-SIMS or BONCAT to this question.

A bottom line from our work in freshwater lakes put the fraction of metabolically-active cells at 35-50%. Quite remarkably, Couradeau et al. (2019) reported that 90% of soil bacterial cells were active, using the BONCAT method. In my opinion, substantial work on this topic is warranted, to resolve hypotheses related to dormancy and assess the state of microbial populations rather than merely cataloging them by 16S rDNA gene sequences. Would using H₂¹⁸O as a “universal substrate” for active bacteria (as it is currently used for stable isotope probing), coupled with Raman spectroscopy of individual cells provide a relatively convenient means of assaying the fraction of active cells?

Dead / Dormant / VBNC

Jay Lennon and his colleagues have done some excellent work on dormancy over the past 15 years. (Lennon and Jones, 2009). If those cells do not immediately resuscitate they will not be ‘counted’ in any viability assay. The phenomenon of “Viable but nonculturable” (VBNC) has also been described for a number of cultures of Gram-negative bacteria (Colwell, 2009). Whether due to in-situ conditions or the ‘shock’ of inoculation into a culture medium, living cells do not give rise to replicating propagules. A resuscitation method is needed to convert these cells back to active growth. Even 30 years after the VBNC state was identified, the mechanisms of VBNC state induction and resuscitation are poorly understood.

Slava Epstein has developed a general theory for this – Scout theory. The premise is that in resource-limited environments, most cells of a specific population are in a dormant state but there is stochastic activation of a few cells that can ‘scout’ if current conditions are favorable. These active cells may then use a chemical signaling system to resuscitate their kin.  

It's the culture medium, stupid!!

(with apologies to James Carville)

Jim Staley’s go-to medium for isolating aquatic bacteria was dilute (0.01%) peptone) and he was successful for groups such as prosthecate bacteria, Hyphomicrobium and caulobacters. That medium contains ~30 mg C l^-1, much lower than traditional media. But a seminal revelation was Don Button’s ², that he could grow marine bacteria in filter-sterilized sea water (dissolved organic C ~ 1 mg l^-1). Steve Giovannoni and his colleagues took this observation and ran with it to great effect, isolating members of Pelagibacterales (SAR11), an order in the Alphaproteobacteria that are very highly abundant in the ocean. With careful effort, investigators have increased the biomass yield several orders of magnitude beyond what unamended sea water can sustain. This has provided his lab the means to generate material for experiments that interrogate important physiological properties relevant to their in situ lifestyle.

This is excellent stuff, but it is unlikely there is any ‘general’ medium for propagation – many organisms will be unique problems to resolve and that needs creativity and effort – the things at which Jim Staley excelled. This is opposed to simply isolating and sequencing DNA from an environmental sample. The relevant question is whether such effort is worth it. I argue it is, if the discipline is to ever make progress on the ‘causings’ of ecological phenomena as opposed to merely describing the parts.

Cultivation? Impossible!

My comments here are primarily an act of faith. Throughout the history of microbiology there have been a few individuals with a particular genius for cultivating microbes. We think of Martinus Beijerinck and Sergei Winogradsky from the early days of the science. From the latter half of the 20^th Century, Norbert Pfennig for photosynthetic bacteria, Fritz Widdel with sulfate-reducing bacteria, John Breznak on spirochetes and Jim Staley for prosthecate bacteria and Planctomycetes were all wizards. Slava Epstein has developed several innovative methods to begin isolation of bacteria under near-in situ conditions. Stephanie Connon and Steve Giovannoni developed a high-throughput method to apply Button’s concept of growing bacteria in unamended seawater.

Perhaps the classic case of the ‘impossibility’ of pure culture are syntrophic associations of anaerobes fermenting organic acids (with H₂ production) and methanogens that serve to poise the partial pressure of H₂ low enough that organic acid oxidation remains thermodynamically favorable. But after some effort and a basic understanding of their physiology, pure culture is feasible for Syntrophobacter isolates under alternative culture regimes.

Even if most bacteria in non-human habitats have so far not been cultivated (Lloyd et al., 2018), I suggest that the Great Plate Count Anomaly is not the product of an impossibility but a lack of interest by funding agencies / grant proposal reviewers in the topic.¹ I can’t fault individual scientists for proposing work that they perceive will have a greater chance for grant awards. Jim Staley had a very successful scientific career, but he did struggle to fund cultivation efforts at a high level. Targeted programs in U.S. science have abounded and produced impressive results. There are testable hypotheses regarding why natural populations are so recalcitrant to easy cultivation – those require validation as do substantial support for innovative programs at cultivation. Providing support to laboratories with demonstrated expertise in creative cultivation would be a wise investment to push forward a mechanistic understanding of microbial ecology.  

Notes:

¹ I intend to amplify on several of these comments in a future post on “the science of microbial ecology science.” I have followed the science of science peripherally the past few years – it is a relatively new pursuit that aims to look for patterns that govern the working of science. Topics include impact, roles of collaboration, productivity and creativity, what gets funded and why. (Fortunato et al., 2018).

² If you are interested in how cells deal with nutrient-limiting conditions, you could do no better than reading some of Don Button’s papers. This one is a particularly classic and deep paper:
Button DK (1994) The Physical Base of Marine Bacterial Ecology. Microb. Ecol. 28:273-285 

Zen for the day

Jim loved the Pacific Northwest and resided there for 55 years. Local field trips included nearby lakes, Spirit Lake near Mount St. Helens 6 months after it exploded, and a few days scurrying down caves near Mount Adams with an arachnologist, searching for microbial slime.

Pisaster ochraceus, the ochre sea star, from near Point of the Arches on the Olympic Peninsula

References

Button, DK, Schut F and Robertson BR (1993) Viability and Isolation of Marine Bacteria by Dilution Culture: Theory, Procedures, and Initial Results. Appl Environm Microbiol 59: 881-891.

Colwell RR (2009) Viable but Not Cultivable Bacteria. In Uncultivated Microorganisms, SS Epstein ed. pp 121-129.

Couradeau, E et al. (2019) Probing the active fraction of soil microbiomes using BONCAT-FACS. Nature Commun 10: https://doi.org/10.1038/s41467-019-10542-0

Epstein, SS (ed) (2009) Uncultivated Microorganisms 208 pp. Springer-Verlag Berlin Heidelberg

Fortunato, S et al. (2018) Science of science. Science 359: DOI: 10.1126/science.aao0185

Giovannoni SJ (2017) SAR11 Bacteria: The Most Abundant Plankton in the Oceans. Ann Rev Mar Sci 9: 231-255. doi: 10.1146/annurev-marine-010814-015934

Lloyd KG, Steen AD, Ladau J, Yin J, Crosby L. (2018) Phylogenetically novel uncultured microbial cells dominate Earth microbiomes. mSystems 3:e00055-18. https:// doi.org/10.1128/mSystems.00055-18.

Kapinusova G , Lopez Marin Marco A. and Uhlik O (2023) Reaching unreachables: Obstacles and successes of microbial cultivation and their reasons. Front Microbiol 14: https://doi.org/10.3389/fmicb.2023.1089630

Lennon, JT and Jones, SE (2011) Microbial seed banks: the ecological and evolutionary implications of dormancy. Nat Rev Microbiol 9: 119-130.

Staley JT and Konopka A (1985) Measurement of in situ activities of nonphotosynthetic microorganisms in aquatic and terrestrial habitats. Annu Rev Microbiol 39: 321-346.  DOI: 10.1146/annurev.mi.39.100185.001541