Historical Background

In 1898, an Austrian microbiologist Heinrich Winterberg made a curious observation: the number of microbial cells in his samples did not match the number of colonies formed on nutrient media (Winterberg 1898). About a decade later, J. Amann quantified this mismatch, which turned out to be surprisingly large, with non-growing cells outnumbering the cultivable ones almost 150 times (Amann 1911). These papers signify some of the earliest steps towards the discovery of an important phenomenon known today as the Great Plate Count Anomaly (Staley and Konopka 1985). Note how early in the history of microbiology these steps were taken. Detecting the Anomaly almost certainly required the Plate.  If so, then the period from 1881 to 1887, the years when Robert Koch and Petri introduced their key inventions (Koch 1881; Petri 1887), sets the earliest boundary for the discovery, which is remarkably close to the 1898 observations by H. Winterberg. Over a century old, the Great Plate Count Anomaly today is arguably the oldest unresolved microbiological phenomenon.

In the years to follow, the Anomaly was repeatedly confirmed by all microbiologists who cared to compare the cell count in the inoculum to the colony count in Petri dish (cf., Cholodny 1929; Butkevich 1932; Butkevich and Butkevich 1936).   By mid-century, the remarkable difference between the two counts became a universally recognized phenomenon, acknowledged by several classics of the time (Waksman and Hotchkiss 1937; ZoBell 1946; Jannasch and Jones 1959).

https://upload.wikimedia.org/wikipedia/commons/8/85/Great-plate-count-anomaly.svg

https://upload.wikimedia.org/wikipedia/commons/8/85/Great-plate-count-anomaly.svg

Surely the “missing” microbial diversity was as large then as it is now.  However, reading the earlier papers leaves an impression that throughout most of the 20th century the “missing” aspect was not viewed as a particularly important problem or as an exciting opportunity.  A casual mention was typical of many publications. “Missing” cells were not necessarily considered missing species, let alone signs of novel classes of microbes. Besides, the unexplored microbial biodiversity was a purely academic issue; the hunt for novel species as a resource for biotechnology had not yet begun. It is also important that the reasons for the Anomaly appeared rather simple at the time.  Counting errors, dead cells, and later damaged cells were continuously considered significant components of the disparity.  Also, it had been obvious at least since Koch’s time that no single nutrient medium could possibly satisfy all microorganisms (Koch 1881), and so the finger was always pointing to media deficiencies.  Indeed, imperfections in media design was such a simple and intuitive explanation for the refusal of the microbial majority to grow in vitro that many microbiologists began viewing it as sufficient.  The triviality of the explanation generated a perception of the Anomaly as a purely technical issue that could be resolved by bettering the media compositions and incubation conditions.

This view began to change towards the end of the 20th century.  Cultivation efforts during the preceding decades did produce success stories; yet even as the manuals for media recipes grew into thick volumes, the overwhelming majority of microorganisms still eschewed the Petri dish. The progress in recovering missing species was rather incremental and did not change the overall picture. And, it was going to get worse.

The rRNA approach (Olsen et al. 1986) was a truly spectacular development: it provided insight into microbial world missed by traditional cultivation.  Novel microbial divisions were discovered by the dozen (Giovannoni et al. 1990; Ward et al. 1990; DeLong 1992; Fuhrman et al. 1992; Liesack and Stackebrandt 1992; Barns et al. 1994; Hugenholtz et al. 1998; Ravenschlag et al. 1999; Dojka et al. 2000). From the molecular surveys of the 1990’s emerged an image of the biosphere with millions of novel microbial species waiting to be discovered (Tiedje 1994; Allsopp et al. 1995). What microbiologists had been able to cultivate and catalogue throughout the entire history of microbiological exploration (Staley et al. 1989) appeared to be an insignificant portion of the total. Successes in cultivation notwithstanding, the gap between microbial richness in nature and that of culture collections just would not close. Even today, most of the known microbial divisions have no single cultivable representative (Rappe and Giovannoni 2003; Schloss and Handelsman 2004). This gap was called “extraordinary” in 1932, and given the same description in 2000 (Butkevich 1932; Colwell 2000), as if the countless cultivation studies during these seventy years never existed.  But, the realities of our age are different from the 1930s, and the Great Plate Count Anomaly is no longer “just” an academic observation.  The need to close the gap is an urgent practical issue, as biotech and pharmaceutical industries appear to have exhausted what the limited number of cultivable species have to offer (Osburne et al. 2000).  Today, the resolution of the phenomenon of microbial uncultivability is recognized as a top research priority for microbial biology (Young 1997; Hurst 2005).  The principal challenges are to understand why uncultivated microorganisms are uncultivated, and to describe, access, and utilize their seemingly infinite diversity.

 

REFERENCES

Allsopp D, Colwell RR, Hawksworth DL (1995) Microbial Diversity and Ecosystem Function. Wallingford UK: CAB International

Amann J (1911) Die direkte Zählung der Wasserbakterien mittels des Ultramikroskops. Centralbl. f. Bakteriol. 29:381-384

Barns SM, Fundyga RE, Jeffries MW, Pace NR (1994) Remarkable archaeal diversity detected in a Yellowstone National Park hot spring environment. Proc Natl Acad Sci U S A 91:1609-1613

Butkevich NV, Butkevich aVS (1936) Multiplication of sea bacteria depending on the composition of the medium and on temperature. Microbiology (Moscow) 5:322-343

Butkevich VS (1932) Zür Methodik der bakterioloschen Meeresuntersuchungen  und einige Angaben über die Verteilung der Bakterien im Wasser und in den Büden des Barents Meeres. Trans. Oceanogr. Inst. Moscow 2: 5-39 (in Russian with German summary) 2:5-39

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Hurst CJ (2005) Divining the future of microbiology. ASM News 71:262-263

Jannasch HW, Jones GE (1959) Bacterial populations in seawater as determined by different methods of enumeration. Limnol. Oceanogr. 4:128-139

Koch R (1881) Zur Untersuchung von pathogenen Organismen. Mitth. a. d. Kaiserl. 1:1-48

Liesack W, Stackebrandt E (1992) Occurrence of novel groups of the domain Bacteria as revealed by analysis of genetic material isolated from an Australian terrestrial environment. J Bacteriol 174:5072-5078

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Osburne MS, Grossman TH, August PR, and , MacNeil IA (2000) Tapping into microbial diversity for natural products drug discovery. ASM News 66:411-417

Petri RJ (1887) Eine kleine Modification des Koch’schen Plattenverfahrens. Centralbl. f. Bakteriol. 1:279-280

Rappe MS, Giovannoni SJ (2003) The uncultured microbial majority. Annu Rev Microbiol 57:369-394

Ravenschlag K, Sahm K, Pernthaler J, Amann R (1999) High bacterial diversity in permanently cold marine sediments. Appl Environ Microbiol 65:3982-3989

Schloss PD, Handelsman J (2004) Status of the microbial census. Microbiol Mol Biol Rev 68:686-691

Staley JT, Bryant MP, Pfennig N, Holt JG (1989) Bergey’s manual of sytematic bacteriology.

Staley JT, Konopka A (1985) Measurement of in situ activities of nonphotosynthetic microorganisms in aquatic and terrestrial habitats. Annu Rev Microbiol 39:321-346

Tiedje JM (1994) Microbial diversity: of value to whom? ASM News 60:524-525

Waksman SA, Hotchkiss M (1937) Viability of bacteria in sea water. J. Bacteriol. 33:389-400

Ward DM, Weller R, Bateson MM (1990) 16S rRNA sequences reveal numerous uncultured microorganisms in a natural community. Nature 345:63-65

Winterberg H (1898) Zur Methodik der Bakterienzahlung. . Zeitschr. f. Hyg. 29:75-93

Young P (1997) Major microbial diversity initiative recommended. ASM News 63:417-421

ZoBell CE (1946) Marine microbiology: a monograph on hydrobacteriology. Chronica Botanica Co., Waltham, MA, U.S.A.

 

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