Microbiological Methods
Setting the Stage for Discovery of the Human Microbiome
Antonie van Leeuwenhoekâa cloth merchant by tradeâis credited for the discovery of single-celled microorganisms, which he called âwee animalculesâ (little animals) (Dobell, 1932). With a simple, personally handcrafted microscope, in the late 1600s, he documented the presence of microorganisms in samples collected from a variety of sources. Leeuwenhoek was the first to observe microorganisms in the human body; he found them in dental plaque and in a stool sample on one occasion when he was ill with diarrhea. About two centuries would pass before techniques were developed to explore the significance of Leeuwenhoekâs observations.
Robert Kochâs extraordinary research career spanned the greater part of an era dubbed the âgolden age of bacteriology,â 1876â1906 (Blevins et al., 2010). In 1876, Koch published a paper demonstrating that anthrax was caused by the bacterium Bacillus anthracis, providing the first proof for the germ theory of disease (Blevins et al., 2010). But his original methods for laboratory cultivation of bacteria were crude and inadequate for routine use, hindering his further progress. To obtain pure culturesâthat is, cultures composed of a single bacterial speciesâhe required a solid medium that would support bacterial growth. His attempts to grow bacteria on the surface of slices of potato or on media solidified with gelatin were unsuccessful. The breakthrough occurred when Fannie Angelina Hesse, the wife of Kochâs associate Walther Hesse, suggested the use of agar to solidify liquid bacteriologic media (Hesse & Gröschel, 1992). Armed with this new medium, Koch and his colleagues developed methods for isolating and studying pure cultures of bacteria. The impact on medical microbiology was immediate, and between 1878 and 1906, nineteen new bacterial pathogens were linked to specific infectious diseases. These techniques, augmented and supplemented with advances in microscopy and microbial biochemistry, endure in modern microbiology laboratories. They not only have formed the basis for the culture-dependent microbiology but also have fostered the expansion of microbiology beyond pathogenicity into diverse fields like biochemistry, genetics, ecology, and biotechnology.
By the 1980s, however, the growing awareness of the great abundance, diversity, and environmental ubiquity of microorganisms (Whitman et al., 1998) prompted a shift in research strategy. The complexity of microbial communities in their natural habitats was exemplified by the observation that most of the microscopically observable microorganisms in an environmental sample could not be cultured in the laboratory. This discrepancy between microbes that could be observed and those that could be cultured was a phenomenon termed the âgreat plate count anomalyâ (Staley & Konopka, 1985). Usually, between 1.0% and 0.1% of the total bacteria could be accounted for by the standard plating method. Thus, scientists realized that culture-dependent methods alone would be completely inadequate for studying complex populations such as those populating the human body. This prompted a search for alternative methods.
Culture-Independent Microbiology for Exploring the Human Microbiome
Several significant discoveries paved the way for the development of culture-independent methods, which, for the first time, allowed access to the unculturable fraction of natural microbial populations like the human microbiota. The most significant early contribution was made by Carl Woese (Pace et al., 2012). In the 1960s, Woese began studying the evolution of microorganismsâasking seemingly intractable questions that could not be answered by classic paleontology methods. Microbes, after all, not only were unicellular and microscopic but also were soft-bodied and left no fossil record except in a few extremely rare instances. Even if they were successfully fossilized, they would hardly ever display unique recognizable morphological characteristics distinctive enough to permit species identification. Woese consequently used a molecular phylogenetic approach for tracing evolutionary history. In this approach to tracking microorganismsâ evolution, he took cellular ribosomes (the most abundant organelles in all forms of cellular life, performing the essential function of protein biosynthesis) and undertook a comparative analysis of the sequences of one component: the small subunit ribosomal RNAs or SSU rRNAs. Woese reasoned that the similarities and differences between these sequences (i.e., the order of the four chemical basesâadenine, uracil (or thymine in DNA), cytosine, and guanine) would reflect the phylogenetic relationships of the organisms from which they were obtained.
Over many years, Woese and his associates collected and comparatively analyzed the sequences of SSU rRNAs from numerous species of microorganisms. SSU rRNA turned out to be, in Woeseâs own words, âthe ultimate molecular chronometerâ (Woese, 1987). There are two forms of SSU rRNAs, designated 18S and 16S. Eukaryotic cells, characterized by genomes enclosed within nuclear membranes, have 18S rRNA, and the morphologically simpler prokaryotic cells that lack nuclear envelopes have 16S rRNA. From analyses of 16S rRNA sequences, Woese and his coworkers discovered that there were actually two distinct groups of prokaryotic cells: the bacteria (originally named eubacteria) and a newly recognized group that was named the archaebacteria (Woese & Fox, 1977). In 1990, the group proposed a new taxonomic scheme to cover all forms of life on Earth, composed of three domainsâthe domain Eucaryota that included all eukaryotic cells and the two prokaryotic domains, the Bacteria and the Archaea (Woese et al., 1990). The SSU rRNA sequences not only contained unique short sequences that defined the three domains but also contained unique sequences that permitted assignment of cells to specific ...