TABLE WO-1Some Major Methods for Studying Individual Microbes Found in the Environment

MethodSummaryComments
MicroscopyMicrobial phenotypes can be studied by making them more visible. In conjunction with other methods, such as staining, microscopy can also be used to count taxa and make inferences about biological processes.The appearance of microbes is not a reliable indicator of what type of microbe one is looking at.
CulturingSingle cells of a particular microbial type are grown in isolation from other organisms. This can be done in liquid or solid growth media.This is the best way to learn about the biology of a particular organism. However, many microbes are uncultured (i.e., have never been grown in the lab in isolation from other organisms) and may be unculturable (i.e., may not be able to grow without other organisms).
rRNA-PCRThe key aspects of this method are the following: (a) all cell-based organisms possess the same rRNA genes (albeit with different underlying sequences); (b) PCR is used to make billions of copies of basically each and every rRNA gene present in a sample; this amplifies the rRNA signal relative to the noise of thousands of other genes present in each organism’s DNA; (c) sequencing and phylogenetic analysis places rRNA genes on the rRNA tree of life; the position on the tree is used to infer what type of organism (a.k.a. phylotype) the gene came from; and (d) the numbers of each microbe type are estimated from the number of times the same rRNA gene is seen.This method revolutionized microbiology in the 1980s by allowing the types and numbers of microbes present in a sample to be rapidly characterized. However, there are some biases in the process that make it not perfect for all aspects of typing and counting.
Shotgun genome sequencing of cultured speciesThe DNA from an organism is isolated and broken into small fragments, and then portions of these fragments are sequenced, usually with the aid of sequencing machines. The fragments are then assembled into larger pieces by looking for overlaps in the sequence each possesses. The complete genome can be determined by filling in gaps between the larger pieces.This has now been applied to over 1,000 microbes, as well as some multicellular species, and has provided a much deeper understanding of the biology and evolution of life. One limitation is that each genome sequence is usually a snapshot of one or a few individuals.
MetagenomicsDNA is directly isolated from an environmental sample and then sequenced. One approach to doing this is to select particular pieces of interest (e.g., those containing interesting rRNA genes) and sequence them. An alternative is ESS, which is shotgun genome sequencing as described above, but applied to an environmental sample with multiple organisms, rather than to a single cultured organism.This method allows one to sample the genomes of microbes without culturing them. It can be used both for typing and counting taxa and for making predictions of their biological functions.

SOURCE: Eisen (2007).

From: Workshop Overview

Cover of The Science and Applications of Microbial Genomics
The Science and Applications of Microbial Genomics: Workshop Summary.
Institute of Medicine (US) Forum on Microbial Threats.
Washington (DC): National Academies Press (US); 2013.
Copyright © 2013, National Academy of Sciences.

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