1. Methods in Microbial Ecology
CHAPTER 18
Methods in Microbial Ecology
Microbial diversity – isolation, identification, and quantification
Microbial activity – in the habitat
Methods
Enrichment and isolation
Cell staining
Gene isolation and characterization
Enrichment culture technique- medium and culture conditions favors growth of desired organism and countersellect for undesired organisms
Azotobacter (N2-fixing bacterium) was first bacterium isolated by enrichment techniques

2. Culture-Dependent Analyses of Microbial Communities  Enrichment and Isolation
Microbial ecology deals with how microorganisms interact with one another and their environment.
The enrichment culture technique is a means of obtaining microorganisms from natural samples. Hundreds of different enrichment strategies have been devised (Table 18.1).

8. A classic enrichment strategy is shown in Figure 18.1.
Azotobacter selection

9. The Sergei Winogradsky column is a miniature anoxic ecosystem that can be used as a long-term source of bacteria for enrichment culture purposes (Figure 18.2).
Gradient of H2S

10. Thiosperillum Chromatium Chlorobium
Jenense okenii limicola
Winogradysky columns used for isolation of sulfate reducers

11. Although the enrichment culture is a powerful tool, in most enrichments there exists a bias, and sometimes a very severe bias, in the outcome. Enrichment bias can be demonstrated by comparing the results obtained in dilution cultures with classical liquid enrichment.
Dilution eliminate rapidly growing but quantitatively insignificant organisms

12. Isolation in Pure Culture
Once a successful enrichment culture has been established, a pure culture can be obtained by conventional microbiological procedures, including streak plates, agar shakes, and dilution methods.

13. In the most probable number (MPN) technique (Figure 18
In the most probable number (MPN) technique (Figure 18.4), pure cultures can be obtained from repeated serial dilutions.

14. Laser tweezers allow one to "pick" a cell from a microscope field and literally move it away from contaminants (Figure 18.5).

16. Molecular (Culture-Independent) Analyses of Microbial Communities   Viability and Quantification Using Staining Techniques

17. DAPI is a general stain for identifying microorganisms in natural samples. It stains DNA.
DAPI stained cells
Some stains can differentiate live versus dead cells, and fluorescent antibodies that are specific for one or a small group of related cells can be prepared.
Live = green
Dead = red
P. fluorscens tagged with GFP
The green fluorescent protein makes cells autofluorescent and is a means for tracking cells introduced into the environment. Unlike in pure cultures, morphologically similar cells may actually be quite different genetically in natural samples.

18. Genetic Stains
A variety of fluorescent-staining methods employ the power of nucleic acid probes and thus are highly specific in their staining properties. These include phylogenetic staining, chromosome painting, and reverse transcription fluorescent in situ hybridization (FISH).
Ammonia oxiding bacteria = red
Nitrite oxiding bacteria green
Sewage sludge - stained with three probes, red, green and purple
Confocal micrograph

19. Linking Specific Genes to Specific Organisms Using PCR

20. The polymerase chain reaction (PCR) can be used to amplify specific target genes such as small subunit ribosomal RNA genes or key metabolic genes (Figure 18.13).

21. The polymerase chain reaction (PCR) can be used to amplify specific target genes such as small subunit ribosomal RNA genes or key metabolic genes.

22. Denaturing gradient gel electrophoresis (DGGE) can be used to resolve slightly to greatly different versions of these genes present in the various species inhabiting a natural sample.
First PCR – six distinct rRNA sequences
Separated by second PCR
And DGGE

23. Environmental Genomics (Metagenomics)
Environmental genomics involves shotgun sequencing and analysis of the collective genomes of the organisms present in a microbial community.
In environmental genomics, all genes in the microbial community—the metagenome—are sampled.

24. Measuring Microbial Activities in Nature  Radioisotopes and Microelectrodes

25. The activity of microorganisms in natural samples can be assessed very sensitively using radioisotopes and/or microelectrodes (Figure 18.18a).

26. In most cases, measurements are of the net activity of a microbial community rather than of a population of a single species (Figure 18.16).

27. Radioisotopes can be used as measures of microbial activity in a microscopic technique called microautoradiography (MAR).

28. Stable Isotopes
Isotope fractionation can reveal the biological origin of various substances.
Fractionation is a result of the activity of enzymes that discriminate against the heavier form of an element when binding their substrates.

29. 12C is preferentially fixed compared to 13C by the enzyme systems