CHAPTER 18 Methods in Microbial Ecology Microbial diversity

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 (N 2 -fixing bacterium) was first bacterium isolated by enrichment techniques

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).

• A classic enrichment strategy is shown in Figure 18. 1. • Azotobacter selection

• 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 H 2 S

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

• 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

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.

• In the most probable number (MPN) technique (Figure 18. 4), pure cultures can be obtained from repeated serial dilutions.

Laser tweezers allow one to "pick" a cell from a microscope field and literally move it away from contaminants (Figure 18. 5).

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

• DAPI is a general stain for identifying microorganisms in natural samples. It stains DNA. • 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. DAPI stained cells 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.

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

Linking Specific Genes to Specific Organisms Using PCR

• 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).

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

• 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 r. RNA sequences Separated by second PCR And DGGE

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.

Measuring Microbial Activities in Nature Radioisotopes and Microelectrodes

• The activity of microorganisms in natural samples can be assessed very sensitively using radioisotopes and/or microelectrodes (Figure 18. 18 a).

• 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).

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

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.

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