《微生物学》（英文版） 4 Methods in Microbial Ecology

Methods in Microbial Ecology Microbial ecology focus on two major issues: (1) biodiversity, including the isolation, identification, and quantification of microorganisms in various habitats, and (2 microbial activity, that is, what are microorganisms doing in their habitats We begin here with a consideration of assessing biodiversity through enrichment and isolation, and then consider nonculture methods of identification and enumeration based on fluorescent antibodies and nucleic acid probes

Methods in Microbial Ecology • Microbial ecology focus on two major issues: (1) biodiversity, including the isolation, identification, and quantification of microorganisms in various habitats, and (2) microbial activity, that is, what are microorganisms doing in their habitats. • We begin here with a consideration of assessing biodiversity through enrichment and isolation, and then consider nonculture methods of identification and enumeration based on fluorescent antibodies and nucleic acid probes

Enrichment and isolation methods In this method a medium and a set of incubation conditions are used that are selective for the desired organism and are counterselective for the undesired organisms 1. The Winogradsky column: for isolation of purple and green phototrophic bacteria and other anaerobes, the winogradsky column has traditionally been used Winogradsky column have been used to enrich for a variety of prokaryotes both aerobes and anaerobes The great ad vantage of a column, besides the cultures, is that it can be spiked with a particular compound whose degradation one wishes to study and then allowed to select from the inoculum for an organism or organisms that can degrade it 2. From enrichments to pure cultures: the objective of an enrichment culture study is usually to obtain a pure culture Pure cultures can be obtained in many ways, but the most frequently employed means are the streak plate, the agar shake, and liquid dilution methods

Enrichment and Isolation Methods • In this method, a medium and a set of incubation conditions are used that are selective for the desired organism and are counterselective for the undesired organisms. • 1. The Winogradsky column : for isolation of purple and green phototrophic bacteria and other anaerobes, the Winogradsky column has traditionally been used. Winogradsky column have been used to enrich for a variety of prokaryotes, both aerobes and anaerobes. The great advantage of a column, besides the cultures, is that it can be spiked with a particular compound whose degradation one wishes to study and then allowed to select from the inoculum for an organism or organisms that can degrade it. • 2. From enrichments to pure cultures : the objective of an enrichment culture study is usually to obtain a pure culture. Pure cultures can be obtained in many ways, but the most frequently employed means are the streak plate, the agar shake, and liquid dilution methods

Foil cap or Algae and cyanoba Purple nonsulfur bacteri supplemented Patches of purple sulfur or with organIc green sulfur bacteria nutrients and Anoxic decomposition and sulfate reduction H2s Figure 14.5 The Winogradsky column (a) Schematic view of a typical column. The column is placed so as to receive sub- dued sunlight. Chemoorganotrophic bacteria grow through out the column, aerobes and microaerophiles in the upper region ns, anaero obes in the zones containing H,S. Anoxic composition leading to sulfate reduction creates the gradi ent of H S Green and purple sulfur bacteria stratify according to their tolerance for H,S(b) Photo of Winogradsky columns that have remained anoxic up to the top where blooms of three different phototrophic bacteria have occurred in the lud and up into the water column. Left to right: Thiospirillum jenense, Chromatium oken, and Chlorobium limicola

。·。 ci七 Figure 14.6 Agar shake technique for isolation of anaerobic bacteria in pure culture. A dilution series was established from right to left, eventually yielding well-isolated colonies The tubes are sealed with a sterile mixture of paraffin and mineral oil to maintain anaerobiosis

Identification and Quantification: Nucleic Acid Probes fluorescent Antibodies and viable counts Staining and fluorescent antibodies: Microorganisms can be identified and enumerated by direct microscopic examination of the habitat. However special procedures are needed to make microorganisms visible in opaque habitats Nucleic acid probes: A very powerful approach to the identification and quantification of microorganisms in nature is the use of nucleic acid D robes

Identification and Quantification: Nucleic Acid Probes, Fluorescent Antibodies, and Viable counts • Staining and fluorescent antibodies: Microorganisms can be identified and enumerated by direct microscopic examination of the habitat. However, special procedures are needed to make microorganisms visible in opaque habitats. • Nucleic acid probes: A very powerful approach to the identification and quantification of microorganisms in nature is the use of nucleic acid probes

Visualization of bacterial microcolonies(bacterial cells appear as greenish-yellow dots)on the surface of soil particles by use of the fluorescent antibody technique. Cells are about 1 um in diameter

Visualization of bacterial microcolonies (bacterial cells appear as greenish-yellow dots) on the surface of soil particles by use of the fluorescent antibody technique. Cells are about 1 μm in diameter

Microbial activity Measurements Radioisotopes and microelectrodes Radioisotopes are very useful in measuring specific microbial processes at high sensitivity and also in obtaining information on the turnover rates of chemical species in nature Microbial ecologists have used small glass electrodes, referred to as microelectrodes, to study the activity of microorganisms in their microenvironments

Microbial Activity Measurements: Radioisotopes and Microelectrodes • Radioisotopes are very useful in measuring specific microbial processes at high sensitivity and also in obtaining information on the turnover rates of chemical species in nature. • Microbial ecologists have used small glass electrodes, referred to as microelectrodes, to study the activity of microorganisms in their microenvironments

Photosynthesis Use of radioisotopes to Light 14CO2 measure microbia rporate activity in nature Dark (a) photosynthesis measured in natural Sulfate reduction in mud seawater with 14c02 H2 present H35S (b) Sulfate reduction in mud measured with 35SO4 H≥ absent () Time (c)Methanogenesis Methanogenesis in mud measured in mud with 14CH. COO acetate labeled in either 14cH4 the methyl(CH3 c00-) or the carboxyl CH14CO0 Time (CH3 4C00-)carbon

Use of radioisotopes to measure microbial activity in nature. (a) Photosynthesis measured in natural seawater with 14CO2 (b) Sulfate reduction in mud measured with 35SO4 2- (c) Methanogenesis measured in mud with acetate labeled in either the methyl (14CH3COO- ) or the carboxyl (CH3 14COO- ) carbon

Platinum Microelectrodes (a) Schematic drawing of an Oxygen Glass microelectrode (b)Photo of microelectrodes being used in a microbial mat Gold 5 um (b)

Microelectrodes. (a) Schematic drawing of an oxygen microelectrode. (b) Photo of microelectrodes being used in a microbial mat

Microbial Activity Measurements: Stable Isotopes Enzyme substrates Enzyme that Fixed carbon fixes Co 12 Figure 14 13 Mechanism of isotopic fractionation using car- bon as an example. Although the ratio of natural abundance of 12CO, to 13CO2 is about 19: 1, enzymes that fix CO2 prefer entially fix the lighter isotope(12C). This results in fixed car bon being enriched in 12C and depleted in 13C relative to the starting substrate. The degree of 1C depletion is calculated as an isotopic fractionation (see legend to Figure 14.14 for calculation)

Microbial Activity Measurements: Stable Isotopes