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