Chapter 3: Methods for Analyzing Diversity

• Microbial diversity - refers unequivocally to biological diversity at three levels: within species (genetic), species number (species) and community (ecological) diversity.
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  A) total number of species

  B) distribution of individuals among these species

  
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  A) quantitative variation among species

  B) total number of species

  C) individuals among species

  D) evenness of equability

  
• In the past, diversity has been determined based on taxonomic species, which may limit the scope of information and relationship obtained.
• The diversity of Operational Taxonomic Unit (OTU) or even communities may give us a better estimation of the functioning of an ecosystem.
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  A) abiotic

  B) biotic

  
• Abiotic factors - include both physical and chemical factors such as water availability, salinity, oxic/anoxic conditions, temp, pH, pressure, chemical pollution, heavy metals, pesticides, antibiotics etc.
• Biotic factors - include plasmids, phages, transposons that are types of accessory DNA that influence the genetic properties and in most cases, the phenotypes of their host and thus have a great influence on microbial diversity.
• The morphological characteristics such as cell shape, cell wall movement, flagella, gram staining, etc. may not be adequate for establishing a detailed classification of microbes.
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  A) Biochemical techniques

  B) Molecular techniques

  
• The diversity can be described using physiological diversity measures too, which avoid the difficulties that may arise in grouping of similar bacteria into species or equivalents.
• Conventional and Biochemical Methods

  • Plate Counts
  • Community level Physiological Profiling (CLPP)/Sole-CarbonSource Utilization (SCSU) Pattern
  • Phospholipid fatty acid (PLFA) analysis/Fatty acid methyl ester analysis (FAME)
• Plate counts - most traditional method for assessment of microbial diversity is selective and differential plating and subsequent viable counts.
• If some bacteria may be clumped together when doing the plate count technique, it is determined by Colony-Forming Units (CFUs) in that known dilution.
• A plate having 30-300 colonies is chosen because this range is considered statistically significant.
• Community level physiological profiling (CLPP)/ Sole-Carbon Source Utilization (SCSU) Pattern - was initially developed as a tool for identifying pure cultures of bacteria to the species level, based upon a broad survey of their metabolic properties.
• Different communities can be compared and classifies based on sole carbon source utilization patterns (SCSU) gathered using BIOLOG microplates.
• Phospholipid Fatty Acid Analysis - is done through a chemical extraction process and analyzed on a gas chromatograph.
• Mole Percentage guanine + cytosine (mol% G+C) - first property of DNA used for taxonomical purpose; can be determined by thermal denaturation of DNA.
• Nucleic acid hybridization - can be done on extracted DNA or RNA, or in situ.
• DNA Microarrays - valuable in bacterial diversity studies since a single array can contain thousands of DNA sequences with high specificity.
• DNA Microarrays:

  • Specific target genes coding for enzymes: nitrogenase, nitrate reductase, naphthalene dioxygenase
• Denaturing and Temperature Gradient Gel Electrophoresis (DGGE and TGGE) - DNA fragments of same length but with different base-pair sequences can be separated. DNA is extracted from natural samples and amplified using PCR with universal primers targeting part of the 16S and 18s rRNA sequences.
• TGGE - employs the same principle as DGGE but in this method , the gradient is temp rather than chemical denaturant.
• Restriction fragment length polymorphism (RFLP) - another tool used to study microbial diversity. This method relies on DNA polymorphisms. Have also been applied to estimate diversity and community structure in different microbial communities.
• Terminal Restriction Fragment Length Polymorphism (T-RFLP) - an extension of the RFLP/ARDRA analysis, and provides an alternate method for rapid analysis of microbial community diversity in various environments. Follows the same principle as RFLP except that one PCR primer is labeled with a fluorescent dye.
• Ribosomal Intergenic spacer analysis (RISA)/Automated RISA/Amplified ribosomal DNA restriction analysis (ARDRA) - similar in principle to RFLP and TRFLP, it provides ribosomal-based fingerprinting of the microbial community. In RISA and ARISA, the intergenic spacer (IGS) region between 16s and 23s ribosomal subunits is amplified by PCR, denatured and separated on polyacrylamide gel under denaturing conditions.
• Single strand conformation polymorphism (SSCP) - also relies on electrophoretic separation based on differences in DNA sequences and allows differentiation of DNA molecules having the same length but different nucleotide sequences.

  • Was originally developed to detect known or novel polymorphisms or point mutations in DNA.