Marine carbonate Calvin cycle plants Pet troley Methane Cyanobacteria Purple sulfur bacteria Figure 14 14 Carbon isotopic com- Green sulfur bacteria positions of various substances. The values were calculated using the Recent marine sediments formula c/12C sample 3.5 billion-year-old rocks (13C/12C standard) (13C/12C standard) 100 10 +10 613c(0 The standard is a belemnite sample from the pee Dee rock formation
Table 28.8 Methods Used to Study Microorganisms in Different Environments Characteristic Evaluated Technique Employed or Property Measured Marine Freshwater Sewage Soil Nutrients Chemical analysis (e. g, C, N. P COD(chemical oxygen demand) BOD(biochemical oxygen demand icrobial biomass ration and dry weight Measurement of chemical constituents(e. g,, ATP. id, lipopolysaccharides) Microscopy and biovolume conversion to biomass using conversion factors Fumigation incubation/extraction Glucose-amended respiration Microbial numbers/types Microscopic procedures-epifluorescence/phase contrast 十十 十十 Flow cytometry Immersion/insertion slides and films Viable enumeration procedures(culture, microscopy) ++ Direct microorganism isolation Thin sections of samples electron microscopy Direct DNA extraction and analysis Polymerase chain reaction(PCR) with species-specific primers DNA probes and hybridization techniques for DNA and RNA n situ PCR with 16S or 18S primers Micromanipulation and single-cell PCR/phy logenetic analysis 中 Microbial viability and turnover Nalidixic 十十 prevents cell division, resulting in elongated active cells): direct viable counts Bioluminescence assessment(green fluorescentprotein) + Stable and radioactive isotope studies Microbial activity Microscopy with reducible dyes Autoradiography Enzyme activity assays RNA-based function probes Gas exchange (O, Ce CH ASSessment of fungal acterial contributions by of selective ar tic inhibition Substrate utilization rate Fluorescent substrate hydrolysis Community structure Microscopic analyses of diversity sity of microbial isolates 16S or 18S ribosomal RNA analysis (including SsCP Isingle-strand conformation polymorphism], DGGE. [denaturing gradiant gel electrophoresis I. TGGE Temperature gel electrophoresis DNA amplification fingerprinting (DAF) 中中 十中 十 Major uses are noted with two plus aigns (++) umd minor uses are noted with one plus (a)i minimal or no use noted with (
a) Suspension containing desired bacterium to be isolated from a mixture (b)Desired bacterium is taken up into the sterile micropipette tip Recovery of Single Cells or cell Organelles from Complex Natural Mixtures Using Micromanipulation by use of an inverted phase-contrast microscope and a micromanipulator, a microorganism can be recovered for direct molecular analysis. (a) The bacterium to be isolated is placed below the micromanipulator tip(diameter 5 to 10 um)and a slight vacuum is drawn.(b) The desired single bacterium is drawn up into the micropipette tube and is ready for molecular analysis
Recovery of Single Cells or cell Organelles from Complex Natural Mixtures Using Micromanipulation. By use of an inverted phase-contrast microscope and a micromanipulator, a microorganism can be recovered for direct molecular analysis. (a) The bacterium to be isolated is placed below the micromanipulator tip (diameter 5 to 10 μm) and a slight vacuum is drawn. (b) The desired single bacterium is drawn up into the micropipette tube and is ready for molecular analysis
Mycoplasma alvi Mycoplasma muris Mycoplasma penetrans Ureaplasma cati Mycoplasma sp, str STOL Mycoplasma sp str, BAWB Mycoplasma sp str BVK Mycoplasma pulmonis Mycoplasma arginini Mycoplasma bovocull F Spiroplasma apis Ct Mycoplasma mycoides Clostridium ramosum Clostridium ce/lulovorans Bacillus anthracis Lactobacillus acidophilus Combining Micromanipulation for Isolation of Single Cells or Organelles with the Polymerase Chain Reaction(PCr).(a) Recovery of an endosymbiotic mycoplasma from single cell of the flagellate Koruga bonita by micromanipulation(bar=1Oum)and(b) phylogenetic analysis of the recovered mycoplasma following PCr amplification and sequencing of the PCr products, with the bar indicating 10%o estimated sequence divergence. Lactobacillus acidophilus is the outgroup reference. This approach makes it possible to link a specific microorganism or organelle, isolated from a natural environment, to its molecular sequence and phylogenetic information. Flagellate (F), capillary tube(Ct)
Combining Micromanipulation for Isolation of Single Cells or Organelles with the Polymerase Chain Reaction (PCR). (a) Recovery of an endosymbiotic mycoplasma from single cell of the flagellate Koruga bonita by micromanipulation (bar=10μm) and (b) phylogenetic analysis of the recovered mycoplasma following PCR amplification and sequencing of the PCR products, with the bar indicating 10% estimated sequence divergence. Lactobacillus acidophilus is the outgroup reference. This approach makes it possible to link a specific microorganism or organelle, isolated from a natural environment, to its molecular sequence and phylogenetic information. Flagellate (F), capillary tube (Ct)
Sequence CGUAGACCUGA Preparing a phylogenetic distance-matrix CCUAGAGCUGGC tree fromm 16s ribosomal rna CCA AGACGUGGC uences For illustrative purposes, only GCUAGAUGUGCC short sequences are shown but it should Evolutionary distance Corrected evolutionary distance be considered that the sequences in(a) O.25 0.30 are representative of the entire 16srrNa C0.33 0.44 The evolutionary distance(Ep) in(b)is 0.61 calculated as the percentage of B一c025 0.30 nonhomologous sequence between the 0.33 0.44 RNAS of any total two organisms. The 033 0.44 corrected en is a statistical correction Phylogenetic tree(computer-generated best fit to corrected necessary to account for either back mutations to the original genotype or additional forward mutations at the same site that could have occurred. The tree(c) B is ultimately generated by computer 0.15 analysis of the data to give the best fit The length of the branches separating any two organisms is proportional to the evolutionary distance between them D
Preparing a phylogenetic distance-matrix tree fromm 16S ribosomal RNA sequences. For illustrative purposes, only short sequences are shown, but it should be considered that the sequences in (a) are representative of the entire 16SrRNA. The evolutionary distance (ED) in (b) is calculated as the percentage of nonhomologous sequence between the RNAs of any total two organisms. The corrected ED is a statistical correction necessary to account for either back mutations to the original genotype or additional forward mutations at the same site that could have occurred. The tree (c) is ultimately generated by computer analysis of the data to give the best fit. The length of the branches separating any two organisms is proportional to the evolutionary distance between them