Methods in Microbial Ecology Chapter 22 Methods in Microbial Ecology

I. Culture-Dependent Analyses of Microbial Communities 22.1 Enrichment and Isolation 22.2 Isolation in Pure Culture

22.1 Culture Dependant Microbial Community Analysis Isolation The separation of individual organisms from the mixed community Enrichment Cultures Select for desired organisms through manipulation of medium and incubation conditions Inocula The sample from which microorganisms will be isolated

The Isolation of Azotobacter Figure 22.1

Some Enrichment Culture Methods

22.1 Enrichment and Isolation Enrichment Cultures Can prove the presence of an organism in a habitat Cannot prove an organism does not inhabit an environment The ability to isolate an organism from an environment says nothing about its ecological significance Animation: Enrichment Cultures

22.1 Enrichment and Isolation The Winogradsky Column An artificial microbial ecosystem Serves as a long-term source of bacteria for enrichment cultures Named for Sergei Winogradsky First used in late 19th century to study soil microorganisms

Schematic View of a Typical Winogradsky Column Figure 22.2a

Photo of Winogradsky Column: Remained Anoxic Up to Top A bloom of different phototrophic bacterium 1: Thiospirillum jenense 2: Chromatium okenii 3: Chlorobium limicola 1 2 3 Figure 22.2b

Some Enrichment Culture Methods

Some Enrichment Culture Methods

22.1 Enrichment and Isolation Enrichment bias Microorganisms cultured in the lab are frequently only minor components of the microbial ecosystem Because the nutrients available in the lab culture are typically much higher than in nature Dilution of inoculum is performed to eliminate rapidly growing, but quantitatively insignificant, weed species

22.2 Isolation in Pure Culture Pure cultures contain a single kind of microorganism Can be obtained by streak plate, agar shake, or liquid dilution Agar dilution tubes are mixed cultures diluted in molten agar Useful for purifying anaerobic organisms Most-probable number technique Serial 10X dilutions of inocula in a liquid media Used to estimate number of microorganisms in food, wastewater, and other samples

22.2 Isolation in Pure Culture Animation: Serial Dilutions and a Most Probable Number Analysis

Procedure for a Most-Probable Number Analysis Figure 22.4

22.3 Pure Culture Methods Figure 22.3

22.2 Isolation in Pure Culture Axenic culture can be verified by Microscopy Observation of colony characteristics Tests of the culture for growth in other media Laser tweezers are useful for Isolating slow-growing bacteria from mixed cultures

Principle of the Laser Tweezers Figure 22.5a

The Laser Tweezers for the Isolation of Single Cells Figure 22.5b

II. Culture-Independent Microbial Community Analysis 22.3 General Staining Methods 22.4 FISH 22.5 Linking Specific Genes to Specific Organisms Using PCR 22.6 Environmental Genomics

22.3 General Staining Methods Fluorescent staining using DAPI or acridine orange (AO) DAPI (4’-6-diamidino-2-phenylindole) stained cells fluoresce bright blue AO stained cells fluoresce orange or greenish-orange DAPI and AO fluoresce under UV light DAPI and AO are used for the enumeration of microorganisms in samples DAPI and AO are nonspecific and stain nucleic acids Cannot differentiate between live and dead cells

Nonspecific Fluorescent Stains: Photomicrograph of DAPI Figure 22.6a

Nonspecific Fluorescent Stains: Acridine Orange Figure 22.6b

22.3 General Staining Methods Viability stains: differentiate between live and dead cells Two dyes are used - Green dye: penetrates all cells - Red dye: penetrates only dead cells Based on integrity of cell membrane Green cells are live Red cells are dead Can have issues with nonspecific staining in environmental samples

Viability Staining Figure 22.7

22.3 General Staining Methods Fluorescent antibodies can be used as a cell tag Highly specific Making antibodies is time consuming and expensive Green fluorescent protein (GFP) can be genetically engineered into cells to make them autofluorescent Can be used to track bacteria Can act as a reporter gene

Fluorescent Antibodies as a Cell Tag Sulfolobus acidocaldarius attached to the surface of solfatra soil particles. Figure 22.8

The Green Fluorescent Protein Pseudomonas fluorescence attached to barley roots. Figure 22.9

22.4 FISH Nucleic acid probe is DNA or RNA complimentary to a sequence in a target gene or RNA FISH (fluorescent in situ hybridization) Phylogenetics of microbial populations Used in microbial ecology, food industry, and clinical diagnostics ISRT-FISH (in situ reverse transcription-FISH) - Use cDNAs as probes CARD-FISH (catalyzed reported deposition FISH) - Peroxidase is attached to the probe - Treat with tyramide after hybridization : converted into a very reactive intermediate that binds to adjacent proteins and fluoresces

Morphology and Genetic Diversity Phase contrast Phylogenetic FISH Figure 22.10a

FISH Analysis of Sewage Sludge: Nitrifying Bacteria Red: ammonia-oxidizing bacteria Green: nitrite-oxidizing bacteria Figure 22.11a

FISH Analysis of Sewage Sludge Figure 22.11b

In-situ Reverse Transcription Stained with DAPI Stained with ISRT probe Figure 22.12b

22.5 Linking Genes to Specific Organisms Using PCR Specific genes can be used as a measure of diversity PCR, DGGE, molecular cloning, and DNA sequencing and analysis are tools used to look at community diversity DGGE (denaturing gradient gel electrophoresis) separates genes of the same size based on differences in base sequence Denaturant is a mixture of urea and formamide Strands melt at different denaturant concentrations - Use gels with different gradient of the denaturant

Steps in Single Gene Biodiversity Analysis Figure 22.13

PCR and DGGE Gels Figure 22.14a

PCR and DGGE Gels Figure 22.14b

22.5 Linking Genes to Specific Organisms Using PCR T-RFLP (Terminal Restriction Fragment Length Polymorphism) Target gene is amplified by PCR using a primer set in which one of the primers is end-labeled with a fluorescent dye Restriction enzymes are used to cut the PCR products Molecular methods demonstrate that less than 0.5% of bacteria have been cultured Phylochip: microarrays that focus on phylogenetic members of microbial community Circumvents time-consuming steps of DGGE and T-RFLP

Phylochip Analysis of Sulfate-Reducing Bacteria Diversity Figure 22.15

22.6 Environmental Genomics Environmental Genomics (metagenomics) DNA is cloned from microbial community and sequenced Idea is to detect as many genes as possible All genes in a sample can be detected Yields picture of gene pool in environment Can detect genes that would not be amplified by current PCR primers Powerful tool for assessing the phylogenetic and metabolic diversity of an environment

Single Gene Versus Environmental Genomics Figure 22.16

III. Measuring Microbial Activities in Nature 22.7 Chemical Assays, Radioisotopic Methods, and Microelectrodes 22.8 Stable Isotopes

22.7 Chemical Assays, Radioisotopes, & Microelectrodes In many studies direct chemical measurements are sufficient Higher sensitivity can be achieved with radioisotopes Proper killed cell controls must be used Radioisotopes can also be used with FISH FISH microautoradiography (FISH-MAR) Combines phylogeny with activity of cells

Microbial Activity Measurements Figure 22.17

An autotroph using 14CO2 as a carbon source. FISH-MAR An autotroph using 14CO2 as a carbon source. Figure 22.18a

FISH-MAR FISH MAR with 14C-glucose Figure 22.18b

22.7 Chemical Assays, Radioisotopes, & Microelectrodes Can measure a wide range of activity pH, oxygen, CO2, and others can be measured Small glass electrodes, quite fragile Electrodes are carefully inserted into the habitat (e.g., microbial mats)

Schematic Drawing of an Oxygen Microelectrode Figure 22.19a

Microelectrodes Being Used in a Hot Spring Microbial Mat Figure 22.19b

Microbial Mats and the Use of Microelectrodes Figure 22.20a

Oxygen, Sulfide, and pH Profiles in Hot Spring Microbial Mat Figure 22.20b

22.8 Stable Isotopes Stable isotopes: non-radioactive isotopes of an element Can be used to study microbial transformations in nature Isotope fractionation Carbon and sulfur are commonly used Lighter isotope is incorporated preferentially over heavy isotope Indicative of biotic processes Isotopic composition of a material reveals its past biology (e.g., carbon in plants and petroleum)

Mechanism of Isotopic Fractionation Using Carbon Figure 22.21

Isotopic Geochemistry of 13C and 12C

Isotopic Geochemistry of 34S and 32S

22.8 Stable Isotopes Stable isotopes probing (SIP): links specific metabolic activity to diversity using a stable isotope Microorganisms metabolizing stable isotope (e.g., 13C) incorporate it into their DNA DNA with 13C can then be used to identify the organisms that metabolized the 13C-labelled substrates SIP of RNA can be done instead of DNA

Stable Isotope Probing