Chapter 22 Methods in Microbial Ecology. I. Culture-Dependent Analyses of Microbial Communities ...

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

Figure 22.1

The Isolation of Azotobacter

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 CulturesAnimation: Enrichment Cultures

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

22.1 Enrichment and Isolation

Figure 22.2a

Schematic View of a Typical Winogradsky Column

Figure 22.2b

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

Some Enrichment Culture Methods

Some Enrichment Culture Methods

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

22.1 Enrichment and Isolation

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 AnalysisAnimation: Serial Dilutions and a Most Probable Number Analysis

Figure 22.4

Procedure for a Most-Probable Number Analysis

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

Figure 22.5a

Principle of the Laser Tweezers

Figure 22.5b

The Laser Tweezers for the Isolation of Single Cells

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

Figure 22.6a

Nonspecific Fluorescent Stains: Photomicrograph of DAPI

Figure 22.6b

Nonspecific Fluorescent Stains: Acridine Orange

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

Figure 22.7

Viability Staining

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

Figure 22.8

Fluorescent Antibodies as a Cell Tag

Sulfolobus acidocaldarius attached to the surface of solfatra soil particles.

Figure 22.9

The Green Fluorescent Protein

Pseudomonas fluorescence attached to barley roots.

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

Figure 22.10a

Morphology and Genetic Diversity

Phase contrast Phylogenetic FISH

Figure 22.11a

FISH Analysis of Sewage Sludge: Nitrifying Bacteria

Red: ammonia-oxidizing bacteria

Green: nitrite-oxidizing bacteria

Figure 22.11b

FISH Analysis of Sewage Sludge

Figure 22.12b

In-situ Reverse Transcription

Stained with DAPI

Stained with ISRT probe

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

Figure 22.13

Steps in Single Gene Biodiversity Analysis

Figure 22.14a

PCR and DGGE Gels

Figure 22.14b

PCR and DGGE Gels

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

Figure 22.15

Phylochip Analysis of Sulfate-Reducing Bacteria Diversity

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

Figure 22.16

Single Gene Versus Environmental Genomics

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

Figure 22.17

Microbial Activity Measurements

Figure 22.18a

FISH-MAR

An autotroph using 14CO2 as a carbon source.

Figure 22.18b

FISH-MAR

FISH MAR with 14C-glucose

22.7 Chemical Assays, Radioisotopes, & Microelectrodes

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)

Figure 22.19a

Schematic Drawing of an Oxygen Microelectrode

Figure 22.19b

Microelectrodes Being Used in a Hot Spring Microbial Mat

Figure 22.20a

Microbial Mats and the Use of Microelectrodes

Figure 22.20b

Oxygen, Sulfide, and pH Profiles in Hot Spring Microbial Mat

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)

Figure 22.21

Mechanism of Isotopic Fractionation Using Carbon

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