Module 4. Microbial Ecology Techniques

 

Topics

        Enrichment and culturing

        Sampling from the environment

        Detection of microorganisms in the environment

        Determination of microbial numbers

 

Objectives

        Understand the factors involved in examining environmental samples

        To be familiar with methods used for isolating and enumerating microorganisms from environmental samples

        Determine techniques for measuring microbial activity in the environment

        Understand the application of molecular biology as a tool to study microbial environments

 

 

 

 

Topic 1. Enrichment and culturing

 

Culturing

 

Culturing microorganisms remains an essential and central part of microbiology. The best way to find out information about the physiology, capabilities and phylogenetic characteristics of microorganisms is to isolate and grow them in pure culture. Full characterisation and the ability to utilise the properties of a microorganism cannot be achieved without culturing.

 

Pure culture methods:

        streak plate

        agar shakes

        roll tubes

        liquid dilution

 

However, when studying the interactions between microorganisms and their surrounds it is best to undertake in situ studies or simulate the environment such as in the Winogradsky column.

 

 

 

 

 

 

Enrichment cultures

 

Developed by Beijerinck, the enrichment culture is the provision of favourable specific conditions for the growth of a particular type of microorganism, eg. to isolate a nitrogen-fixing bacterium the enrichment medium would not contain a fixed nitrogen source.

 

Enrichment cultures help to isolate microorganisms that can metabolise a particular substrate or can live under certain conditions which may be present in very small numbers in the original sample, eg a naphthalene-degrading bacterium from soil, a medium would be prepared where naphthalene was the only source of carbon. The Winogradsky column (link to Module 4) is an example of a method to enrich for a variety of prokaryotes.

 

The growth media is vital to the successful isolation and cultivation of microorganisms in the laboratory. The correct components will select for the types of microorganisms that are able to grow under the media and incubation conditions.

 

Non-culturable microorganisms

 

It is likely that fewer than 1% of bacteria can be cultured in the laboratory using conventional culturing techniques (viable but nonculturable organisms). These microorganisms are growing and are metabolically active in the environment. They are not capable of growing under the conditions provided and require some factor(s) that was in the environment but not provided in the laboratory. Alternatively they may be living as symbionts in plants and animals. Methods and techniques have been developed to detect these microorganisms in the environment.

 

 

Topic 2. Sampling from the environment

 

Sample collection

 

A critical step in any study within microbial ecology. Specialised devices are often required eg deep subsurface, deep waters, air, biological samples. There are a number of important factors to consider in sample collection:

        Representative of the ecosystem

        Numbers and activity of the microorganisms are not altered

        No contamination

        Correct storage conditions

 

Sample processing

 

Microorganisms in environmental samples are usually present both in inappropriate numbers (too low or high) and with many other microorganisms. They may require concentration, dilution or enrichment. The aim is to gain optimum recovery of the microorganisms.

 

 

Topic 3. Detection of microorganisms in the environment

 

1.      Phenotypic detection

 

Growing microorganisms in laboratory cultures based on phenotypic properties. Detecting individual or groups of microorganism that can use a particular substrate or energy source, or live under certain physico-chemical conditions.

For example, the provision of acetate as sole carbon source and iron oxide in media allows for the detection of microorganisms that can metabolise acetate and use iron oxide as an energy source:

Geobacter metallireducens: CH3COO- + 8Fe3+ + 4H2O 2HCO3- + 8Fe2+ + 9H+

 

2.      Lipid profiling

 

Lipid compositions of cytoplasmic membranes vary, with each microorganism having a characteristic pattern of lipid composition and proportions. Microbial communities can be examined in the environment without relying on culturing.

1.      Fatty acid methyl ester (FAME) analysis can be used to detect microbial species.

FAME: esterification of lipids, then identification and quantification by gas chromatography

2.      Phospholipid-linked fatty acid (PLFA) analysis provides information about the composition and abundance of microbial populations

 

3.      Molecular detection

 

The application of molecular techniques has greatly advanced the study of microbial ecology. Molecular methods have given a new perspective on biodiversity, community structure and allowed the detection and identification of many novel microorganisms. Molecular techniques have shown that greater than 99% of microorganisms in the environment remain uncultured and uncharacterized.

 

Nucleic acid probes

 

A DNA or RNA oligonucleotide complementary to a sequence in a target gene in a particular organism(s) and can be radiolabeled or labeled with fluorescent dyes, eg fluorescent in situ hybridisation (FISH).

 

Using FISH and depending on the specificity of the probe, particular organisms or groups of organisms can be targeted and identified in an environmental sample. Using multiple probes, each designed for particular organisms, on a sample an entire habitat can be characterised. Nucleic acid probes can also be used to determine the phylogenetic make-up of a microbial community. Optical tweezers have been developed for isolating pure organisms identified by FISH.

 

4.      Microbial community analyses

 

PCR

Amplification of DNA, eg 16S rRNA genes, using specific primers

 

DGGE

Electrophoresis technique for separating out nucleic acid fragments eg 16S genes of the same size but different sequence. Resolves the number of phylotypes present in a particular habitat. Can be used with metabolic genes.

 

5.      Microscopy

 

Light microscopy

Used for most routine microbiology studies where specific lenses magnify the image of a cell (up to 1000x). In microbial ecology, fluorescent microscopy has most application, where specimens that fluoresce can be visualised. A number of fluorescent staining methods have been devised for detecting and enumerating microorganisms in environmental samples (these will be discussed in the next Topic)

 

Electron microscopy

Widely used for studying the detailed structure of cells (up to 100,000x). These microscopes use electrons instead of light rays and electromagnets function as lenses under vacuum. There are 2 main types:

TEM: For cells, thin sections are taken to view internal structures

SEM: Scans across the surface and allows external features to be seen

 

Confocal laser microscope

A method of observing through layers of an opaque environment. It is a computerised microscope that couples a laser source to a light microscope to generate 3-dimensional digital images.

 

 

 

Topic 4. Determination of microbial numbers

 

1.      Total counts

 

Direct counts

Microscopic determination of the numbers of microorganisms which generally give the highest estimates. Usually applied with staining, in particular fluorescent staining is used with environmental samples.

Acridine orange: binds to nucleic acids and fluoresce either green or orange

DAPI: fluoresce bright blue

INT: stains respiring microorganisms (viable)

Counting chamber: counting numbers cells in known volumes eg. haemocytometer

 

Others

 

Turbidity (OD)

A rapid method of obtaining an estimate of cell numbers is where the turbidity of a cell suspension is measured using a spectrophotometer. However it is only useful for laboratory cultures with no interference from other particulate matter.

 

Dry weight

A measurement of cell mass, where a culture is filtered or centrifuged then the solids are weighed. Again it is only useful for laboratory cultures with no other particulate matter.

 

Fluorescent antibody staining

Adds specificity to fluorescent staining by preparing antibodies against a surface constituents of a particular organism.

 

 

2.      Viable counts

 

Methods that count only live cells. A viable cell is able to divide and form offspring.

 

Plate counts

The plate count requires the separation of cells and the formation of colonies from the cells on agar plates. It usually involves the dilution of a sample and each dilution is applies to an agar plate either by spread plate or pour plate.

 

MPN (most probable number)

A similar process without using agar by serially diluting a sample in liquid medium until the final tubes show no growth. It is done either in sets of three or five replicates. A statistical estimate of the microbial numbers in a sample is gained.

 

Colony hybridisation

A combination of nucleic acid hybridisation and viable plating to select microorganisms with specific genetic properties.

 

 

 

Topic 5. Microbial activity measurements

 

Numerous components and products of microbial cells have been used to gain information about microorganisms in the environment. These measurements can be used to estimate the numbers of microorganisms, microbial biomass and find out what the organisms are doing in their environment. As well, information can be gained on microbial community structures. Some of these are listed below:

 

ATP

        A luciferin-luciferase assay is used to detect ATP

        The light emitted in the assay is directly proportional to the ATP concentration.

luciferin(red) + O2 + Mg + ATP luciferin(ox) +light (enzyme: luciferase)

 

Cell wall components

        Muramic acid is component of bacterial cell walls:used to estimate bacterial levels

 

 

Enzyme assays: general, specific

        Assaying enzymes, substrates or end products should correlate to biomass

        Can be specific for total community or specific groups with particular (rarer) enzyme reactions or substrates

        Enzymes: dehydrogenase, phosphatase, lipase, nitrogenase

        Substrates: carbohydrate, oxygen, nitrogen etc

 

Metabolic end products

        End products: organic acids, CO2, etc

 

DNA

        DNA relatively constant

        Reaction with fluorescent dyes (ethidium Br), measured spectrofluorometrically

        Purity important

 

Protein

        Easily quantified, colourimetric or chromogenic reaction, used for a long time (Lowry, Bradford)

        Best suited to pure cultures because of variations between organisms

        Problems with background proteins (extracellular)

 

Lipids

        All microbial cells enclosed in membranes containing polar lipids

        Phospholipid ester-linked fatty acids and FAME analysis

 

 

Radiolabelled substrates and components

        Radiolabelled substrates (14C, 3H incorporation eg carbohydrates, amino acids, organic acids, CO2, nucleic acids)

        Measuring uptake of radiolabelled substrates or release of radiolabelled products

        Measurement: GC-MS or scintillation counter

 

Microarrays

        Monitor patterns of gene expression

        Specific DNA fragments on inert support and used to hybridise to labeled probes/mRNA of an organism