· Enrichment and culturing
· Sampling from the environment
· Detection of microorganisms in the environment
· Determination of microbial numbers
· 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 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.
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.
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
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
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.
Amplification of DNA, eg 16S rRNA genes, using specific primers
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.
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)
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
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
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
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.
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.
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.
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.
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:
· 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 relatively constant
· Reaction with fluorescent dyes (ethidium Br), measured spectrofluorometrically
· Purity important
· 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)
· 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
· Monitor patterns of gene expression
· Specific DNA fragments on inert support and used to hybridise to labeled probes/mRNA of an organism