The main sampling problems in soil microbiology are usually a result of the complexity of the medium being sampled. If the type of method being used requires a "generalized" sample of the soil, the problem is to determine what soil horizons to sample, how many samples to take (to estimate variability), exactly where to take the samples (to determine spatial variation), how often to take the samples (to determine temporal variation) and what size of samples should be used. These are often interrelated problems - e.g. a larger sample subdivided into smaller ones after mixing is different to many small independent samples. The first will show experimental or procedural error, while the second will show a combination of procedural error plus natural field variation.
Microorganisms can have many different growth patterns in soil. Some bacteria form microcolonies on the surfaces of mineral or organic particles, some fungi and Actinomycetes form visible and distinct diffuse colony forms over many soil particles, some bacteria exist as single cells and others form colonial structures. These different morphologies may be at many different scales - some fungal colony structures can be kilometres in extent, while some bacterial colonies an exist on a clay particle. In addition, individual cells in one of these structures can vary in morphology depending on the stage in the growth cycle, nutrient status, physical conditions, etc. Arthobacter species often exhibit polymorphism - the cell shape changes based on its growth rate and age. They range from coccoid cells to bacilli.
It is often necessary to associate microorganisms with other structures or objects in soil. Plant roots, organic materials, mineral grains, fungal mycelium and arthropods are all colonized by particular types of bacteria and Actinomycetes.
Many types of microbial activities are only possible under certain conditions. The distribution of the cells in soil often reflects their activities on certain substrates (above) or their responses to chemical or physical conditions. Some anaerobic cells such as Clostridium will only be found in anaerobic regions (or regions that have been anaerobic). Formation of resistant spores is a common response to adverse conditions and can be used to indicate previous growth habits or arrangements.
Using standard techniques, it is usually impossible to both locate spatially and also identify a microorganism in a soil sample (but see fluorescent antibody methods below). To identify microorganisms, the cells are usually cultured in some way to permit colony formation for subsequent identification.
Techniques to detect form, pattern and arrangement in soil can be broken down into:
Microscopic methods plus culturing
Many older methods using direct microscopic examination of soil samples are still in use today because of their simplicity. They are especially useful when examining smaller soil samples such as pieces of organic materials or mineral grains. There are two main types of methods used to visualize the microorganisms in these samples; classical stains such as phenol aniline blue and fluorescent stains such as fluorescein isothiocyanate. The first can be examined after staining with any bright-field, white light microscope assuming that light can be transmitted through the object being examined. The second uses a stain that emits light at a visible wavelength when illuminated with far-violet or ultraviolet light. This can be incident illumination that does not have to pass through the object (see diagram below).The mercury arc lamp is a strong source of ultraviolet light that is filtered though an excitation filter (to exclude all but ultraviolet light)and passed to a dichroic mirror. This mirror is coated with a very thin metal film that reflects ultraviolet light but does not allow it to pass through. It does allow visible light to go through and not be reflected. The ultraviolet light is passed down through the objective lens and focussed onto the object from the top (this is why the object can be opaque). If the fluorescent dyes staining the microorganisms fluoresce in the visible spectrum, the emitted light is collected by the objective lens and passes through the dichroic mirror to the eyepiece lens and the eye of the observer. The eyepieces always contain a barrier filter (usually yellow) that prevents any of the ultraviolet light from reaching the eyes of the observer.
The most common fluorescent stains are acridine orange, fluorescein isothiocyanate (FITC) and rhodamine (fluoresces red). They both react with parts of protein molecules - the -sulfhydryl groups - and attach strongly to the protein molecules. Another example is Calcofluor; it reacts with cellulose, chitin and similar polysaccharides and it useful for staining fungi and Actinomycetes. It is also relatively non-toxic and can be used as a vital stain to examine living cells. Other fluorescent stains include europium chelate (europium (iii) thenoyltrifluoroacetonate) that stains nucleic acids and DAPI (4'-6'-diamidino-2-phenyl-indole), ethidium bromide and Hoechst 33258 (bisbenzimide) that all stain DNA.
Another group of fluorescent stains are the fluorescent probes. They differ from FITC and rhodamine in that they are not fluorescent until they come into contact with the correct environment. Typically this is the lipid within microbial cells. Only then do they fluoresce and emit visible light. Examples of this group are DANSYL chloride and the 8-anilino-1-naphthalene sulfonic acid salts (Mg-ANS and Na-ANS). Their major advantage is that they can be applied to soil samples and immediately examined without removing excess, unreacted stain. FITC and rhodamine need extensive washing to remove unreacted stain.
Lake Ontario water stained with Mg-ANS fluorescent probe. The red fluorescence is chlorophyll a in algal cells, the green fluorescence is cellulose and bacterial cells.
Soil stained with Mg-ANS to show bacterial colonies and fungal mycelium
Electron microscopy can be used in a manner similar to light microscopy to examine very small areas within soils. Both scanning and transmission electron microscopy has been used, but the major disadvantage of both is the effort required to examine even relatively small volumes of soil. Another problem is the fixation and coating/mounting processes involved. They often destroy some of the features of the microbial cells. The process is tedious and can only examine very small regions of a soil sample.
Microscopic methods plus culturing
Soil samples can be impregnated with agar or polyacrylate resins, sectioned into thin "plates" Soil and examined by direct microscopy. Diamond microtomes are needed to section through mineral grains but the resulting very thin sections can be examined by transmission light microscopy.
Direct culture methods
If soil can be removed intact, placed on a nutrient medium and incubated, the resulting small colonies can be examined macroscopically or microscopically. Using selective media (see later), particular types of microorganisms can be selectively cultured and identified. One technique uses Scotch tape to take successive samples from an exposed soil surface. The samples are transferred to a selective agar medium and incubated. The position of colonies on the plate reveals the location and distribution of the original cells in the soil. This technique can be combined with replica plating (see later) of the agar plates to test the reactions of the colonies to different media thus aiding in identification of the colonies.
Fluorescent antibody and related methods (immunofluorescence methods)
The fluorescent antibody technique is the only one that can simultaneously locate and identify microorganisms in intact soil samples or sections.
The antibodies to microbial cells are produced by injecting the cells under study into a suitable animal (guinea pigs or rabbits are commonly used). After incubation, the animals produce antibodies to the microbial cells that can be isolated from serum samples of the animals. The antibodies are proteins so can be reacted with FITC to produce FITC-antibody conjugates. These FITC-antibodies will only adhere to the correct microbial cells if applied to a soil sample. When excess FITC-antibody conjugate has been removed by washing, only those microbial cells will fluoresce and they can be simultaneously located and identified by epifluorescence microscopy (as for FITC staining).
This technique has been extensively used in medical microbiology to identify Neisseria gonorrhoeae, enteropathogenic Escherichia coli, Salmonella spp., beta-haemolytic streptococci and viruses in tissue samples. It has been used in soil microbiology to identify nitrogen-fixing Rhizobium spp., Bacillus spp., various fungal genera such as Aspergillus, and a few Actinomycetes.
One problem is the relatively non-specific nature of many antibody preparations. Many bacteria in the same general taxonomic group have similar chemical structures on their cells and produce a complex of antibodies from those structures that overlap with the complex produced by the other similar bacteria. Thus if the antibody complex is used to form the conjugate with FITC, that FITC-Ab will cross-react with the other bacteria as well as the target species. Usually this reaction will be weaker but still significant. One way to "purify" the complex is to remove the cross-reacting antibodies by reacting them with the actual bacterial cells from the unwanted cross-reacting species. Any common antibodies (the cross-reacting group) will be adsorbed onto the surfaces of the added cells and removed from the complex. The resulting remaining antibodies will be much more specific to the target species.
A more recent modification of the method usesmonoclonal or polyclonal antibodies produced in other microbial cells to obtain larger quantities of antibody for conjugation with FITC. Many of these antibodies are now available commercially from suppliers and some are available already conjugated and therefore labelled with FITC and/or rhodamine.
Enzyme-linked immunosorbant assays
(ELISA assays) have found some use in soils when populations sought for exceed 10,000 cells per ml. The technique has been applied mainly to Rhizobia in soil and roots of legumes (see later chapter on nitrogen fixation). The major difficulty is removal of the microbial cells from the substrates; both direct lysis in situ and removal of cells followed by lysis have been used.
Gene probe and nucleic acid hybridizationtechniques rely on detecting specific sequences of nucleic acids in the organisms under study. If the sequences used are carefully chosen to be diagnostic, this technique can find specific organisms in soils and other environmental samples. The gene probe is a short segment of nucleotides that binds specifically with the homologous sequence in the target microorganism. If the segment is labelled with radioactive 32P, any binding to the target nucleotides can be detected by the presence of the radioactivity after reaction.
The Polymerase Chain Reaction (PCR) has recently been applied to soil microbial ecology.(More references material on PCR) In this technique, extracted DNA is melted to form single strands, annealed with primers, and the DNA is extended from the primers by nucleotide addition using DNA polymerase enzyme. The primers are chosen to link to regions of DNA of interest (close to a diagnostic target sequence).
Bioluminescence marker genes, typically the lux gene of Vibrio fischeri. This gene causes photoluminescence in the bacterium (emits light). If the gene can be inserted into the target organisms, they become photoluminescent and this property can be used to detect them and track their fate in soil and water samples. This technique has been used with E. coli, and Pseuodomonas target organisms. It has been extended by fusing other genes with the lux gene and inserting both into cells. Naphthalene degradation is promoted by a gene (nah) which has been combined with the lux gene to make a diagnostic pair. This can track both the bacteria and their activity in soil samples.
Various methods have been used by individual investigators to determine microbial distribution in soils. Rossi-Cholodny slides are simple microscope slides buried in soil and left for various times before microscopic examination. Strips of transparent chitin and cellulose materials have also been used. Pedoscopes are small-bore glass capillary tubes used in a similar manner.
A few workers have used direct micromanipulation of soil under a microscope to remove and incubate individual pieces of soil or even individual microorganisms. Using that technique, it was shown that the basidiomycete fungi were much more common than previously thought, They are under-represented in dilution plates because of the rapid growth of spores from other fungi. When pieces of fungal mycelium were isolated directly using micromanipulation tools, the basidiomycetes were much more numerous.
If specific chemical compounds are added to soil in the field or in the laboratory and incubated under specific conditions, the organisms capable of growing under those conditions will multiply and will come to comprise a greater percentage of the total microbiota. Alternatively, if specific inhibitory chemicals or incubation conditions are used specific parts of the microbiota will be decreased in overall percentage. This very simple concept is the basis for a large fraction of the industrial uses for soil microbiology such as isolating hydrocarbon-degrading bacteria, isolating bacteria that degrade various pollutants such as pesticides, PCBs, organochlorines, isolating bacteria (including actinomycetes) or fungi that produce new antibiotics, and many other examples. Because of the diversity of the microbiota in soil, this technique can often be applied to isolate microorganisms with any desired property.
For example, to isolate a bacterial isolate that can degrade cellulose under acid, anaerobic conditions, it is only necessary to add cellulose (cotton, paper, etc.) to a soil sample that has been acidified (with acetic acid, addition of sulfur, etc.) and incubate it anaerobically. A more sophisticated approach might be to find a soil that has been naturally subjected to conditions similar to those that you wish to use and use that as a sample source. Thus a peat bog would have a microbial population already adapted to acidic and anaerobic conditions with the presence of cellulose. Using that as a source of material would probably lead to more successful isolations. In a similar manner, soil from an oil refinery contains more hydrocarbon degrading bacteria than farm field soils.
Despite this, it is often found that many soils contain a very large variety of organisms that do not seem to be related to that soils obvious chemical or physical environment; most farm field soils do contain hydrocarbon degrading bacteria. This could be a reflection of the large degree of diversity in soil leading to many microbial niches, a result of the biochemical diversity even within individual species of microorganisms (many bacteria can degrade a wide variety of compounds), or movement of bacteria through wind or water transport mechanisms.
All media used in microbiological laboratories are selective to some degree or another; there are no truly non-selective media. It is possible to make media and incubation conditions very selective by chemical and physical modifications. This is often very useful to isolate and count particular groups of bacteria or other organisms from soil samples.
There are routine ways to produce selective media:
a). Add compounds used by an organism as a nutrient source.
b). Omit compounds required by most other organisms(omit nitrates and other fixed nitrogen sources to isolate nitrogen fixing bacteria)
c). Add selectively biocidal compounds(penicillin to inhibit gram-positive bacteria, neomycin and streptomycin as general bacterial inhibitors, actidone and nystatin as general fungal inhibitors)
d). Change physical properties (pH, pE [redox potential], etc.)
e). Alter incubation conditions (temperature, water content, osmotic pressure, light, etc.)
Using combinations of these techniques it is possible to design very selective media; Pseudomonas isolation media is very specific for its named bacterial group. In theory, almost any physiological group of organisms can be cultured selectively.A single stage isolation from a soil sample can be changed to a multistage isolation process by replica plating. Individual colonies are transferred in their original orientation on the plate by pressing a pad of sterile velvet cloth onto the surface of the plate, removing a small sample of each colony and pressing it onto the surface of a fresh plate. This can be a different growth medium so that only a part of the original population is able to grow on the new medium. In this way, progressive selective media can be used to isolate bacteria with combinations of properties. To enhance the detection of the particular group of microorganism under study, it is also possible to improve the diagnostic precision of the media by using some properties of the organisms (pigment, fluorescence under ultraviolet, biochemical reactions with extra, added substrates, etc.)
So-called "non-selective" media are only media and incubation conditions designed to isolate as large a part of the microbiota in soil as is possible. Truly non-selective media do not exist. The least selective media today isolate maybe 1% to 10% of the total soil bacteria and maybe 5% to 15% of the fungal population of soils. The media used for bacteria and fungi are different.
General activity measurements
Respiratory activity as a general activity measurement is well established. The Biometer flask is a common piece of apparatus used to measure the activity of soil samples. Soil is incubated at the required temperature, moisture content, etc. in the main flask and air can be added through the CO2absorber in the tube on top of the main flask. During incubation the CO2 produced by respiration is absorbed into the alkali in the side tube.
This is then titrated to discover the amount of alkali neutralized by the evolved CO2 More air can be added and the alkali replaced to continue the process of analysis.
To obtain reproducible results from assays of respiration rate the soils are usually sieved to remove plant materials, insects, etc. This destroys aggregate structure and may stimulate activity due to release of nutrients previously protected inside the aggregates. Disturbance of any kind usually causes a temporary burst in respiratory activity.
Cell division rate
Fungi grow by extension of mycelium; if this can be observed , it can be used to estimate growth rates. Direct observation or extraction from soil (followed by observation and measurement) can be used.
Enzyme activity or content
The dehydrogenase enzymes can be extracted from soil samples, reacted with tetrazolium dyes to form colouerd products and the colour measured by colorimertic methods. This gives an estimate of the dehydrogenase activity in the saoil ample and thus an estimates of the organisms present. Various sieving techniques can be used to separate small soil animals, fungi and bacteria from soil samples, but theyare all somewhat unsatisfactory - i.e. they do not completely separate the organisms even in a well-mixed soil suspension.
Substrate utilization rate
Any compound that is a substrate for microoroganisms can be used to estimate their activity if the decrease in substrate concentration can be measured.
2. Determination of ATP content of soils - ATP is extracted from the cells in the soil and measured by its reaction with the enzyme system luciferin + luciferinase. The enzyme luciferinase is extracted from firefly tails and emits light when ATP reacts with its substrate luciferin. The emitted light is measured in a scintillation counter. Specific machinery is available for this ATP determination that integrates the reaction/enzyme/substrate system into one vial of dedicated small scintillation counter.
The amount of ATP per gram a cell material varies, but averages 10.0 moles g-1resting biomass. It has been proposed that the ratio of ATP to biomass C content is 1:120 in soil samples and this is very close to the ratio in exponentially growing microorganisms and eucaryotic cells.
3. Determination of cell wall components of bacteria - Bacteria contain specific cell wall components such as muramic acid that can be released by acid hydolysis and analyzed by High Pressure Liqiud Chromatography (HPLC). The amounts of muramic acid in bacterial cell walls varies depending on whether the cells are gram negative or gram positive (12 g mg-1 average in Gram negative and 44 g mg-1in Gram positive cells). Thus, unless the proportion of Gram positive to Gram negative cells is known, this technique has serious limitations. Bacterial spores also contain up to 4 times the normal levels of muramic acid.
4. Dilution plate counts and direct microscopical counting - Dilution plates usually only are able to culture between 1 and 10% of the viable organisms in soil samples. Direct microscopic observation methods (FITC, acridine orange staining, etc.) usually overestimate the number of cells because they include dead organisms or other particles in their count.