Measuring and Identifying Soil Organisms

How can the amount of microorganisms in a soil be measured?

The whole mass of living organisms in soil is called its microbial biomass. It can be estimated by measuring the amount of carbon, nitrogen, phosphorus or sulfur that is present in the microorganisms, small animals and algae in the soil. There are a number of different ways of measuring microbial biomass.

For example:

1. The fumigation-incubation technique: Two samples of soil are collected. One is not treated; this is called the untreated soil. The other sample is treated with fumigant to kill all the organisms in it. Living organisms are then added back into the fumigated soil and the sample is left to incubate. During incubation, the living organisms degrade a proportion of the dead organisms. The difference between the amount of carbon dioxide released from the untreated soil and the treated soil samples is used to estimate the amount of respiration that has occurred during the degradation process. Respiration is then used to estimate the amount of carbon degraded which correlates to a certain biomass of microbes. (Only a proportion of the organisms will be degraded, so this is taken into account).
2. The fumigation-extraction method: Two soil samples are collected, one remains untreated and the other is fumigated to kill all the organisms. Chemical tests are done on the fumigated soil to extract the ninhydrin-positive compounds. Carbon, nitrogen, or phosphorus that has been released from the dead microorganisms is then measured to determine microbial levels.
3. The substrate-induced respiration method: Two soil samples are collected, one remains untreated and the other is fumigated to kill all the organisms. A source of energy (eg sugar) is added to the other sample, so that the organisms in the soil can be more active. When they are more active, they respire (breathe) more and so release more carbon dioxide into the soil. The amount of carbon dioxide released by the organisms in the treated soil is then compared to the untreated soil to calculate the probable mass of microorganisms in the soil.
4. Using ATP or enzyme activity: ATP is the name given to one of the molecules that carries energy inside living things. Enzymes are proteins that help reactions to go faster. The amounts of ATP and specific enzymes in soil indicate how many organisms are in that soil.

How many soil animals are there in the soil?

The number of individual soil animals in the soil is enormous. In all but the driest environments there are billions of protozoa per square metre (m²), millions of nematodes/m² and 100 000’s of mites/m². Data from east Beverley in Western Australia indicates that there were approximately 800 million protozoa/m², 900 000 nematodes/m² and 130 000 mites/m² in a soil under pasture.

What techniques are used for identifying soil organisms?

Soil organisms are identified by looking at:

• Morphology (their structure and shape)
• Physiology (what happens inside them)
• Genetic characteristics (their DNA structure)
• Ecological characteristics (where they live, how they interact with other living things), and
• Molecular characteristics (whether they have particular parts of genes and the type of proteins or molecules they produce)

Methods used to identify and assess organisms include: · Molecular markers (DNA profiles) · Serological techniques · Protein and enzyme profiles · Embedding soil with resin · Rossi-Cholodny slides · Sieving, heating and floating to extract animals · Artificial media · MPNs (Most Probable Number technique) · Baiting techniques · FAME tests (Fatty Acid Methyl Ester assay). The type of method used to characterise the organisms in soil depends on how you want to use the information.

How are rhizobia identified?

Rhizobia are bacteria which can grow and multiply on artificial food sources. They are very difficult to identify from their shape or size alone because all forms are extremely small, short rods with rounded ends.

Recently, new methods of rhizobia characterisation have been developed using DNA patterns from known rhizobia grown under laboratory conditions. Comparisons are made between sections of the known DNA molecule that code for particular functions (eg. nitrogen fixation) with samples of DNA from soil or from a root nodule. This tells the scientists whether there are genes for that function in the soil sample or root nodule.

Serological techniques can also be used to identify rhizobia. Samples of known bacteria are injected into an animal under specific laboratory conditions. The animal's body produces antibodies against the bacteria. (A process that is similar to vaccination). The component of the blood containing the antibodies is the serum and this is separated from the red blood cells. This is called antiserum.

The antiserum produced by the animal can be collected and used to help locate bacteria in a sample. The antibodies in the antiserum can be joined to a fluorescent dye or to an enzyme with an attached dye. When the antibody is added to the corresponding bacteria it attaches firmly. The bacteria can then be identified using a fluorescence microscope or in a solution that contains a molecule that can interact with the enzyme and cause a colour change in the solution. This method is called the ELISA method.

The antiserum can be chosen to be very specific for particular types of rhizobia, so it is a good way to identify whether those rhizobia are present in a root nodule. It is often important to know whether the rhizobia that were added with the seed have actually been responsible for forming the nodules on the legume or whether other rhizobia already in the soil formed the nodules.