1. OVERVIEW OF SAMPLE COLLECTION AND STORAGE
Obtaining a representative soil sample is essential when determining microbial soil parameters such as soil ecosystem functions in relation to specific soil microbes, the presence of plant pathogenic microbes, and the survival and distribution of unmodified or genetically modified microbial populations. Without proper representative sampling, results can be skewed and be deemed meaningless statistically. Since soils are inherently very heterogeneous it is essential to determine what is to be sampled and devise a sampling strategy to determine what microbes are present as well as individual microbial populations within the soil ecosystem.
Soil microbiota are usually localized in close association with clay-organic matter complexes and because soils are very heterogeneous, often times there exists differences in microbial populations even within a few inches. This is caused by differences in microhabitats and is one reason why it is so difficult to obtain a true representative sample. Usually, when samples are taken from a site they should be viewed as a composite sample of these varying microecological environments.
Sample storage is another concern when sampling for microbial biota. In a perfect world, storage of samples should be avoided whenever possible because vast changes in microbial populations could occur overtime and the representative sample obtained may not be at all representative by the time it is analyzed. If CFU counts or enzymatic activities are to be derived then storage invariably alters the microbiology within the soil. When storage is deemed necessary one should store samples in polyethylene plastic bags which will be discussed in more detail below.
As mentioned above, before sample collection should even
be considered one should ask themselves several questions to determine
the sampling strategy. It would be inadequate to simply travel to
a site; collect a few samples and composite them. For example, if
one sample was taken from a floodplain and mixed with a sample obtained
from a summit, results could invariably be skewed because microbial populations
are sure to be different in a moist alluvial soil compared to that of a
residual summit soil. Below is a diagram that could be used when
determining how and where to sample. By following the flowchart,
one can determine there aim of sampling which will aid in identifying where
to sample. After a sampling strategy is determined then one can begin
to determine how they will sample. Four common methods of sampling
are show in the adjacent figure.
2. OVERVIEW OF SAMPLE COLLECTION AND STORAGE PROCEDURES
Procedures for sample collection and storage are very straightforward once a sampling strategy is determined. Thus strategy determination is by far the most important step in the process. However, when sampling for microbial populations or soil viruses one should make sure to sterilize the equipment used and do everything possible to prevent cross contamination.
Once a method of sampling has been derived, one needs to determine the number of samples to be acquired over a given area and how much soil should be sampled. In general, samples for microbial analysis require relatively small samples to be obtained at each locale. For a relatively homogenous site on the macroscale, it is recommended to obtain 15 subsamples for one total composite sample over a 2 ha area and 15 sub samples for each of 5 total composites over a 10-20 ha area.
Upon reaching each sampling locale, it is imperative to collect physical data about the site because this may provide valuable knowledge when studying the microflora. The following items should at least be collected at each subsampling location:
Texture Moisture status Animal presence Field History
Bulk Density Temperature range Rainfall amounts Weather
Often times, the easiest and most efficient way to gather samples is to simply use a soil auger as pictured below. One's already determined sampling plan will decide on how deep samples must be taken. By simply using an augur, representative samples can be obtained with little or no downward contamination. A study in 1918, revealed that statistically speaking very little contamination occurred from upper horizons as the auger passes downward. The soil microflora are just so large, so characteristic, and firmly established that the introduction of relatively small numbers of contaminants has no noticeable effect. This means that one could take samples of both the A horizon and Bt horizon to provide two subsamples for each sampling locale.
Once samples are obtained they should immediately be put
into two polyethylene bags and taken to the lab for analysis. The
bags are used because they allow the passage of Oxygen and Carbon Dioxide
but hold water within the samples adequately. In our environment,
once samples are gathered, and a composite made if desired, precautions
during transport include; keeping the samples on ice as well as avoiding
sunlight. If samples must be stored for prolonged periods the best
thing to do is ensure they stay at the same moisture content as when sampled
and remain at 4 degrees C. It is important to note, that ones sampling
strategy could alter these commonly used storage techniques.
3. PROS AND CONS
These methods of sampling for microbiota have proven to be effective and will provide statistically reliable results when followed properly. As mentioned above, the most important step is to figure out what kind of data or measurement is to be made and design your sampling strategy around that. Once that is obtained and the samples are taken, very real and valuable data can be generated. A simple rule of thumb however, is once samples are taken get them analyzed as soon as possible.
That is perhaps the one major con about sampling. Often times, is just is not feasible to analyze the samples rapidly and even in polyethylene bags changes can occur within the microflora. This causes concerns with not only plate counts, but all other quantitative, biological soil analyses as well. Research has shown that storing samples for more than a few hours can cause both quantitative and qualitative changes within the soil microflora. Therefore, the data generated could be skewed simply due to storage of the soil for prolonged periods.
4. ADDITIONAL SOURCES OF INFORMATION
Grey, T.R.G., and Parkinson, D. 1968. The Ecology of Soil Bacteria. University of Toronto Press. pp.106-108.
Hurst, C. et al. 1997. Manual of Environmental Microbiology. American Society for Microbiology Press, Washington D.C. pp.383-404.
Johnson, L.F., and Curl, E.A. 1972. Methods for Research on the Ecology of Soil-Borne Plant Pathogens. Burgess Publishing Company, Minneapolis, MN. pp.1-5.
Paul, E.A., and Clark, F.E. 1996. Soil Microbiology
and Biochemistry 2nd Edition. Academic Press, New York, NY. pp.35-41.
Ben Meadows --This link is to Ben Meadows Company which supplies numerous products for sample collection and storage including: augers, probes, sample bags, sample rings etc.
Fisher Scientific --This site provides additional sampling and storage equipment as well as numerous materials for microbial studies.
VWR Scientific Products -- This site also offers a wide range of soil sampling products and storage containers.
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