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Ian L. Pepper, Charles P. Gerba, and Raina M. Maier
How many samples and what sampling strategy would you use to characterize a portion of land of area 500 km? Assume that the land is square in shape with a river running through the middle of it which is contaminating the land with nitrate.
A treatment sampling strategy is outlined below showing three transects that would allow examination of the influence of the river on the land mass. Number of samples actually taken depends on funding available and degree of homogeneity of soil.
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Discuss the reasons that deep subsurface sampling is more difficult than surface sampling.
Surface soil sampling can be conducted easily with a shovel, trowel or hand auger. Soil cores can also be obtained relatively easily to a depth of 150 cm using soil augers. Sampling at deeper depth requires specialized equipment for excavation or deep soil coring. In addition, it is difficult to prevent subsurface samples from being contaminated with surface soil material.
A soil is extracted for its community DNA and is found to contain 0.89 μ g DNA per gram soil. How many bacterial cells does this theoretically involve?
Community DNA concentration = 0.89 μ g DNA/g soil. If each cell has 4 fg of DNA, then:
4 fg DNA/cell (1 x 10 -9 ug/fg) = 4 x 10 -9 ug DNA/cell
(0.89 ug DNA/g soil)/( 4 x 10 -9 ug DNA/cell) = 2.2 x 10 8 cells/g soil
When would one utilize electropositive filters for concentrating viruses from environmental samples?
For concentrating enteric viruses from large volumes of tapwater, groundwater or freshwater.
When would one utilize electropositive filters for concentrating viruses from environmental samples?

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For concentrating enteric viruses from large volumes of seawater.
In what ways is it easier to sample water for subsequent microbial analysis than it is to sample soil?
Strategies and problems associated with subsurface soils are outlined in question (2). Sampling water from streams, lakes or rivers is potentially easier since hoses can be dropped beneath the water surface to any desired depth and water at that depth obtained via a pump. Sampling large bodies of water is also essentially non destructive.
If you collected a surface soil sample from a desert area in the summertime, when daytime temperatures were in excess of 40 C, how would you store the soil and how would you get the soil ready for microbial experiments to be conducted 1 month later? Discuss the pros and cons of various strategies.
Of course, ideally one would not collect the soil sample until immediately before analysis. Typically prior to analysis, soils are stored at 4 C to reduce microbial metabolism. Studies have shown that storage for up to 3 weeks at 4 C does not substantially change the microflora. If the soil samples that were collected were essentially dry, then samples could be stored at room temperature. The advantage of this would be that it precludes inactivation of thermophilic microbes in the soil that could occur with storage at 4 C.
What sampling strategy would you use to give the most complete picture of all bacteria found in a soil sample?
An effective strategy would be to take multiple samples from each treatment being evaluated. Remember, single samples from multiple replicates are more effective than multiple samples within one replicate. Precision can be enhanced by taking composite samples. The most complete picture of all bacteria in a soil could be obtained by utilization of microscopic, cultural, molecular, and physiological menthols.
Microscopic:
Cultural: HPCs
Molecular:
Physiological: total counts via acridine orange staining community DNA extraction, gene cloning and sequencing for diversity studies
CO
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evolution and dehydrogenase activity; potential biochemical transformations, e.g. nitrification, sulfur oxidation
How do electropositive filters concentrate viruses from water? Why are they not effective in concentrating viruses from seawater or from water with a pH above 8.5? What is the principle behind eluting viruses from filter surfaces?
The filters have a net positive charge on their surface and attach the negatively charged viruses to their surface by electrostatic attraction. At a pH above 8.5 the electropositive filter becomes less positively charged and does not attach the virus as well to its surface. Viruses are eluted from the filters by increasing the pH of the eluent making the filters less positively charged and releasing the virus. The use of organic matter in the eluent, such as beef extract, competes with the virus for adsorption sites on the filter surface, also causing additional release of the virus.