Soil Microbial Ecology - Soil Molecular Ecology Laboratory

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6. Continue this procedure on out to the 10 -8 dilution using a new pipette for each
transfer; however, to conserve pipettes, when you reach the 10 -2 dilution use the
pipette that you transfer the sample with to also deliver 1 ml samples to each of
the labeled sterile Petri plates. Taking 1 ml from a dilution tube to a plate
represents an additional 10 -1 dilution, but this factor is canceled by the fact that 10
g of soil were added to the original dilution.
7. Each student will have 10 plates: 5 will receive fungi medium (1 each at 10 -2 , 10 -
3 , 10 -4 , 10 -5, and control from sterile water blank) and 5 will receive bacteria
medium (1 each at 10 -5 , 10 -6 10 -7 , 10 -8 and control).
8. When all samples have been distributed among the plates, carefully pour
approximately 10-15 ml of warm (50°C) melted medium into each plate. Pour
just enough to cover the plate - do not fill the plate! All media are kept in a hot
water bath until ready for use.
9. Gently swirl the plates clockwise and then counterclockwise to distribute the
inoculum. When the medium in each plate has solidified, invert the plates and
place them in the incubator (28°C).
10. Count the colonies after 3 days (either before or after class on Thursday) and
again after 1 wk.
11. Choose the dilutions, which yield between 30 and 300 colonies per plate and
make total counts on both media. The instructor will assist you in differentiating
bacteria cultures. Refer to the figure on next page for an example of how to arrive
at the final count of bacteria or fungi per gram of dry soil. Note the
characteristics of the colonies i.e. pigments, texture, and form. Compare your
results to the overall class data.
SOIL WATER CONTENT (PERCENTAGE MOISTURE) ESTIMATION
1. Weigh fresh subsample soil in aluminum containers
2. Oven-dry them (105°C for 72 hours)
3. Reweigh them
4. Water content calculation, use the formula:
Total wet weight - total dry weight
% water content = ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯ X 100
Total wet weight - weight of container