The Soil Ecology WebPage of

Charlotte, Lizzie, and Margot


                As part of the Environmental Summer Science Research Experience for Young Women (ESSRE), we spent the last two-and-half weeks (July 9-25, 2001) researching the soil ecology of the Roland Park woodlands in Baltimore, Maryland--an urban deciduous forest. To allow us to perform an in-depth analysis of the soil health, we first performed a general biota survey. Our actual laboratory research was made possible only by first understanding the soil composition and its living contents. Aside from the numerous run ins with poison ivy and oak, ticks, and other hellish conditions, we had some fun times! So sit back, relax, and hear about the experience.

Description of Research

                For our biota survey, we tested for a number of bioindicators, which told us how “healthy” the soil was:

1.        Soil texture: the varying components of sand, clay, loam

2.        heterotrophic bacteria count

3.        pH levels: how acidic or alkaline the soil is

4.        earthworm count 

5.        nematode count: of the visible variety, unsegmented worm

6.        salt tests for presence of ferric iron, calcium, potassium, chloride, manganese, aluminum, and magnesium

7.         zoosporic fungi count

8.        protozoa count

9.        arthropod count, identification, and biodiversity

10.     vascular plant count and biodiversity

11.     algae count

Our group performed these survey tests on Microclimate 2, while the other groups handled three nearby sites. We became interested with protozoa levels throughout the four sites. Such a dramatic difference in amount of protozoa between sites 2 and 3 combined with our curiosity about organic matter levels (broken down, formerly living matter composed of carbon) led us to speculate: could differing amounts of organic matter account for the differing amounts of protozoa found in our survey?  We wondered if site 3, which had about 10 times as much protozoa as site 2, would also have higher levels of organic matter (or, humus).

How to do it

How we explored the question, Does the quantity of protozoa in any given area vary with the quantity of organic matter?

1.Go to sites 2 and 3 to collect 32 samples, 4 within each quadrant. To collect each soil sample, use core sampler up to the “first mark” (down to a depth of 13.5 cm).

2.Repeat step 1 in another time period (i.e., day 2), until there is a total of 64 soil samples

3.Perform the following protozoa test on each of the samples :

a. Place 10g of the sample soil into a labeled  (by site, quadrant, and sample #) into a petri dish.

b. Gradually add 14 mL of sterile water until you have saturated the soil (hint:  tilt the petri plate at a 45 degree angle to allow the water to run off, creating a soil liquid solution).

c. Cover petri dish and allow it to sit for 2 days or, approximately 48 hours, at room temperature (25-30degrees Celsius) .

 Preparing petri dishes for protozoa count.

d. After 48-hour waiting period, collect 1 drop of each sample’s soil-water solution  and place it on a depression slide. Add 1 drop of diluted methyl green dye solution (50 percent distilled water, 50 percent pure methyl green dye) to each depression slide, and cover with a cover slide.

e. After preparing each of the 64 protozoa slides, examine each under a light microscope. First examine the slide at 10x and then 40x and, finally, 100x. Under the 100x magnification, count and record the number of protozoa in 5 individual field views. Then convert protozoa counts for each sample to number per effluent extract by summing the 5 field views (per sample) and then dividing this by 2.025. To convert the number of protozoa per effluent extract to per gram of soil, we multiplied the former data by 1400.

  Preparing depression slide.

4. Using the core samples also used for the protozoa count, test for humus presence.:

a.     For each sample, measure 2 level measures (i.e., 2 grams) of the soil, and place into a soil testing tube.

b.     Add demineralized water until the mixture reaches the 14 mL mark, and shake gently for 5 seconds.

c.     Add 1 gram of Humus Screening Regeant to each tube, and then shake vigorously for 60 seconds.

d.     Finally, complete the original humus solution by adding 15 drops of Soil Flocculating Regeant and shake gently for 5 seconds.  Allow this solution to settle for a couple of minutes.

e.        Filter the settled solution into another soil testing tube through use of a funnel and quartered filter paper.

f.         Match the resulting color of the new humus liquid solution with a corresponding color on the Humus Color Chart (provided by a chemical soil testing kit). Although the Humus Color Chart only provided a “key” of colors in terms of integers 1-5 (5 being a most intense orange, 1 the lightest), we used not only the whole numbers but also data like 1.5, 2.5, and so on. We added 0 to our color scale since occasionally no color change was observed. Record this data.

Sample Results

    Firstly, we looked for a general correlation between humus and protozoa levels, spanning both sites. A regression analysis indicated that little correlation existed, as an r-value of 0.14 was found.

Since we found only a weak (if any actual) correlation between humus and protozoa levels, we decided to isolate each variable and compare their presence in site 2 versus site 3. We performed a 2 sample t-test  for the humus presence, which ultimately indicated that even for a 0.2 t-value, the sample t-value (0.779) was less than the table value (1.296). Thus, there is not even an 80 percent chance that we can refute the Null Hypothesis; and the 2 populations are most likely from the same population in terms of humus levels.

    A comparison of protozoa levels in each site was also performed. While site 2 had an average of 12 885 protozoa, site 3 had 29 703. We then performed a 2 sample t-test as with the humus data. The found t-sample value of 8.989 was greater than the t-alpha value of 4.169 (for a 0.0001 degree of error), signifying a 99.999 percent likelihood that the Null Hypothesis could be refuted. Hence the 2 sites are almost certainly statistically from 2 different populations in terms of protozoa levels.

    Since our research indicated that humus and protozoa levels are unrelated ecological factors, we have begun to look elsewhere for factors affecting the size of a protozoa population. Our initial biota survey between sites 2 and 3 indicated that bacteria, ferric iron, and potassium levels are higher when those of protozoa are as well. 

Troubleshooting suggestions

                We learned a few things “the hard way.” So listen to us! Never use cheese (a fungi-based product) as a fungi bait in your test! Something like bread may be a wiser bait choice. Always label the bags in which you collect soil samples (bitterly, Otherwise, you may have to repeat nearly your entire procedure.) Never sit in poison ivy—it results in an irksome rash. 

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                further info (bibliography with links)

        1. Anderson, O. Roger; Darbyshire, John F.; & Rogerson, Andrew. (1996) “Protozoa”. Methods for the Examination of Organismal Diversity in Soils and Sediments; ed. G.S. Hall. Paris: CAB INTERNATIONAL. Pgs 79-90.

        2. Samuels, Myra L. (1998) Statistics for the Life Sciences. Englewood Cliffs: Prentice Hall.

        3. Ingham, Elaine R.; Tugel, A.J.; & Lewandowski, A.M. eds. "Soil Protozoa," [Online]a,” Soil Quantity Institute, Oregon State University.. February 2001.