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Step by Step Instructions:

1. Find a space of land that is 7m by 1.4m and is on a slope.

2. Acquire 36 small construction flags, and 9 small construction flags of a different color.

3. Make a plot with 3 rows and 3 columns. Each plot in a row is 30 cm by 30 cm with 15 cm of space in between. Each row is 3 meters apart. There should be 9 plots total.

4. Label each of the 9 different colored flags with the name of its respective plot. (See site map on right for plot labeling assistance.)

          a.

5. Place a flag on each corner of each plot, with a labeled flag in the center of the plot.

6. Label 27 plastic plastic bags with the row number, plot letter, and sample number. (i.e. 0a1 = first row, plot a, sample 1. (See soil samples (blue circles) on site map on right.)

7. For each square, place a soil cylinder with a 2.5cm diameter 15 cm into the ground and pull up the soil, placing it into the corresponding labeled plastic bag. Repeat this process three times at different locations in the same square, placing it in a different corresponding plastic bag for a total of three samples per plot. (note: all soil samples must be taken at the same time on the same day.)

           a.

8. After getting each soil sample, evenly sprinkle 1 liter of tap water into each of the letter B plots and 2 liters for all the letter C plots. (note: no water is added to the letter A plots)

           a.

9. Make 27 aluminum "boats" (something that can hold soil)

           a.

           b.

10. Make a data table for each of the soil samples. (i.e. 0a1, 0a2, 0a3, 1a1, 1a2, 1a3...) You should have 27 total rows in your data table.

11. Weigh each sample in its "boat" and record the weight into data table. (note: complete immediately after step 12.f.)

           a.  

12. Test the soil in the bag labeled 0a1 for the number of fungi (note: step 11 and this step must be completed at the same time on the same day)

          a. Use a clean, new transfer pipette to add 10 ml of sterile water to a 15 ml culture tube.

           b. 

           c. Label the tube and cap“0a1 100

           d. Use the same pipette to add 9 ml of sterile water to a second 15 ml culture tube. Label the tube and cap “0a1 10-1

           e. Repeat step two three more times to one additional 15 ml culture tubes, only label the tube and cap “0a1 10-2” respectively

           f. For the rest of the steps, look at the following website. http://essre.rpcs.org/ESSREProtocols.htm 

           g. (note: when plating 100 μL samples from the 0a1 100, 0a1 10-1 and 0a1 10-2 tubes onto their own separate, correspondingly labeled fungal nutrient plates) (look at our trouble shooting page to see what type of plates we used)

           h. Repeat all the steps for 0a2, 0a3, 0b1, 0b2, 0b3, 0c1, 0c2, 0c3, 1a1, 1b2, 1b3, 1c1,1c2, 1c3, 2a1, 2a2, 2a3, 2b1, 2b2,  and 2b3 labeling the tubes to the corresponding bags and fungal nutrient plates

14. Allow to grow for 48 hours

           a. Examine each of the plates for individual yeast and mold colonies by looking at the dilution plate with the lowest dilution. Count the yeast which are small solid dots which are blue, green, gray. Count the mold which are fuzzy larger dots of the same colors as yeast on the plate using a magnifying glass.

               b.

           c. If there are yeast but not mold, count the yeast and move to the next dilution to look for mold and vice versa.

15. Use the equation below to calculate the number of yeast and mold then add those number together for the total of fungi.

  

16. Record data in data table.

           a.

17. Weight the aluminum boat with the soil in it (W1)

           a.

18. Record data into a data table

19. After all the boats have been weighed and recorded place them in an oven pan and put them into an oven that is between 105 °C (221 °F) and 110 °C (230 °F).

20. After the boats of soil have been in the oven for 16-24 hours, take them out and weigh them. (W2)

21. Record data in a data table.

22. Calculate the percentage of water in the soil using the equation below.

Percentage of Water Content=(W1-W2)/W1×100

23. Repeat steps 7-22 (24 hours after collecting the soil samples)