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Materials

 

Meter stick

10 Wire screen (35 X 35 cm 0.25 mm2)

16 wooden sticks (20 cm)

Saran wrap

Scissors

Sharpie

10 mL serological pipettes

 

3 15 mL culture tubes with caps

1-cc scoop

28 mL of Sterile water for each sample

Disposable dropper or pipettes

P200 micro-pipette with tips

Soil core samplers

Chemical Test Kit

 

Procedure

 

1)      Cut out 10 (35 by 35 cm) wire screen squares (using ¼ of 1 mm screening)

2)      Cut 16 (20 cm long) wooden sticks

3)      Take the wooden sticks (1 mm diameter) and roll them in saran wrap so that the ends are closed. This way mold cannot form/ attach to the wooden sticks.

4)      Label half way mark on the wooden sticks

5)      Use a whole punch to cut four holes on the corners of one 35 mm by 35 mm square. Then place the saran wrapped wooden sticks through the holes, down to the half way mark.

 

                        

6)     Label three areas of excavation on the squares

7)  Go outside and find areas with adequate sunlight

8)  Take soil core samples (15 by 2 mm) for each quadrant (three per quadrant) for five quadrants

9)  Put soil samples in plastic bags and label the bags according to their respected areas.

10)  Plant the newly constructed canopies in relatively the same area (the first plot should have 0 canopies, Plot 2 should have one canopy, Plot 3 should have two canopies, Plot 4 should have 3 canopies, Plot 5 should have four canopies)

                       

11)  take soil samples inside and run tests

12)  set up for soil extractions test and serial dilution

13)  Perform the soil extractions test (on each sample) at exactly the same time as serial dilution in order to get accurate results.

14)  For the first sample start with the serial dilution test; use a clean, new transfer pipette to add 10 mL of distilled water to a 15 mL culture tube.  Label the tube “100.”

         

15)  Use the same pipette to add 9 mL of distilled water to a second 15 mL culture tube.  Label the tube 10-1.

16)  Use that same pipette to add to the a third 10-2 culture tube, label the tube 10-2.

17)  Place 1 cc of your soil sample into the “100” culture tube.

             

18)  Cap the tube and shake vigorously.

19)  Using a new clean pipette, remove 1 mL of the soil/water mixture from the “100” tube and place into the “10-1” tube.

20) Cap and shake vigorously.

21)  Using the same pipette from step 19, remove 1 ml of the soil/mixture from the “10-1” tube and place into the “10-2” tube.

22)  Cap and shake vigorously.

23)  You should now have a total of 3 cultural tubes.

24)  Using the micropipette, extract 100 µl liquid from each tube and put on to a corresponding mold growth medium, repeat steps 14 through 24 for the remaining soil samples

25)  In total there should be 45 mold sheets.

26)  Allow to grow for 48 hours.

27)  Examine each of the plates for individual mold colonies and choose the plate that is the most dilute with the least amount of mold colonies in order to make sure your estimates of the number of mold in the original 1 cc soil sample using the following formula:

            # Microbes in 1 cc of soil = # Colonies on sheet and 102 x 10 |dilution # at which these colonies were found|

28)  At the same time as serial dilutions, test all soil samples for iron

 

29)  After finishing the serial dilution and iron tests for the raw soil samples, allow the sunlight to shine on the canopies for 24 hours.

33)  Go back to your plots and take a second set of soil samples

34)  Put the soil samples in plastic bags and label the bags according to their respected areas (make sure to label “Day 1 samples” on the plastic bags)

35)  Next follow steps 14- 28 for these samples

36)  Allow the sunlight to shine on the canopies for 48 hours.

37)  Go back to the plots and take a third set of soil core samples (filled to the first line) from underneath the canopies.

38)  Put the soil samples in plastic bags and label the bags according to their respected areas (make sure to label “Day 2 samples” on the plastic bags)

39) Next follow steps 14 to 28 on these samples

40)  Allow the sunlight to shine on the canopies for 72 hours.

41)  Go back to your plots and take a fourth set of soil core samples (filled to the first line) from underneath the canopies.

42)  Put the soil samples in plastic bags and label the bags according to their respected areas (make sure to label “Day 3 samples” on the plastic bags)

43)  Next follow steps 14 to 28 on these samples

 

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