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Fungi Serial Dilutions

1. Put on gloves and goggles.

2. Use a clean, new, serological transfer pipette to add 10 mL of distilled water to a empty, clean, new, 15 mL culture tube with a cap. Label the tube "100".

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

4. Repeat step 2 one more time to one additional 15 mL culture tube. Label the tube "10-2"         

5. Place 1 cc of your soil sample into the "100" culture tube.

6. Cap tube and shake vigorously.

7. Using a new clean pipette, remove 1 mL of the soil/water mixture from the "10 0" culture tube and place into the "10 -1" tube.

8. Cap and shake vigorously.

9. Using the same pipette in step 5, remove 1 mL of the soil/water mixture from the "10-1" tube and place into the "10-2" tube.

10. Cap and shake vigorously. 

11. Plate 100 micro milliliters of the samples from the tubes onto its corresponding yeast and growth media. For each soil sample, label media "10-0", "10-1", "10-2". Remember to label each count media with its corresponding location number, sample number, and experimental trial number.

12. Allow the yeast and mold to grow at room temperature on the yeast and mold media for 48 to 72 hours before you count the number of Fungi.

13. Make preparations to count the Fungi by creating an organized table including a column for Location/ Sample, Dilution #, Number of Yeast, and Number of Mold. Make sure to label the Experimental Trial Number of sample collection for the table (four days of sample collections).

A.) When counting Fungi: Place the yeast and mold media in front of you. If you are counting the Fungi for Experiment trial 2, Location 1, Sample A, place the media with the soil sample in front of you. Now examine media plate with the lowest dilution (i.e "10-2") and count for individual Yeast (brown/red dots) and Mold (tan or green spots) colonies with a magnifying glass. If no colonies are found on the lowest dilution media plate, then move onto the second lowest dilution.

14. Once all the data is recorded, use the following formula to determine the number of microbes in 1 cc of soil.:            

# Microbes in 1 cc of soil = # Colonies on sheet x 102 x10 I dilution # at which these colonies were found I .

Repeat this Serial Dilutions procedure for every single sample. You need to complete this entire procedure for ever single one of your samples.