Phosphorus

1. Use a transfer pipet (0364) to fill a "Phosphorous B" Tube (0244) to the mark with the general soil extract. 

2. Add 6 drops of *Phosphorous Test Reagent #2 (5156). Cap and shake to mix. 

3. Add one *Phosphorous Reagent #3 Tablet (5157). Cap and shake until dissolved. 

4. Immediately compare the color that develops in the test tube against the Phosphorous Color Chart (1312). Hold the tube about one inch in front of the white surface in the center of the color chart. View the chart and sample under natural light for optimum color comparison. The test result is read in pounds per acre Available Phosphorous, which needs to be converted to ppm (divide by two)

Sulfate/Sulfur

1. Use a transfer piper (0364) to transfer five drops of the general soil extract to a flat-bottomed turbidity vial (0242). 

2. Add one drop of *Sulfate Test Solution (5171). Swirl gently to mix. Compare the turbidity of the sample to the turbidity standards of the Sulfate Chart (1314). Lay the chart flat under natural light and hold the turbidity vial one-half inch above the black strip in the middle of the chart. View the black strip down through the turbid sample and compare the resulting shade of gray with the six standard shades. The test result is read in parts per million sulfate.

Serial Dilutions for Fungi Materials 

60 15 mL culture tubes with caps

60 3MM Petrifilm TM Aerobic Count Plates 

36 3M Petrifilm Yeast and Mold Count Plates 

Sharpie and tape (labeling) 

1 cc scoop 

13 serological transfer pipettes (1 for each soil sample and 1 for distilled water) 

Gallon of distilled water

P200 micropipette Box of micropipette tips 

Magnifying glass 

Alcohol squirt bottle 

Directions 

1. Label 60 15 mL culture tubes, 3 for each of the 12 soil samples. Label 36 3M Petrifilm Yeast and Mold Count Plates, 3 for each of the 12 soil samples. 

2. Use a clean, new transfer pipette to add 10 mL of sterile water to the 15 mL culture tube labeled "10^0". Use the same pipette to add 9 mL of sterile water to a second 15 mL culture tube labeled "10^-1". 

4. Repeat step 3 one more time to one additional 15 mL culture tube labeled "10^-2". 

5. Place 1 cc of your soil sample into the 10^0 culture tube.

 6. Cap the tube and shake vigorously. 

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

8. Cap and shake vigorously. 

9. Using the same pipette in step 7, 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. Using the same pipette in step 7, remove 1 mL of the soil/water mixture from the 10^-2 tube and place into the "10-3" tube. 

12. Cap and shake vigorously. 

13. Using the same pipette in step 7, remove 1 mL of the soil/water mixture from the "10-3» tube and place into the "104» tube. 

14. Cap and shake vigorously. 

15. You should now have all of the 1A.1 culture tubes, five in total, filled with the soil/water mixture in them. 

16. plate 100 ul samples from up to the 10^-2 dilution onto their own correspondingly labeled 3M Petrifilm Yeast and Mold Count Plate using the micropipette and tips, making sure to use a new micropipette tip for each dilution. 

18. Repeat steps 4-19 for remaining soil samples (1A.1 - 4C.1) 

i. Reminder to use a new clean transfer pipette for every soil sample. It may be helpful to label the sleeve of the transfer pipette for whichever soil samples you are diluting at the time to avoid cross-contamination. You should end up with 13 total transfer pipettes after completing the dilutions. 12 for each soil sample and 1 for distilled water. 

ii. Wash the 1 cc scoop between samples to avoid cross contamination. 

iii. TIP: once finished reading through the directions and you fully understand them, it may be easiest to fill all of the culture tubes with distilled water before starting to add soil. 

19. Allow the plates to sit and grow for 48 to 72 hours in a dark place, with no direct sunlight. 

20. After the plates have been sitting for at least 48 hours, examine each of the plates for individual colonies of yeast and mold.

ii. For fungi: choose the plate with the fewest colonies (but at least 3 yeast and 1 mold) to make your estimates of the number of fungi in the original 1 cc of soil starting by examining the most dilute (10^-2) plate and moving up from there.

 21. To find the number of bacteria and fungi found in the original 1 cc soil sample, use the following formula: # microbes in 1 cc of soil = # colonies on sheet x 10^2 x 10 ^dilution # at which these colonies were found 

22. If there are not individual colonies but still a "lawn" at the 10 dilution, repeat the dilution adding a 5th (105) and 6th (10-5) dilutions, etc. as necessary until individual colonies are observed. 

23. Cleaning Procedures:

 i. Make sure to alcohol any surfaces that came into contact with the petrifilm plates and dispose of alcohol paper towels in the flammable trash can. 

ii. Dump out soil/water mixture from culture tubes into and bucket and then dump it down the big sink. Regular lab sinks can be clogged by too much dirt. 

iii. Wash out culture tubes with water and alcohol then set out to dry and reuse in the future. 

iv. Dispose of any petrifilm plates in the autoclave.

21. Repeat for every day's collection of samples. (i.e. 1A.2-4C.2 and 1A.3-4C.3)