NeurosporaCrassa

Procedure Number One

The first procedure of this experiment will involve attempting to reproduce the data obtained from my Genetics experiment. If the data is not reproducible, then it is worthless.

*Procedure
Step 1. Using a sterile loop, transfer conidia from Neurospora crassa stock cultures, albino and wild-type, to slant tubes of minimal agar. Label tubes “a” (albino) and “o” (orange, wild-type).
Step 2. Incubate at 35 degrees C for two days
Step 3. Place the tubes under fluorescent light for two to three days to promote conidial growth
Step 4. Using a moistened sterile swab, collect conidia from the two strains and place in separate test tubes containing 10 mL of sterile distilled water.
Step 5. Using a sterile funnel and receptacle, filter the conidial suspensions to assure that no mycelium were transferred
Step 6. Using a hemacytometer (microscope slide), count Neurospora conidia and adjust the volume of water added to each experimental flask.
Step 7. Transfer 2 mL of the conidial suspension from the albino culture into each of three sterile flasks containing 35 mL of sterile liquid minimal media. Also transfer 2 mL of the wild-type in the same manner. These are the six experimental flasks.
Step 8. Add 2 mL of sterile distilled water to each of three sterile flasks containing sterile liquid minimal media. This is to ensure that all flasks contain the same volume of liquid. These are the three control flasks.
Step 9. Record the initial optical rotation of the minimal media (with and without sucrose)
Step 10. After 24 hours, using a sterile pipette, remove 5 mL of media from each of the nine flasks
Step 11. Filter each sample of minimal media form experimental flasks through a Pasteur pipette to ensure that conidia do not enter the polarimeter tube. Analyze 1 mL of media using Perkin Elmer Automatic Digital Polarimeter model 342
Step 12. Perform a Benedict’s Test on all nine samples using water as a negative control and glucose as a positive control
a. Add 3 mL of Benedict’s solution to 1 mL of each test tube containing minimal media
b. Heat the test tube in a boiling water bath for two to three minutes
Step 13. Repeat steps 10-12 after 48 hours
Step 14. Place labeled filter paper in the oven at 80 degrees C to drive off moisture.
Step 15. Weigh filter paper to the nearest 0.001 g
Step 16. Suction filter the six Neurospora cultures onto the filter paper
Step 17. Place six filter papers into the oven at 80 degrees C overnight
Step 18. Weigh the filter paper plus Neurospora to the nearest 0.001 g

*Media preparation and sterilization requirements
• Minimal agar, 2 x 8 mL, screw-cap slant tubes
~author's edit--slant tubes with cotton plugs (required for Neurospora to get oxygen
• Cotton applicators, 8
• Distilled water, 4 x 10 mL, white-capped standard tubes
• Conidial funnels, 2
• Erlenmeyers, 2
• Pasteur pipettes, 2
• 2 mL pipettes, 10
• 5 mL pipettes, 10 x 2
• Liquid minimal, 9 x 35 mL (125 mL Erlenmeyers with cotton plugs)
• Liquid minimal, 5 mL, white-capped standard test tube
• Liquid minimal, no sucrose, 5 mL, white-capped standard test tube

*Schedule of days
Wednesday
• Prepare and sterilize minimal agar tubes
Thursday
• Transfer conidia from Neurospora cultures using a sterile loop
• Incubate for two days (Thursday and Friday) at 35oC
Saturday
• Place tubes under fluorescent light for three days (Saturday through Monday)
Tuesday
• Prepare and sterilize remaining media and equipment
• Complete steps 4-9
Wednesday
• Complete steps 10-12
Thursday
• Complete steps 13-17
Friday
• Complete step 18