TITLE: Isolation of bacteria and fungi from cheese rind microbiomes
AUTHORS: Emily C.P. Weiss

[DESCRIPTION]
This protocol describes how to harvest and stock bacterial and fungal isolates from cheese rind microbial community samples.

[STEPS]
SECTION: Harvesting the cheese rind biofilm
2. Holding the cheese in one (gloved) hand and a sterile razor blade in the other, gently scrape the surface of the cheese to remove the rind biofilm. Avoid applying too much pressure, as you will start to dig into the paste. Scrape the rind biofilm into a weigh boat or other container to clean off the blade as needed.

 

SECTION: Harvesting the cheese rind biofilm
3. After removing the desired amount of rind, use a sterile wooden dowel or a pipette tip to homogenize the harvested rind.

 

SECTION: Resuspension and dilution
4. Add a lentil-sized amount of harvested cheese rind biofilm to 500 μL of sterile 1× phosphate-buffered saline (PBS) + 0.05% Tween in a 1.5 mL microcentrifuge tube.
SECTION: Resuspension and dilution
5. Using a small sterile pestle, gently grind the cheese rind sample until it is completely resuspended in the buffer. 
 

SECTION: Resuspension and dilution
6. Add another 500 μL of PBS + 0.05% Tween to the suspension.
SECTION: Resuspension and dilution
7. Vortex thoroughly to mix the suspension.
SECTION: Harvesting the cheese rind biofilm
1. If you are starting from a glycerol stock of a previously stored rind community, proceed directly to the dilutions in step 8. If starting from an intact cheese, start at step 2.

 

SECTION: Resuspension and dilution
8. Make 10-fold serial dilutions of the cheese rind suspension eight times in PBS + 0.05% Tween to create 10-1, 10-2, 10-3, 10-4, 10-5, 10-6, 10-7, 10-8 dilutions.

 

SECTION: Bacterial isolation
9. Use four LB + 100 μg/mL cycloheximide agar plates to plate 100 μL each of the 10-5, 10-6, 10-7, 10-8 dilutions from step 8.

 

SECTION: Bacterial isolation
10. Leave the plates at room temperature until you can see individual bacterial colonies on at least one of the dilution plates.

 
 
  

SECTION: Bacterial isolation
11. Using a sterile toothpick or other sterile instrument, gently touch a single individual colony and quadrant-streak onto a fresh LB + 100 μg/mL cycloheximide agar plate.
SECTION: Bacterial isolation
12. Leave at room temperature until single colonies appear.

 

SECTION: Bacterial isolation
13. Using a sterile toothpick or other sterile instrument, gently touch a single pure individual colony and inoculate into 2 mL LB broth. 
SECTION: Bacterial isolation
14. Shake the culture at 200 rpm at room temperature until cloudy.
SECTION: Bacterial isolation
15. Add 250 μL of culture to 750 μL of sterile 80% glycerol in a labeled 2 mL cryotube, invert to mix well, and immediately store tube at −80 °C.
SECTION: Bacterial isolation
16. Repeat this process until all desired colonies from the original community plate have been stocked.

 

SECTION: Fungal isolation
17. Use four PCAMS + 50 μg/mL chloramphenicol agar plates to plate 100 μL each of the 10-1, 10-2, 10-3, 10-4 dilutions from step 8.

 

SECTION: Fungal isolation
18. Leave the plates at room temperature until you can see individual fungal colonies on at least one of the dilution plates.

 
  

SECTION: Fungal isolation
19. Using a sterile toothpick or other sterile instrument, gently touch a single individual colony and quadrant streak onto a fresh PCAMS + 50 μg/mL chloramphenicol agar plate. 
SECTION: Fungal isolation
20. Repeat as many times as needed onto a new plate to get a pure single colony.
SECTION: Fungal isolation
21. From a pure single colony, use a sterile toothpick to pick up fungal spores and then streak the spores in horizontal lines back and forth across a new full plate. Then turn the plate 90° and streak in horizontal lines back and forth across the plate. The goal is to spread the fungus evenly over the surface of the plate.

 

SECTION: Fungal isolation
22. Once the fungus is grown and sporulated (~5–7 days), add 1 mL of sterile PBS with 20% glycerol to the plate. Use a cell scraper to gently scrape the surface of the plate to collect the fungal spores into the liquid. 
SECTION: Fungal isolation
23. Use a 1 mL pipette to collect the liquid with the resuspended spores. Vortex to mix thoroughly.
SECTION: Fungal isolation
24. Transfer the liquid to a labeled 2 mL cryotube and store at −80 °C.
SECTION: Fungal isolation
25. Repeat this process until all desired colonies from the original community plate have been stocked.