TITLE: Harvesting and stocking cheese rind community samples
AUTHORS: Emily C.P. Weiss

[DESCRIPTION]
This protocol describes how to harvest and stock cheese rind microbial community samples for a variety of purposes, including spatial imaging, metagenomics, metatranscriptomics, microbial isolation/culturing, and bacteriophage extraction. Processing one small cheese wheel for all of these purposes took about 45 minutes in our hands.

[STEPS]
SECTION: Rind scraping
6. 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: Rind scraping
7. After removing the desired amount of rind, use a sterile wooden dowel or a pipette tip to homogenize the harvested rind.

 

SECTION: Preparing glycerol stocks of full community
13. Prepare a solution of 1× phosphate-buffered saline with 20% glycerol. Filter sterilize with a 0.22 μm filter.
SECTION: Preparing glycerol stocks of full community
16.  Use a sterile wooden dowel to help break up the rind, which may be sticky, into smaller pieces. 
 

SECTION: Preparing glycerol stocks of full community
14. Add 2 mL of 1× phosphate-buffered saline with 20% glycerol to a 2 mL cryotube.
SECTION: Preparing glycerol stocks of full community
17. Vortex for at least 20 s to homogenize the mixture.
SECTION: Preparing glycerol stocks of full community
18. To three new labeled empty cryotubes, distribute 500 μL of the mixture to create a total of four 500 μL glycerol stocks. 
 

SECTION: Preparing glycerol stocks of full community
15. Add ~200 mg of rind into the 2 mL cryotube containing 2 mL of 1× phosphate-buffered saline with 20% glycerol.


SECTION: Preparing glycerol stocks of full community
19. Store the glycerol stocks at −80 °C. These can be used later for microbial isolation.
SECTION: Sampling for spatial imaging
3. Using a cheese lyre, knife, or other suitable cutting instrument, cut off a corner or other small section of the cheese to reveal a clean profile view of the cheese rind biofilm and cheese paste. 

 
  

SECTION: Sampling for metatranscriptomics
8. Add ~200 mg of rind mixture to 1 mL of RNAprotect.   

 

SECTION: Sampling for spatial imaging
4. Take a scalpel and start from the top of the cheese to cut out a small section of the rind plus paste (around 0.5 in × 0.5 in), taking care not to disturb the structure of the rind. If using a cheese core, remove most of the paste and then section.

  

SECTION: Sampling for spatial imaging
5. Store the cheese sections at −80 °C for later imaging.

 

SECTION: Sampling for metatranscriptomics
9. Thoroughly resuspend the rind in the RNAprotect.
SECTION: Sampling for metatranscriptomics
10. Centrifuge at 4 °C for two minutes at 8000 rpm to pellet cells. 

 


SECTION: Sampling for metatranscriptomics
11. Discard the supernatant and snap freeze the sample in liquid nitrogen or a dry ice–ethanol bath.
SECTION: Sampling for metatranscriptomics
12. Store the frozen cell pellets at −80 °C until RNA extraction.
SECTION: Equipment setup
1. Cool a centrifuge that can hold 1.5 mL tubes to 4 °C.

SECTION: Phage and other miscellaneous sample storage
20. If sampling the viral population, add ~200 mg of rind (or more) to a 50 mL conical tube and store at 4 °C until phage extraction.
SECTION: Phage and other miscellaneous sample storage
21. Split the remainder of the harvested rind into 1.5 mL tubes and store at −80 °C for metagenomic DNA extraction and any other desired analyses.

 

SECTION: Equipment setup
2. Have liquid nitrogen or a dry ice–ethanol bath ready for snap freezing samples.