Harvesting and stocking cheese rind community samples

Cool a centrifuge that can hold 1.5 mL tubes to 4 °C.

Have liquid nitrogen or a dry ice–ethanol bath ready for snap freezing samples.

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.

Note

If desired, remember to take a photo of the intact cheese prior to any sampling.You can store the sections of cheese that are cut off (rind and paste) at −80 °C in 50 mL conical tubes for mass spectrometry or other future analyses.If it is necessary to sample non-destructively from a larger cheese wheel, you can use a cheese trier to take a small core out of the cheese.

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.

Store the cheese sections at −80 °C for later imaging.

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.

After removing the desired amount of rind, use a sterile wooden dowel or a pipette tip to homogenize the harvested rind.

Add ~200 mg of rind mixture to 1 mL of RNAprotect.

Thoroughly resuspend the rind in the RNAprotect.

Centrifuge at 4 °C for two minutes at 8000 rpm to pellet cells.

Discard the supernatant and snap freeze the sample in liquid nitrogen or a dry ice–ethanol bath.

Store the frozen cell pellets at −80 °C until RNA extraction.

Prepare a solution of 1× phosphate-buffered saline with 20% glycerol. Filter sterilize with a 0.22 μm filter.

Add 2 mL of 1× phosphate-buffered saline with 20% glycerol to a 2 mL cryotube.

Add ~200 mg of rind into the 2 mL cryotube containing 2 mL of 1× phosphate-buffered saline with 20% glycerol.

Use a sterile wooden dowel to help break up the rind, which may be sticky, into smaller pieces.

Vortex for at least 20 s to homogenize the mixture.

To three new labeled empty cryotubes, distribute 500 μL of the mixture to create a total of four 500 μL glycerol stocks.

Store the glycerol stocks at −80 °C. These can be used later for microbial isolation.

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.

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.