Jun 06, 2023
Isolation of bacteria and fungi from cheese rind microbiomes
- 1Arcadia Science
Protocol Citation: Emily C.P. Weiss 2023. Isolation of bacteria and fungi from cheese rind microbiomes. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzb7b4vx1/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 19, 2023
Last Modified: January 29, 2024
Protocol Integer ID: 75598
Keywords: Bacteria, bacterial, fungus, fungi, microbes, isolate, isolates, cheese, microbial community, community, contaminant, contamination, separate, single, colony, colonies
Abstract
This protocol describes how to harvest and stock bacterial and fungal isolates from cheese rind microbial community samples.
Image Attribution
Arcadia Science
Materials
- 1.5 mL sterile Eppendorf tubes
- Sterile razor blade
- Sterile 96 well plates (flat bottom)
- Multi-channel pipette
- Solution reservoir (sterile)
- 1 mL and 200 μL sterile pipette tips
- Sterile toothpicks or wooden dowels
- Sterile plastic pestles
- Sterile 2 mL cryotubes
- Sterile 1× phosphate-buffered saline + 20% glycerol
- Sterile 1× phosphate-buffered saline + 0.05% Tween
- Sterile 80% glycerol
- LB broth
- LB agar plates with 100 μg/mL cycloheximide
- PCAMS plates with 50 μg/mL chloramphenicol
Recipe for 1 L of PCAMS without antibiotic:
5 g tryptone
2.5 g yeast extract
1 g dextrose
1 g whole milk powder
10 g sodium chloride
15 g agar
965.5 mL deionized water
Harvesting the cheese rind biofilm
Harvesting the cheese rind biofilm
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.
Note
This protocol is focused on obtaining pure bacterial and fungal isolates. For a more detailed protocol on sampling and stocking cheese rind communities (for metatranscriptomics, metagenomics, long-term storage, etc.), see https://www.protocols.io/view/harvesting-and-stocking-cheese-rind-community-samp-ckf2utqe.
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.
Note
If the cheese surface contains nooks/crannies, use the corner of the razor blade to gently scrape the rind from these.
After removing the desired amount of rind, use a sterile wooden dowel or a pipette tip to homogenize the harvested rind.
Note
If not harvesting the entire rind, scraping from multiple locations may help to capture more of the microbial diversity present.
Resuspension and dilution
Resuspension and dilution
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.
Using a small sterile pestle, gently grind the cheese rind sample until it is completely resuspended in the buffer.
Equipment
Disposable Pellet Pestles
NAME
Fisher Scientific
BRAND
12-141-364
SKU
https://www.fishersci.com/shop/products/fisherbrand-rnase-free-disposable-pellet-pestles-2/p-4246531
LINK
Add another 500 μL of PBS + 0.05% Tween to the suspension.
Vortex thoroughly to mix the suspension.
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.
Note
You can do these serial dilutions in a sterile 96-well plate. If you have multiple samples, you can use a multichannel pipette after the initial dilution to serially dilute all samples at once. If plating for both bacteria and fungi, you may want a minimum of 250 μL of each dilution.
Bacterial isolation
Bacterial isolation
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.
Note
When working with new communities, it may take some trial and error to identify the correct dilution to plate for getting single colonies. These recommendations are based on our past experience.
Cycloheximide is an antifungal. It is not always effective against Galactomyces. If trying to remove Galactomyces fungi, you can add 21.6 μg/mL natamycin.
Leave the plates at room temperature until you can see individual bacterial colonies on at least one of the dilution plates.
Note
In our experience with cheese communities, this may take around 3–5 days. Over time, pigments will start to develop in some colonies, which will make identification of different morphologies easier. Leaving the plates exposed to light (e.g. on the bench top) can help encourage pigment production.
Bacterial colonies from a cheese rind community
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.
Leave at room temperature until single colonies appear.
Note
You should store plates inside a plastic bag with a moistened paper towel to prevent them from drying out.
Using a sterile toothpick or other sterile instrument, gently touch a single pure individual colony and inoculate into 2 mL LB broth.
Shake the culture at 200 rpm at room temperature until cloudy.
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.
Repeat this process until all desired colonies from the original community plate have been stocked.
Note
We usually try to get at least one representative from each unique morphology, but this will depend on your downstream purpose.
Fungal isolation
Fungal isolation
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.
Note
When working with new communities, it may take some trial and error to identify the correct dilution to plate for getting single colonies. These recommendations are based on our past experience.
Chloramphenicol is an antibacterial drug.
Leave the plates at room temperature until you can see individual fungal colonies on at least one of the dilution plates.
Note
In our experience with cheese communities, this may take around 3–7 days for fungi. Morphologies may become more distinguishable over time as more color develops.
Fungal colonies from a cheese rind community
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.
Repeat as many times as needed onto a new plate to get a pure single colony.
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.
Note
You should store plates inside a plastic bag with a moistened paper towel to prevent them from drying out.
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.
Use a 1 mL pipette to collect the liquid with the resuspended spores. Vortex to mix thoroughly.
Transfer the liquid to a labeled 2 mL cryotube and store at −80 °C.
Repeat this process until all desired colonies from the original community plate have been stocked.
Note
We usually try to get at least one representative from each unique morphology, but this will depend on your downstream purpose.