After my first experiment plating dregs from commercial bottles on culture media containing Bromocresol green failed, I decided to try another plating media. The goal of these experiments is to isolate and grow up different strains of wild yeast from commercial bottles in the lab and use them for homebrewing. Instead of MYPG plates I decided to make WLN and WLD media that can potentially differentiate between non-wild and wild yeast. WLN stands for Wallerstein Nutrient Broth and is used for detecting both bacteria and yeast. WLD stands for Wallerstein Differential Broth and contains 10 mg/L of cycloheximide, a reversible inhibitor of protein synthesis in eukaryotes (this includes yeast). Interestingly, we routinely use this drug in the lab for biomedical research and it potently kills mammalian cells of any type. Surprisingly, wild yeast (which are eukaryotes) are resistant to cycloheximide.
There weren’t many recipe sources for WLN media as this is usually pre-made at a large company and ordered. However, I did find the recipe from Condalab, a company that provides microbiology culture media. Unfortunately, some of the ingredients will be unavailable to homebrewers and plates made with malt extract (MYPG) would have to suffice. The recipe for 1 liter:
- 50 g of dextrose
- 5.0 g of tryptone
- 4.0 g of yeast extract
- 0.55 g of Monopotassium phosphate
- 0.425 g of Potassium chloride
- 0.125 g of Calcium chloride
- 0.125 g of Magnesium sulfate
- 0.0025 g of Ferric chloride
- 0.0025 g of Mangenese sulfate
- 15.0 g of agar.
–- Wyeast 1056 (negative control – should be no growth on WLD)
- Wyeast Brett B (positive control – should grow on WLN and WLD)
- Wyeast Brett L (another positive control)
- Russian River Temptation dregs (experimental sample)
I’m really loving your blog. Very interesting. Keep up the good work!
Thanks Rob! Keep checking back for more results.
So jealous of your lab and the ability to experiment with wild yeast.
And great job at explaining what you are doing so a person with little organic lab experience can understanding your process.
Thanks!
Actually, I think some of the procedures can be done at home, one just needs to invest in some basic microbiology equipment like petri dishes, flame for sterilizing, etc… The WLN/WLD media might be hard to make at home but MYPG plates are doable especially if you use a pressure cooker for sterilizing.
There are some books out there about creating a yeast lab in your home. I may have to point this out in a future post.
J
Jason,
Not sure why the MYPG plates didn’t work. When making my own WL plates, I use a slightly different recipe, which adds Bromocresol green (22 mg/L). I’m also guessing that your cycloheximide needs to be at least 20 mg/L to inhibit Sacchromyces – I’ve normally used 50 mg/L.
Good luck!
Hi Michael,
It’s not that the bromocresol green didn’t totally work – it did somewhat. The amount of acid produced by the wild yeast buffered the dye very well. However, I think the large proportion of normal Sacch. was so much more than any wild yeast that it might have overgrown the plate.
Actually, 10 mg/L works pretty darn well – I’ll post it in the future with some photos.
Wow… you really are pushing the beer geekyness on this one… love it!
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Hi Jason,
First of all, great blog. Learning a ton of great lab/microbio stuff.
I have a question about cycloheximide usage. Since its pretty toxic to humans, would you recommend pitching brett from a petri that had cycloheximide in it? I would be concerned for even trace amounts of toxins that could end up in my beer.
You are correct, it is pretty toxic since it blocks protein synthesis. However, I never use it in preparation for a batch of beer. I only use it on plates to select for wild strains and one can easily streak one colony onto another plate that has no drug. Basically, its just for quantitation purposes and not used for consumption.
Any suggestions for a source of cycloheximide? There are places to order it online, but they require it be shipped to a business address and they charge a HazMat fee. I could have it shipped to my office, but it’s crazy to pay over $50 in shipping charges for 1gm. Any suggestions would be greatly appreciated.
Hey Nathan, sorry for the late reply. I sent you an email.