This message was posted by a user wishing to remain anonymous
Wild yeast is an ambiguous term sometimes... You are testing for copper resistance, so keep in mind that not all wild yeast have copper resistance, and not all brewer's yeasts are inhibited by copper.
1) Just take a small sample (aseptically) from your next yeast purchase and plate it on the media (~200uL on 100mm petri dish, spread plate) to see if your brewing strain has copper resistance at the dosing that you are using in your preparation. If there is full, raised growth, you have a strain that is resistant, if you have a few distinct colonies your yeast supplier may have contamination in their lines, and if you see no growth then it is not resistant. From a thick yeast slurry, you may see some flat residue of the plated sample on the surface when you read them- this is not new growth. You do not need to rely on your supplier for this info.
2) I believe LCSM is at ~0.05% copper sulfate anhydrous (or 500 mg/L). I'd recommend looking at the original research paper (Lin, J. Inst. Brew, 1981) if you are trying to mimic this product.
3) Cycloheximide with a range of 5-20ppm can also be a technique used to identify cycloheximide resistance, common in some "wild yeast". Resistance varies from weak (5ppm) to very strong (20+ ppm). This will inhibit most brewer's yeast and is often used to remove background noise when plating beer with residual brewing yeast.
4) If you are still getting TNTC results, I would recommend trying some direct plating techniques like a pour plate (you can prepare media at a higher ratio of powder to liquid to account for liquid added from the sample). There's no point filtering 100mL if you are never able to distinguish the contamination.
Original Message:
Sent: 12-13-2024 05:56
From: Anonymous Member
Subject: Copper Sulphate Yeasts and Moulds
This message was posted by a user wishing to remain anonymous
Good day all, our Lab team are currently using 325mg/l Copper Sulphate innoculated into Yeast & Moulds Agar to detect Wild Yeasts. The Copper Sulphate is made up, then autoclaved and added to the Autoclaved Media at about 70 degrees before the plates are poured below 60 degrees c.
Our Bright Tanks hold a cell density of 3,000 cells per ml after filtration. And 210,000 per ml in conditioning before filtration.
Our current method for pre and post-filtration is to filter 100ml of sample onto Membrane Filtration membranes along with WL and Raka Ray plates. Culture Yeast is growing on both plates most of the time to TNTC levels.
Would the cell density above be too high for the sample size at this stage of production? To cause the Yeast growing despite the inclusion of Copper?
Or is it that the Copper Tolerance is high in the strain? The Yeast supplier hasn't been able to tell us whether or not this strain is resistant to Copper.
I am proposing reducing the Y&M +Cu sample down to 10mls to "see the wood from the trees" so to speak. Any guidance / experience to share on this?
Regards, Seán