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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 

This message was posted by a user wishing to remain anonymous

I will bring up the Copper Sulphate Solution to 500mg/l and the sample volume down for the Conditioning Stage. Should this not suppress Culture Yeast I will aseptically make up a stock dilution of the Culture Yeast to confirm its Copper tolerance. The strain being used is AY3 from AEB. 

Original Message:
Sent: 12-13-2024 13:36
From: Cullen Dwyer
Subject: Copper Sulphate Yeasts and Moulds

Hey Sean,

Are you getting TNTC yeast colonies on your bright tank samples, or just conditioning tanks?  You want to put about 1 million cells on the plate, so I would think that 3K cells/ml x 100ml = 300,000 cells would be fine.  But 20 million cells would be too much for both your membrane filtration and the cupric sulfate medium.  For the conditioning tank, I would skip the membrane and just spread 0.25ml directly onto the agar.

If you want to dig a little deeper, you could easily prepare serial dilutions of a known concentration of culture yeast.  Direct-plate them and check your rate of recovery, and see how many/few cells you can put on the plate to get negative results.

If you are getting yeast colonies forming with less than 1 million cells on the plate, you may need to try a different concentration of cupric sulfate.  At 325mg/liter, Lin still got about 15% growth of culture yeast; only 500mg/liter totally suppressed it (Lin, J. Inst. Brew 1981, Vol 87, pp 151-154).  Also, as you said, your culture strain could be a little "wild", and resistant to certain suppressants.  Your yeast supplier may have some insight and advice on this.

Cullen Dwyer
Cellarman-Brewing-QA

Finestkind Brewing, LLC

 

chris.hamer@finestkindbrewing.com">Cullen@wachusettbrew.com">Cullen@wachusettbrew.com

o: (978) 874-9965 x1160

c: (505) 795-3599

 

175 State Road East

Westminster, MA 01473

Makers of

Smuttynose Brewing Co. & Restaurant

Five Boroughs Brewing Co.

Island District Cocktails

Wachusett Brewing Co.

 

 

For clarity, is 325mg/L of Cu the final media concentration or the concentration of the Cu solution before adding it to the YM?  If that is the final media concentration, English ale yeast can grow at the ppm you've indicated, especially if the cell density is higher than recommended.  Raising the final media concentration to 400-500mg/L of Cu should inhibit the base English yeast, however, if not, you could try increasing it further but you will undoubtedly miss out on some of the lower threshold wild yeasts that grow as low as 200mg/L.  If you continue to see growth, a simple test you can perform is a colony pick subculture into sterile wort to determine if it's your base yeast.  

------------------------------
Kory Davis
QC and Propagation Specialist
The Brewing Science Institute
Woodland Park, Co
719-482-4895 ext. 3
------------------------------

Original Message:
Sent: 12-16-2024 10:36
From: Anonymous Member
Subject: Copper Sulphate Yeasts and Moulds

This message was posted by a user wishing to remain anonymous

I will bring up the Copper Sulphate Solution to 500mg/l and the sample volume down for the Conditioning Stage. Should this not suppress Culture Yeast I will aseptically make up a stock dilution of the Culture Yeast to confirm its Copper tolerance. The strain being used is AY3 from AEB. 

Original Message:
Sent: 12-13-2024 13:36
From: Cullen Dwyer
Subject: Copper Sulphate Yeasts and Moulds

Hey Sean,

Are you getting TNTC yeast colonies on your bright tank samples, or just conditioning tanks?  You want to put about 1 million cells on the plate, so I would think that 3K cells/ml x 100ml = 300,000 cells would be fine.  But 20 million cells would be too much for both your membrane filtration and the cupric sulfate medium.  For the conditioning tank, I would skip the membrane and just spread 0.25ml directly onto the agar.

If you want to dig a little deeper, you could easily prepare serial dilutions of a known concentration of culture yeast.  Direct-plate them and check your rate of recovery, and see how many/few cells you can put on the plate to get negative results.

If you are getting yeast colonies forming with less than 1 million cells on the plate, you may need to try a different concentration of cupric sulfate.  At 325mg/liter, Lin still got about 15% growth of culture yeast; only 500mg/liter totally suppressed it (Lin, J. Inst. Brew 1981, Vol 87, pp 151-154).  Also, as you said, your culture strain could be a little "wild", and resistant to certain suppressants.  Your yeast supplier may have some insight and advice on this.

Cullen Dwyer
Cellarman-Brewing-QA

Finestkind Brewing, LLC

 

chris.hamer@finestkindbrewing.com">chris.hamer@finestkindbrewing.com">Cullen@wachusettbrew.com">Cullen@wachusettbrew.com

o: (978) 874-9965 x1160

c: (505) 795-3599

 

175 State Road East

Westminster, MA 01473

Makers of

Smuttynose Brewing Co. & Restaurant

Five Boroughs Brewing Co.

Island District Cocktails

Wachusett Brewing Co.

 

 

Hi Sean,

  Your sampling density should be around 100k cells/plate.  If you are plating lagers, incubate them at 37C, as they have copper tolerance.  At the ppm you've indicated, some ales still exhibit copper tolerance.  Which strain are you having growth issues with?

------------------------------
Kory Davis
QC and Propagation Specialist
The Brewing Science Institute
Woodland Park, Co
719-482-4895 ext. 3
------------------------------

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.