Ask The Brewmasters

Hello everyone,

Need some help with identification of growth on a WLD agar plate. Some back ground, Were a small brewery that has been can packaging for about 1 year. We had recently experienced the dreaded "exploding can phenomenon" and quickly realized we had a major issue. We suspected wild yeast causing secondary fermentation, however, after sending off samples of product from both the "canned bombs" and a sample from another batch in production, we found out it was ​​​S. cerevisiae var. 

Since our discovery of diastatic yeast we have completely changed our CIP/SIP procedures and to date have not had an issue (knock-on-wood). I have upped my lab game and test every point in our process within my means to ensure a clean fermentation. This brings me to the reason for this post. During a recent brew we transferred a small amount of the finished wort to a pilot FV so we could try some variation (yeast strain/dry hops) to a core beer we make and plated with WLD at various points from the HX to FV. The plates from the main batch has no growth, but the plate taken from the zwickel of the pilot FV has two micros growing. From what I have researched I know cyclohexamide inhibits most yeast growth and is mainly a selective media for bacteria, but does the white colonies on the plate indicate yeast and if so could it be diastatic yeast?

I hate asking this question because it shows how naïve I am at all this but I have come to a loss at finding the answers through research alone. I thank everyone in advance who takes the time to respond. 

PS. for those wondering, our pilot FV CIP/SIP regime was not changed and this was the first time the FV was used since prior to the discovery of diastatic contamination. We took for granted the vessel was clean and just did a simple sani rinse. Totally my fault for the oversight.     

------------------------------
Troy Rassmussen
Brewer
Gaston Brewing Company
Faytteville NC
(910) 920-4071
------------------------------

Troy,

Having been through diastaticus hell I sympathize.  I keep several different diastatic and Brett strains around now as routine controls for our media, and I have not found any of either that will grow on our LMDA+chx.  I've read literature stating that Brett spp can grow in the presence of cycloheximide but I have trialed many different strains and I haven't found any that will grow against our chx dosing, which may be a little high at around 8ppm.  I would recommend doing a quick wet mount on a microscope and checking the morphology of both the green and white colonies.

All this said our current media solution for wild yeast is a very slightly modified Cupric Sulfate Starch Medium (CSSM) Agar recipe based on the paper by, I believe, Priess et al, which is itself a tweak on Laura Burns' modified LCSM.  I can't locate the paper at the moment so apologies if this is misattributed.  It is the absolute best media I have found yet as far as being specific for starch degrading wild yeast, both diastaticus and Brett, as well as some wild Pichia strains I've collected.  It needs to be made fresh each week, but if you have an autoclave and/or a lab capable of making media it is totally worth the hassle.  If you want any further depth feel free to message me.

------------------------------
Shawn Savuto
QA/QC Specialist
COOP Ale Works
Oklahoma City OK
(405) 842-2667
------------------------------

Original Message:
Sent: 05-30-2022 14:54
From: Troy Rassmussen
Subject: WLD agar plate growth, Diastatic yeast?

Hello everyone,

Need some help with identification of growth on a WLD agar plate. Some back ground, Were a small brewery that has been can packaging for about 1 year. We had recently experienced the dreaded "exploding can phenomenon" and quickly realized we had a major issue. We suspected wild yeast causing secondary fermentation, however, after sending off samples of product from both the "canned bombs" and a sample from another batch in production, we found out it was ​​​S. cerevisiae var.

Since our discovery of diastatic yeast we have completely changed our CIP/SIP procedures and to date have not had an issue (knock-on-wood). I have upped my lab game and test every point in our process within my means to ensure a clean fermentation. This brings me to the reason for this post. During a recent brew we transferred a small amount of the finished wort to a pilot FV so we could try some variation (yeast strain/dry hops) to a core beer we make and plated with WLD at various points from the HX to FV. The plates from the main batch has no growth, but the plate taken from the zwickel of the pilot FV has two micros growing. From what I have researched I know cyclohexamide inhibits most yeast growth and is mainly a selective media for bacteria, but does the white colonies on the plate indicate yeast and if so could it be diastatic yeast?

I hate asking this question because it shows how naïve I am at all this but I have come to a loss at finding the answers through research alone. I thank everyone in advance who takes the time to respond.

PS. for those wondering, our pilot FV CIP/SIP regime was not changed and this was the first time the FV was used since prior to the discovery of diastatic contamination. We took for granted the vessel was clean and just did a simple sani rinse. Totally my fault for the oversight.   

 

------------------------------
Troy Rassmussen
Brewer
Gaston Brewing Company
Faytteville NC
(910) 920-4071
------------------------------

Troy,

Please prepare a wet mount of both colony types and share photos. If you have gram staining capabilities, that could be useful in identifying the bacterial (most likely green) colonies. Sharing your Zwickel sample collection SOP would also help us diagnose the issue.

Cheers,

------------------------------
Jeremy Pryes
Founder & HeadBrewer
Pryes Brewing Company, LLC
Minneapolis MN
(612) 787-7937
------------------------------

Original Message:
Sent: 05-30-2022 14:54
From: Troy Rassmussen
Subject: WLD agar plate growth, Diastatic yeast?

Hello everyone,

Need some help with identification of growth on a WLD agar plate. Some back ground, Were a small brewery that has been can packaging for about 1 year. We had recently experienced the dreaded "exploding can phenomenon" and quickly realized we had a major issue. We suspected wild yeast causing secondary fermentation, however, after sending off samples of product from both the "canned bombs" and a sample from another batch in production, we found out it was ​​​S. cerevisiae var.

Since our discovery of diastatic yeast we have completely changed our CIP/SIP procedures and to date have not had an issue (knock-on-wood). I have upped my lab game and test every point in our process within my means to ensure a clean fermentation. This brings me to the reason for this post. During a recent brew we transferred a small amount of the finished wort to a pilot FV so we could try some variation (yeast strain/dry hops) to a core beer we make and plated with WLD at various points from the HX to FV. The plates from the main batch has no growth, but the plate taken from the zwickel of the pilot FV has two micros growing. From what I have researched I know cyclohexamide inhibits most yeast growth and is mainly a selective media for bacteria, but does the white colonies on the plate indicate yeast and if so could it be diastatic yeast?

I hate asking this question because it shows how naïve I am at all this but I have come to a loss at finding the answers through research alone. I thank everyone in advance who takes the time to respond.

PS. for those wondering, our pilot FV CIP/SIP regime was not changed and this was the first time the FV was used since prior to the discovery of diastatic contamination. We took for granted the vessel was clean and just did a simple sani rinse. Totally my fault for the oversight.   

 

------------------------------
Troy Rassmussen
Brewer
Gaston Brewing Company
Faytteville NC
(910) 920-4071
------------------------------

Hey Jeremy,

I'll get some photos of the wet mount for both sometime today. I'll gram stain them once I have them isolated on their respective plates and keep you posted with photos and such. As for our SOP to zwickel, we clean our zwickels after each use by disassembling them, cleaning, autoclave, reassemble and mount to the tank. We alcohol and flame the zwickel port prior to drawing the sample and use an autoclaved sample tube for sample collection. We wear gloves during the entire process and spray with alcohol pretty much anything around the sampling are to include our gloved hands. I have become paranoid since our diastatic issues and try to ensure everything is sanitized/sterilized as we can make it. Once the sample is collected I place it in my lab space (a stainless steel rolling desk) spray the surface with alcohol wiping excess away, then turn on the Bunsen burner to create an "aseptic" area to work. I ensure no fans are blowing the the area and work in accordance with aseptic technique. I mount the samples on the plates working near the flame at all times and ensure nothing is left open/uncovered longer than I need to for transfer. I use individually sealed (sterilized) pipettes or swabs as needed for the application and I flame the inoculation loop between specimen samples, if that's the tool I'm using. 

I believe our collection method is sound, baring any potential airborne contamination, which given the length of time the sample is exposed to the atmosphere should be very low. Let me know if you see any issues with it and I'll post an update with photos when I get them.

Thanks for your insight!

Cheers

------------------------------
Troy Rassmussen
Brewer
Gaston Brewing Company
Faytteville NC
(910) 920-4071
------------------------------

Original Message:
Sent: 05-31-2022 11:38
From: Jeremy Pryes
Subject: WLD agar plate growth, Diastatic yeast?

Troy,

Please prepare a wet mount of both colony types and share photos. If you have gram staining capabilities, that could be useful in identifying the bacterial (most likely green) colonies. Sharing your Zwickel sample collection SOP would also help us diagnose the issue.

Cheers,

------------------------------
Jeremy Pryes
Founder & HeadBrewer
Pryes Brewing Company, LLC
Minneapolis MN
(612) 787-7937

Original Message:
Sent: 05-30-2022 14:54
From: Troy Rassmussen
Subject: WLD agar plate growth, Diastatic yeast?

Hello everyone,

Need some help with identification of growth on a WLD agar plate. Some back ground, Were a small brewery that has been can packaging for about 1 year. We had recently experienced the dreaded "exploding can phenomenon" and quickly realized we had a major issue. We suspected wild yeast causing secondary fermentation, however, after sending off samples of product from both the "canned bombs" and a sample from another batch in production, we found out it was ​​​S. cerevisiae var.

Since our discovery of diastatic yeast we have completely changed our CIP/SIP procedures and to date have not had an issue (knock-on-wood). I have upped my lab game and test every point in our process within my means to ensure a clean fermentation. This brings me to the reason for this post. During a recent brew we transferred a small amount of the finished wort to a pilot FV so we could try some variation (yeast strain/dry hops) to a core beer we make and plated with WLD at various points from the HX to FV. The plates from the main batch has no growth, but the plate taken from the zwickel of the pilot FV has two micros growing. From what I have researched I know cyclohexamide inhibits most yeast growth and is mainly a selective media for bacteria, but does the white colonies on the plate indicate yeast and if so could it be diastatic yeast?

I hate asking this question because it shows how naïve I am at all this but I have come to a loss at finding the answers through research alone. I thank everyone in advance who takes the time to respond.

PS. for those wondering, our pilot FV CIP/SIP regime was not changed and this was the first time the FV was used since prior to the discovery of diastatic contamination. We took for granted the vessel was clean and just did a simple sani rinse. Totally my fault for the oversight.   

 

------------------------------
Troy Rassmussen
Brewer
Gaston Brewing Company
Faytteville NC
(910) 920-4071
------------------------------