Cell Counting and viability testing - Home Brew Forums

Register Now!
Home Brew Forums > Home Brewing Beer > Fermentation & Yeast > Cell Counting and viability testing

Reply
 
Thread Tools
Old 08-05-2013, 05:28 PM   #1
dstar26t
If it's worth doing, it's worth overdoing
HBT_SUPPORTER.png
Recipes 
 
Jan 2008
Ridley Park, PA
Posts: 1,151
Liked 57 Times on 42 Posts



In the Yeast book, it says to use a .5% solution of sulfuric acid to combat flocculation when looking at a sample under a microscope. I'm typically looking at how a starter is doing on the stir plate after 24 hours so my dilution is usually around 5:1. Some strains will still clump so I'm wondering if a stronger sulfuric acid solution is necessary. Also, I've come across some info lately claiming that cells don't retain the dye for checking viability when the diluent is an acid solution. Is this true? If so, what acid concentration is too high for checking viability?

Nate
__________________
Next: ?
Fermenting: Azacca Oat Pilsner, Egregious-ish, Barrel Fermented Dreg DelcoLambic, Sacch Trois Helles
Drinking: RIS v7, Doppelbock, RIS v6, Carrot Blossom Cedar Mead, Sauerkraut Fermented Gose, Gueuze, Brett Blonde, Kriek, Saison, Sour Blonde
Barrel aged: Rye Barleywine, Tripel, Wee-Heavy

 
Reply With Quote
Old 08-08-2013, 11:36 AM   #2
dstar26t
If it's worth doing, it's worth overdoing
HBT_SUPPORTER.png
Recipes 
 
Jan 2008
Ridley Park, PA
Posts: 1,151
Liked 57 Times on 42 Posts


After some research and an e-mail to White Labs, it seems that the .5% sulfuric acid solution that is recommended in the Yeast book can give erroneous viability results when using dye. Especially if you increase the strength of the solution to get better deflocculation for more clumpy strains. Apparently, the more preferred method is to use disodium EDTA. The Yeast book recommends a 10% w/w solution and also that you centrifuge the slurry first. One of the lab people at White Labs assured me that centrifuging isn't necessary. The EDTA has to be dissolved in water at a pH of 8.0 so the common practice is to add NaOH to raise the pH enough for the EDTA to dissolve.

I recently got a vial of WLP860 which was 4 months old and I wanted to grow it up for a 12 gallon batch of Helles. After putting it in a 300mL starter for 48 hours, there were only 82 Billion cells and they were 95% viable using sulfuric acid as the diluent and Alkaline Methylene Violet as the stain. This seemed impossible which got me concerned about my process. Hopefully the EDTA is a step in the right direction.
__________________
Next: ?
Fermenting: Azacca Oat Pilsner, Egregious-ish, Barrel Fermented Dreg DelcoLambic, Sacch Trois Helles
Drinking: RIS v7, Doppelbock, RIS v6, Carrot Blossom Cedar Mead, Sauerkraut Fermented Gose, Gueuze, Brett Blonde, Kriek, Saison, Sour Blonde
Barrel aged: Rye Barleywine, Tripel, Wee-Heavy

 
Reply With Quote
Old 05-08-2014, 02:39 PM   #3
dstar26t
If it's worth doing, it's worth overdoing
HBT_SUPPORTER.png
Recipes 
 
Jan 2008
Ridley Park, PA
Posts: 1,151
Liked 57 Times on 42 Posts


So I finally got around to making up an EDTA solution. Added 50g EDTA to 400mL of distilled water. Put on stirplate and added NaOH until the pH rose to 8.0 and the EDTA dissolved. Then topped up to 500mL.

I transferred 11.5 gallons of Pre-Prohibition Pilsner fermented with Wyeast 2124 for 3 weeks. Added 1L of sterile water to the fermenter and swirled. Removed and saved 1L of that slurry and kept it cold, dumped the rest. All break material was removed from the wort before pitching so it was pretty clean yeast. After 5 days, the slurry was shaken to resuspend everything and a 1mL sample was pulled (lost about 150mL due to foaming while shaking). The 1mL sample was diluted with 39mL of the EDTA solution and a 1 mL sample of that was pulled and added to 1mL Alkaline Methylene Violet from White Labs. Total dilution is 80:1. Counted 1,248 Billion cells and 80% viability. I know Lager yeast doesn't clump as much as say an English strain but this was the first successful slurry count I've done for repitching. Just thought I'd share.

The issues before have been too thick of a slurry that the pipette couldn't pull from, not enough AMV, poor cell distribution and unrealistic viability. The EDTA and AMV worked great.
__________________
Next: ?
Fermenting: Azacca Oat Pilsner, Egregious-ish, Barrel Fermented Dreg DelcoLambic, Sacch Trois Helles
Drinking: RIS v7, Doppelbock, RIS v6, Carrot Blossom Cedar Mead, Sauerkraut Fermented Gose, Gueuze, Brett Blonde, Kriek, Saison, Sour Blonde
Barrel aged: Rye Barleywine, Tripel, Wee-Heavy

 
Reply With Quote
Old 05-08-2014, 04:35 PM   #4
dbals
Recipes 
 
Jan 2010
Chicago
Posts: 127
Liked 7 Times on 5 Posts


If you have problems with staining clumping yeast you might wanna try another stain. Methylene Blue stain does not stain clumps well, Safranin stain doesn't seem to have that problem.

http://bkyeast.wordpress.com/2013/01...life-vs-death/

 
Reply With Quote
Old 05-08-2014, 05:04 PM   #5
dstar26t
If it's worth doing, it's worth overdoing
HBT_SUPPORTER.png
Recipes 
 
Jan 2008
Ridley Park, PA
Posts: 1,151
Liked 57 Times on 42 Posts


I'm using alkaline methylene violet, works great. I would never use Methylene Blue for saved slurry since I've read it's not accurate under 90% viability. The clumping was an issue with cell distribution, not staining. Thanks for the link. I wonder how AMV compares to Trypan blue for checking Brett?
__________________
Next: ?
Fermenting: Azacca Oat Pilsner, Egregious-ish, Barrel Fermented Dreg DelcoLambic, Sacch Trois Helles
Drinking: RIS v7, Doppelbock, RIS v6, Carrot Blossom Cedar Mead, Sauerkraut Fermented Gose, Gueuze, Brett Blonde, Kriek, Saison, Sour Blonde
Barrel aged: Rye Barleywine, Tripel, Wee-Heavy

 
Reply With Quote
Reply
Thread Tools


Similar Threads
Thread Thread Starter Forum Replies Last Post
Enough for cell counting? rgcalsaverini Fermentation & Yeast 14 05-09-2013 08:01 PM
Cell counting - anyone do it? jat147 Fermentation & Yeast 4 03-20-2013 08:02 PM
Rinsed yeast CELL COUNTING / VIABILITY laserghost Fermentation & Yeast 2 02-06-2013 05:50 PM
Lab testing for slurry cell counts and viability WoodlandBrew Fermentation & Yeast 8 12-19-2012 01:37 PM
Testing yeast viability Chizult Beginners Beer Brewing Forum 3 02-21-2011 12:50 AM


Forum Jump