Cell Counting and viability testing

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

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.

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.

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/2013/01/26/dyeing-yeast-cells-life-vs-death/

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?