Phosphoric acid to dilute highly flocculant slurry

[woohokie]

I'd like to use wlp007 as our house ale strain but due to the difficulty in trying to get a homogenous and non-clumpy sample to actually count cells, I finally tried using phosphoric acid (85%) I normally use to adjust mash ph.

I read about it here:

http://braukaiser.com/blog/blog/2012/10/03/yeast-un-flocculation-for-cell-counting/

I tried it on a tube of wlp007 and it seemed to work great, floccs smoothed out and cells looked normal and not dead - however, it DID require me to dilute the sample with more acid than I normally would havee thought. My procedure for serial dilutions of slurry call for weighing out 40g of slurry and then adding to that slurry exactly (by weight in grams) the same amount of distilled water to create a 1:1 dilution.

My question is: if I used the phosphoric acid for the 1:1 dilution step, would that cause any issues as far as an accurate cell count? It seems to me that it wouldn't given that it's all based on exact weights anyway - any issues with this that you all can see?

[dirtfish]

I have the same problem. I found Braukaiser's page last Friday after lots of suffering trying to get a consistent count-and the method seemed to work.

I collect yeast in a 5 gallon corny and add a quart or so of sterile wort at the time of collection. When I count (1 or 2 days later) I shake and swirl the keg to try and homogonize the contents, then take a 1 ml sample with a sanitized pipette pump and use a 2% acid solution to make a 1:100 dilution. This seemed to break up the clumps and I had what looked like a normal yeast suspension instead of a dough ball in the bottom of a tube of water.

I used the method to establish our count for our brew last Saturday and fermentation seems to be going normally.

I made a bad count the week before and instead of trusting my instinct, pitched what our count said. It was probably more than twice the quantity needed. Came in the next morning to find stout all over the floor and fermenter-took a good while to clean that mess up. Still waiting for the fusel alcohols we created continue to dissipate and hoping for the best....

I sent an email to our yeast supplier (BSI) with the same sorts of questions about the phosphoric acid un-clumping method.

[mmussen]

I've used it a few times - although I've only ever needed a drop of two of 85% phos in a 1:100 dilution to get the yeast to stop clumping. I take my yeast dilute 1:10 with water and then dilute 1:10 again - during the second dilution I'll add a drop of two of acid before adding all the water I need.
With the Chico ale I use I find I rarely have to use acid to declump the yeast.

Adding acid to the slurry will make viability staining useless - so you need to do a separate dilution without acid and use methylene blue or violet to get an approximate viability.

[woohokie]

I tried it out today and did my initial 40g of yeast slurry diluted 1:1 with 40g of 85% phos acid, which seemed to work great. Counts were great and seemed consistent on both sides of the hemacytometer - 1.8 bill cells/ml ... The viability thing did seem odd as I barely noted any dead cells - which leads me to agree that it might have made staining them with methelyene blue useless... If that's correct then the acid somehow doesn't allow for the dead cells to take in the stain?

[mmussen]

I'm not sure as to the exact reason why the acid makes the staining not work correctly. I believe the acid reacts with the dye itself, but I'm really not to sure. Maybe someone else will chime in and correct me

[woohokie]

Did my usual method of cell counting today after I noticed that 007 (this slurry) seemed to not be peanut butter-esque... Sure enough I saw some stained cells, so the acid definitely doesn't show dead cells accurately fwiw.

[CharlosCarlies]

I tried it out today and did my initial 40g of yeast slurry diluted 1:1 with 40g of 85% phos acid, which seemed to work great. Counts were great and seemed consistent on both sides of the hemacytometer - 1.8 bill cells/ml ... The viability thing did seem odd as I barely noted any dead cells - which leads me to agree that it might have made staining them with methelyene blue useless... If that's correct then the acid somehow doesn't allow for the dead cells to take in the stain?

I'm so glad I found this thread because we started using phosphoric for the same reason and couldn't seem to find any dead cells even after microwaving the sample! Good to hear we aren't crazy.

[dirtfish]

What concentration of acid are you using? I see dead cells in my counts and am using a 2.5% solution of phosphoric acid to make up a 1:100 dilution.

[ChadBeer]

Density correction

Hi woohokie,

One thing you should keep in mind - if you're doing your dilution 1:1 by weight then you need to take density into account (because the hemocytometer is based on volume).
The density of 85% phosphoric is huge (1.685), so if you don't correct for it your dilution will be off by a fair bit (a 1:1 weight dilution will be approx. 1:0.6 volume dilution) and your calculated cell counts will come out about 25% too high (for the case of a 1:1 dilution).

Cheers,
Chad