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**gandelf** · 10-18-2012, 01:56 PM

I have been staining with methylene blue and would like to give trypan blue a
go. Do I use a 0.4% solution (source?) and is the procedure the same as with
methylene? Any other advice would be appreciated.

**Johnnyhitch1** · 10-18-2012, 02:09 PM

make sure you add enough aluminum and vent to de-gas

**gandelf** · 10-18-2012, 05:05 PM

Could you elaborate on that; or outline the procedure? I'm not a lab tech.

**Johnnyhitch1** · 10-18-2012, 05:20 PM

Quote:

Originally Posted by gandelf

Could you elaborate on that; or outline the procedure? I'm not a lab tech.

Nowhere near a lab tech
just a play off breaking bads "blue"
sry i figured someone would be able to answer your question...

WoodlandBrew · 11-19-2012, 11:24 AM

Bring a dead thread back to life here to answer the question.

Yes, 0.4% Trypan blue works great. Use equal parts of trypan blue and the sample.

**gandelf** · 11-19-2012, 11:50 AM

Thanks woodlandbrew, better late than never. I have been working on getting more accurate cell counts. I did a count on a fresh unsmacked pack of wyeast 1056 and got 172,000,000. So, I obviously have some work to do yet.

WoodlandBrew · 11-19-2012, 01:11 PM

My brother is a microbiologist, so that's where I learned about trypan blue, but I'm pretty green when it comes to cell counts. 0.4% is the "gold standard" for viability counts of mammalian cells, but it also correlates very well to methylene blue and violet for yeast viability counts.

http://www.coulterflow.com/bciflow/documents/instruments/Vi-Cell/Comparison%20of%20the%20efficacy%20of%20various%20 yeast%20viability%20stains%20(ta-204).doc

I've never taken apart an un-smacked smack pack, but 176 million does seem low. What are you using for making measurements? Baking measuring cups? Pipette and graduated cylinder?

Have you been counting slurries or starters? I'm curious as to how they correlate to the calculators for different peoples processes.

Have you found a good dilution for methylene blue? White Labs suggests 0.01% which seem much lighter than 0.4% trypan blue. I'm using the 2.303% from a pet store because it is cheap and readily available. 1% seems to be what is sold as a ready to use dilution by chemical companies, but it would be nice to hear from someone doing similar work as to what works for them.

**gandelf** · 11-19-2012, 05:10 PM

It should be 172 billion, not million; I was multitasking. At 55 years old, memory is not as reliable has it has been, but I'm not willing to take that lying down.

I have been fiddling around with cell counting and viability for a couple of months now. Two weeks ago I took a weekend course at UWSP; microbiology for brewers. Their was only two homebrewers and about 12 local craftbrewers. It was a great weekend. The professor has been homebrewing for almost 20 years. We did some cell counting and viability. But, the professor's hobby is collecting bacteria and identification, so 95 percent of class time was spent on brewery bugs and there eradication.

I would be interested in exchanging ideas and procedures to improve the reliability and accuracy. I do need to state that I have only been doing this for the past few months.

My brewing process is tight, my fermentation temp control is +/- 1 degree ( liquid, not air contact), so accurate pitch rates are what I'm chasing now.