Yeast measuring for counting

[biodarwin]

First time trying to count cells and get cell viability. I have 4 mason jars from a stepped up 2L starter I am trying to get a count to compare against the calculators. The question is how do you measure yeast? I have a tight yeast "cake" in the mason jars and I need 1ml of yeast to start the proper dilution. It is way too thick to pull with my pipette and pump. I took 20ml of water and mixed it up but this effects my dilution ratio. I am looking to get a 100 to 1 dilution ratio and had planned to do 2, 10-1 ratio dilutions. Any suggestions or am I going about this all wrong from the get go?

 

[ColoHox]

You are doing fine, you just need to keep track of your dilutions in order to get an accurate count.

For example: Specified volume of original sample, say 1ml of sample, well mixed into 9ml of sterile water. That is a 1:10 dilution. If that is still too concentrated, you can make another dilution from the first. 1ml from the 1:10 into 9ml water, makes a total of 1:100 dilution of the original sample.

The count you get just needs to be multiplied by the dilution factor for a cell count of the original (also consider the volume you used in your haemocytometer when calculating). Thorough mixing and multiple counts will make your counts more accurate.

There are a few online calculators that can be helpful until you get used to the process.

 

[biodarwin]

OK, I think I have this figured out, thanks for the help.

I took one mason jar, decanted all liquid and then filled it back up to the 100ml line on the jar. I slowly swirled the jar for a few minutes. I dispensed 1ml of yeast solution after pulling and in out of the pippette 10 times from the sample. I add 10ml of water to this solution and swirled around again. Once again I did 1ml of the new solution into a 10ml of water to make a 121-1 dillusion, I think?

I averaged 120 cells per large square. The equation looks something like this:

Cells x dilution factor x 10-4

120 x 121 X 10-4 = 145.2 Million cells per ml.
My 100ml sample should have ~14.5 Billion cells.

Next step is viability, but I wanted to understand and get my processes repeatable before I moved on.

 

[TrickyDick]

Where did you learn about doing this stuff?
I'm becoming more and more interested in maintaining my OWN yeast "library" and not being forced to rely on commercial sources, HOT summertime shipping prices, etc.
Thanks
TD

 

[biodarwin]

Where did you learn about doing this stuff?
TD

Mostly from the internet. Here are some of the resources I have used:

http://braukaiser.com/wiki/index.php?title=Microscope_use_in_brewing
http://www.microbehunter.com/2010/06/27/the-hemocytometer-counting-chamber

The equation I am using is a bit different now:
A * 1000 / B * C = D

A = Cell Count
B = Volume of the grid you are counting on the hemocytometer.
C = Dilution factor
D = Yeast cells per mL

For me C is normally 100 as I do 100-1 dilutions now. B for me is 1mm x 1mm x .10mm = .10 µL

So if I count 4 large squares and average them out to 130 per square.

130 * 1000 / .10 * 100 = 130 Millon Cells per mL.

 

[biodarwin]

I'm becoming more and more interested in maintaining my OWN yeast "library" and not being forced to rely on commercial sources, HOT summertime shipping prices, etc

Summer time is the main reason I am wanting to have my own yeast bank. While I do wash yeast, I had to order 5 different vials of yeast this summer for different styles I didn't have on hand. Each time they were pretty banged up when I got them.

 

[TrickyDick]

Summer time is the main reason I am wanting to have my own yeast bank. While I do wash yeast, I had to order 5 different vials of yeast this summer for different styles I didn't have on hand. Each time they were pretty banged up when I got them.

5 x $7.99 = $40 not counting shipping, handling, ice packs, etc.
time is money, but money is also obviously money.
Frustration on the other hand is a real pisser.

It doesn't take much time if you brew 10gal 10-12 times per year and have to pay for yeast (especially if you are brewing lager) to justify the equipment necessary to harvest and bank yeast and count accurate cell counts for brewing. This is kind of where I am at. I did about 200% more brewing this year than last 3 years combined. I was a little cheapo on the yeast, preferring dry to liquid when possible. I am still at the point where I like to experiment and have fun with new yeasts though and see how they behave etc in different styles and so forth. I don't mind paying for malt and hops, but some strange quirk makes me reluctant to shell out the cash for yeast.
One reason, as you pointed out is the quality upon receipt for mail order yeasts (a necessity for me) in the summer months.

I also hate it when you need to get particular styles that are the limited release yeasts but aren't offered that month you want to brew it.

I am about 90% of the way, mentally, into doing slants, and washing, and the whole 9 yards it takes to get it done. The other 10% is telling me dry yeast is easy cheap and less time and money but ignore the lack of variety compared to liquid.

Ill be looking into those links. I really wish there were other homebrewers nearby that we could exchange yeasts. I don't think that once a month brewing with varied styles and a mix of ale and lager yeasts plus occasional wheat, and Belgian and kolsch brews, really condones itself to using a single strain and repitching each month. As a hobbyist especially as I have no lure to "turn pro".

TD

 

[TrickyDick]

Mostly from the internet. Here are some of the resources I have used:

http://braukaiser.com/wiki/index.php?title=Microscope_use_in_brewing
http://www.microbehunter.com/2010/06/27/the-hemocytometer-counting-chamber

The equation I am using is a bit different now:
A * 1000 / B * C = D

A = Cell Count
B = Volume of the grid you are counting on the hemocytometer.
C = Dilution factor
D = Yeast cells per mL

For me C is normally 100 as I do 100-1 dilutions now. B for me is 1mm x 1mm x .10mm = .10 µL

So if I count 4 large squares and average them out to 130 per square.

130 * 1000 / .10 * 100 = 130 Millon Cells per mL.

Thanks!

How long does it take you to count the cells in four large squares, including microscope setup time, sample prep and dilution, staining (if done)? Do you use a clicker counter?

 

[biodarwin]

Thanks!

How long does it take you to count the cells in four large squares, including microscope setup time, sample prep and dilution, staining (if done)? Do you use a clicker counter?

I use a click counter app on my phone. The whole process takes less than 30 minutes.

 

[TopFlatBrwr]

Where did you learn about doing this stuff?
I'm becoming more and more interested in maintaining my OWN yeast "library" and not being forced to rely on commercial sources, HOT summertime shipping prices, etc.
Thanks
TD

You could also look into getting the book "Yeast". It has pretty much everything you would need to know to set up your own yeast lab.

 

[biodarwin]

t has pretty much everything you would need to know to set up your own yeast lab.

Definitely. Its a must read!

 

[TrickyDick]

I actually have the book already, and just began reading it a few days ago. Finding time has been an issue however.


TD

 

[dstar26t]

OK, I think I have this figured out, thanks for the help.

I took one mason jar, decanted all liquid and then filled it back up to the 100ml line on the jar. I slowly swirled the jar for a few minutes. I dispensed 1ml of yeast solution after pulling and in out of the pippette 10 times from the sample. I add 10ml of water to this solution and swirled around again. Once again I did 1ml of the new solution into a 10ml of water to make a 121-1 dillusion, I think?

I averaged 120 cells per large square. The equation looks something like this:

Cells x dilution factor x 10-4

120 x 121 X 10-4 = 145.2 Million cells per ml.
My 100ml sample should have ~14.5 Billion cells.

Next step is viability, but I wanted to understand and get my processes repeatable before I moved on.

If you counted an average of 120 cells per one of the 25 grids, you also need to multiply by 25. All 25 grids together are 1/10,000 of a mL.

 

[vinpaysdoc]

Noob with first post, so, take this with a pinch of yeast....

I just started a yeast library for the fun of it.

I took a vial of yeast (100 billion cells) and stepped up the count with starters. After cold crashing and decanting, I placed the slurry in individual 33 ml test tubes that were almost identical in size to the one the yeast came in: http://www.hometrainingtools.com/giant-test-tubes-30-pack/p/CE-TTBSB30/. Once the yeast settled out to cakes in the bottom of the tube, I decanted and filled with sterile water.

Since I had a known quantity of yeast cells when I started with a similar vessel, I made the assumption that I could get a rough estimate of the amount of cells by comparing the yeast cakes in the test tubes. The known quantity with 100 billion cells had a yeast cake of 2 inches from the bottom. I use that as the standard and then approximate the amount of yeast in each of the test tubes. It's not very scientific, but, I think it gets me in the right ball park to make a starter with. Of course, I just used that method yesterday for the first time (fermentation going fine), so, realize there is not much experience behind the advice.....