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Old 05-20-2009, 03:11 AM   #1
sootedpair
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Default How can I determine yeast cell count

I've recently harvested yeast from a Rogue Brutal Bitter using the cool new stir plate that I made via the help of this forum. Now I'm wondering how I can tell how much yeast I've got.

I started with 1 cup of water and an ounce of DME. I added to that, the last couple ounces of the beer after shaking it enough to free the yeast from the bottom of the bottle. A couple days later, it was pretty obvious that the yeast was doing it's thing and multiplying.

What I want to know, is how to tell what the cell count is after the fermentation is complete. Since the first addition of wort, I've twice added the same water/DME combo to the yeast. How can I tell when I have enough to pitch in to a batch of brew?

Also, I want to start a yeast bank, and build starters from smaller vials of yeast. How will I know when I have enough?

TIA,
Scott



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Old 05-20-2009, 04:24 AM   #2
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short of a microscope you will never know exact cell count. They have a slide whos name is escaping me that has gridlines on it. You can then extrapolate how many cells per mm. You can also do a serial dilution of a sample and then smear a sample onto an agar plate. Since its diluted each colony which grows is representative of a single yeast cell. COunt them and scale up and multiply by the volume of your yeast sample...



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Old 05-20-2009, 12:21 PM   #3
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scinerd, i think the slide you're thinking of is a hemocytometer. I don't want to deal with the microscope and all of that quite yet. I was just hoping for a rule of thumb.

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Old 05-20-2009, 01:28 PM   #4
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I think some labs also use densitometers.

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Old 05-20-2009, 04:43 PM   #5
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The only way to determine a good cell count is to use a hemocytometer and stain the cells with methylene blue. If you were to measure the optical density you still would have no idea of the actual viability of the culture. With methylene blue the live cells stay transparent and the dead cells stain blue. Another way would be to serially dilute your culture in sterile PBS, plate 0.1 ml on wort agar, count colonies, and calculate back to determine a live cell count.

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Old 05-20-2009, 05:00 PM   #6
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When we take bacterial cultures in my lab, we take them from culture without taking a cell density measurement. We freeze off into 50% glycerol, so 500 ul (microliters) of glycerol and 500 ul of culture. We also freeze off into a 7% solution of Dimethylsulfide (DMSO) and then they go into a liquid nitrogen freezer at ~ -80*C. The glycerol is the stock we use for general use. The DMSO stock is used when the glycerol stock is either used up, or has gone bad, maybe from too much freeze/thaw.

For starting cultures, we do what's know as a "stab" where we take a pipette tip and stab it directly to frozen glycerol stock, and then drop the tip into about 5 ml of liquid media, let that go for about 24 hours and then use that to seed a larger stock, either 100 ml or 250 ml.

Cell density doesn't matter for taking off cells for culture. just along as there are some happy cells in the glycerol stock, you can culture up to whatever volume you want.

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Old 05-20-2009, 05:10 PM   #7
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Here ya go !

One hundred million one. One hundred million two. One Hundred Million "Honey can you come here please"...

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Old 05-20-2009, 06:22 PM   #8
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Unfortunately, there is no simple approach to finding the cell count. It's microscope or winging it.

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Old 05-21-2009, 12:21 AM   #9
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Quote:
Originally Posted by sootedpair View Post
scinerd, i think the slide you're thinking of is a hemocytometer. I don't want to deal with the microscope and all of that quite yet. I was just hoping for a rule of thumb.
yes! exactly


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