Pulling yeast samples aseptically

[jsvarney5]

Hi all,

I'm getting started into counting and have a pretty good understanding of the process after much research. But one question I have is how are you working to minimize contamination when pulling a yeast sample to count?

I'm thinking that I will be pulling samples right out of a starter that has had time to grow to double check my count before pitching. And I'll also want to pull samples from a thick slurry harvested from each batch to get an adequate count at that stage as well. What processes are you using for doing this when you think of aseptic technique and not introducing unwanted organisms into the slurry/starter before it's next use? Is simply sanitizing a pipette and drawing some out sufficient enough? Flame the lip of the flask?

Also, for thick slurries, are say 5ml pipettes going to work well enough to pick up a dense sample? Thanks for the help!

 

[broadbill]

I'd probably use a sanitized pipette, pre-sterlized single-use ones if you can buy them.

As for pulling a sample of slurry, I think that is a fools errand; Even if you could pull a representative sample (and how would you know if you did?) the sample will be so dense with cells you'll never be able to count it.

You will always need to do your counts on resuspended yeast samples (cells floating in media), possibly diluted and do a back calculate to slurry amounts.

 

[jsvarney5]

I'd probably use a sanitized pipette, pre-sterlized single-use ones if you can buy them.

As for pulling a sample of slurry, I think that is a fools errand; Even if you could pull a representative sample (and how would you know if you did?) the sample will be so dense with cells you'll never be able to count it.

You will always need to do your counts on resuspended yeast samples (cells floating in media), possibly diluted and do a back calculate to slurry amounts.

Thanks for the suggestion on the pipettes. Quick question on those. Dont they come in large bags containing a bunch of them? If so, how do you keep them sterilized once you open the bag? Wouldn't that expose them and defeat the purpose? I question that because I've seen those on Cynmar.

As far as a sample of slurry, yes I would have to dilute it. What would you suggest for a process if i have a few mason jars of collected yeast and want to know how much I have to be able to pitch? I sometimes do 5 or 10 gallon batches and want to start getting a more precise idea on what I'm pitching. I'll also be staining to test viability on these counts too.

 

[Anatidaephobic]

Thanks for the suggestion on the pipettes. Quick question on those. Dont they come in large bags containing a bunch of them? If so, how do you keep them sterilized once you open the bag? Wouldn't that expose them and defeat the purpose? I question that because I've seen those on Cynmar.

As far as a sample of slurry, yes I would have to dilute it. What would you suggest for a process if i have a few mason jars of collected yeast and want to know how much I have to be able to pitch? I sometimes do 5 or 10 gallon batches and want to start getting a more precise idea on what I'm pitching. I'll also be staining to test viability on these counts too.

Yeah, pipette tips usually come in big bags. For sterile purposes, labs put a bunch of them in a plastic box and autoclave them. You could could probably boil them for a minute or two to sterilize them though.

You might have to cut the end of the 5mL tip to draw really thick slurry. It shouldn't affect your dilution factor as long as you add the water with the same tip and volume setting. Make sure to mix your slurry and dilutions well at every stage, preferably you'd have a vortex mixer.

As for sterilization, yeah flame the lip and maybe use a spray bottle filled with isopropanol or starsan to clean nearby surfaces.