Growing yeast for homebrewers.

[gyst]

I have plans on purchasing a microscope and hemocytometer I'll add a gram stain kit to the list. I don't think I will encounter very much mutation as I am using a flask type of media and I pull a 10ul loop through the culture for propagation. When the flask becomes low (or infected) I will pull a colony off and reinoculate a new flask. So I don't foresee much mutation with such low amount of manipulation.y.

You need to freeze your original cultures in glycerol stocks and store them in the -80C. You will get changes over time in the flask, even without manipulation. According to Verstrepen et al, "Flocculation: What Brewer's Should Know" (Appl Microbiol Biotechnol (2003) 61:197–205): "Storage at lower temperatures (4C or 10C) resulted in a reduced flocculation, independently of yeast agitation and starvation."

The FLO (flocculation) genes are are near the telomeres, and as such are very unstable and show high mutation rates (due to silencing effects and recombination rates). If you freeze, you can always go back to the original - unfrozen, not so much.

 

[gyst]

There are commercial medias for propagation of fermentation yeast. UBA with cyclohexamide being one choice.

Am. J. Enol. Vitic. 38:4:273-276 (1987)

While the article is obviously aimed at the wine industry based on the journal, I believe the general methods are applicable to any fermentation strain of yeast. It should be stated that I have no first hand experience with these tests and have only given the article a cursory glance.

I concur with the posters that pointed out the possibility that White Labs or Wyeast may hold some proprietary rights to their yeast strains. It would definitely be in your best interests to check out what types of legal holds the company(ies), that you originally obtained the yeast strains from, have on their commercial distribution. If you believe that there is a significant difference in the geno-/phenotypes of your yeast strains, it may be prudent to get your strains sequenced or in some other way characterized (outside of the qualitative observations regarding fermentation characteristics). Although, I bet DNA sequencing would be cost prohibitive without a significant funding source or the available lab space/supplies to do it yourself.

*begin microbiology-beer geek talk*
Depending on how far you want to take this venture, I would be interested in a qualitative assessment of the flocculation characteristics, enough of this high, medium, medium-low, business). I have only worked with adherent cell lines so I don't how this is properly done, but it seems reasonable that this property could be determined by measuring the optical density at various stages of the cell cycle and comparing those values to sugar concentration (as a reporter for fermentation progress) and cell count/viability (via a hemocytometer and colony counting) to normalize the data.

I wonder to what degree temperature effects flocculation characteristics. Will a "high" flocculating yeast settle out of solution faster at 62F than 70F after fermenation is complete?
*end beer-geek talk*

Just some thoughts, good luck.

Flocculation assay: "Cells were harvested and washed twice in deflocculation buffer [20 mM citrate (pH 3.0), 5 mM EDTA]. After washing, cells were suspended in deflocculation buffer to an optical density (absorbance
at 600 nm; A600) of 2. An aliquot of 800 ll of this cell suspension was pipetted into a 1-ml cuvette, and 200 ll of 100 mM calcium chloride was added to initiate flocculation. The cuvette was vigorously shaken and the absorbance (A600) was measured immediately and at 30-s intervals for 5 min using a spectrophotometer." (J.C. Bayly et al. / FEMS Yeast Research 5 (2005) 1151–1156)

Cyclohexamide: I've worked with this, its very bad ****. It isn't just a fungal poison: its a eukaryotic (ie you and me too) poison. Wikipedia entry: "significant toxic side effects, including DNA damage, teratogenesis, and other reproductive effects (including birth defects and toxicity to sperm)." Make frozen stocks, go back to them regularly, and use sterile technique, and you won't have to poison yourself figuring out what the hell is contaminating your yeast.

 

[pabloj13]

Flocculation assay: "Cells were harvested and washed twice in deflocculation buffer [20 mM citrate (pH 3.0), 5 mM EDTA]. After washing, cells were suspended in deflocculation buffer to an optical density (absorbance
at 600 nm; A600) of 2. An aliquot of 800 ll of this cell suspension was pipetted into a 1-ml cuvette, and 200 ll of 100 mM calcium chloride was added to initiate flocculation. The cuvette was vigorously shaken and the absorbance (A600) was measured immediately and at 30-s intervals for 5 min using a spectrophotometer." (J.C. Bayly et al. / FEMS Yeast Research 5 (2005) 1151–1156)

Cyclohexamide: I've worked with this, its very bad ****. It isn't just a fungal poison: its a eukaryotic (ie you and me too) poison. Wikipedia entry: "significant toxic side effects, including DNA damage, teratogenesis, and other reproductive effects (including birth defects and toxicity to sperm)." Make frozen stocks, go back to them regularly, and use sterile technique, and you won't have to poison yourself figuring out what the hell is contaminating your yeast.

Oh come on. Cyclohexamide isn't that bad. That's the same stuff they say about all the other wussy lab chemicals like acrylamide, ethidium bromide, etc.

 

[gyst]

LD50 is 2mg/kg for a rat. Assuming scale-up (not a great assumption, mind you), and a 70 kg human, that's what, 140 mg? I wouldn't want someone noodling around with that at home. And I don't know about you, but I'm pretty cavalier about gloves, but I use them for EtBr, acyrlamide (unpolymerized), sodium azide, cyclohexamide, and MMS. Everything else is negotiable.

Aaaand, I just realized this is a zombie thread, dammit. Sorry, folks.

 

[pabloj13]

LD50 is 2mg/kg for a rat. Assuming scale-up (not a great assumption, mind you), and a 70 kg human, that's what, 140 mg? I wouldn't want someone noodling around with that at home. And I don't know about you, but I'm pretty cavalier about gloves, but I use them for EtBr, acyrlamide (unpolymerized), sodium azide, cyclohexamide, and MMS. Everything else is negotiable.

Aaaand, I just realized this is a zombie thread, dammit. Sorry, folks.

Well I TRY not to eat it.

 

[gyst]

Dude, the entirety of any lab safety training course can be boiled down to: don't eat the chemicals. Yet they are routinely at least an hour...