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Yeast Culturing Techniques

dharalson

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Bootleg
Attached are some references I have collected that I have based my culturing techniques on. 

This website is where I learned most of the techniques and procedures for handling yeast
http://www.alsand.com/beer/yeast/index_E.html
The four attachments are Word Docs, one is an article on storing yeast in sterile water, the other three are Kansas State U class articles that the professor posted online.  I gleaned some yeast data and handling techniques from there.

I have six strains in 12 ml vials about half full of sterile distilled water.  There is enough yeast to just cover the bottom with visible cells and it gets slightly cloudy when shook up.  To start a culture, I smear a couple of drops from an inoculation loop on an Agar Slant.  I don't try to isolate a single cell colony as these are already "pure" cells.  The original cells into storage were streaked on Agar Petri Dishes and single cell colonies were harvested.  The current batch (this is the first batch) are about 10 months old and based on the current starter in work are still viable.

All the best, David
 

Attachments

  • Sterile Distilled Water Yeast Storage.doc
    53 KB · Views: 366
  • Yeast_Article_ksu_Part A.doc
    63.5 KB · Views: 366
  • Yeast_Article_ksu_Part G.doc
    134.5 KB · Views: 415
  • Yeast_Article_ksu_Part Ga.doc
    113 KB · Views: 490
thank you for that link! as soon as I clicked on it and saw the background, I remembered that page from when  was learning about canning wort and streaking a petri dish. Also of interesting note is that your article from Dave Whitman is what I used when I first started slanting yeast in 2001.

My personal culture techniques are using slants. I currently maintain a bank of about 10 strains and re-culture them about every 6 months. I only keep up to 4 generations before I go back to the original source. I don't typically isolate colonies either since my source is always a vial of White Labs.

  • For a 5.5 gallon batch I typically start with a 25ml vial of 1.030 wort that I inoculate by scraping a bit of yeast from the slant and then transferring into the vial.
  • I let this grow for 2  days at room temp and then transfer to a 500ml flask filled with 1.040 wort. I let this grow for 2 days, on a stir plate.
  • Lastly I transfer to a 2000ml flask filled to 1600ml of 1.040 wort and keep on a stir plate for 2 days.

Here is a great site with pictures on how to make slants at home:
http://www.jimsbeerkit.co.uk/forum/viewtopic.php?f=12&t=35033

Some other ways to store yeast are by freezing them. Here are a few links:

http://www.homebrewtalk.com/wiki/index.php/Yeast_bank
http://www.homebrewtalk.com/wiki/index.php/Freezing_yeast
 
Bootleg

Are you storing the yeast on the Slant?  Do you keep the slant refrigerated?  You re-culture every six months? from a White Labs each time?

I have two 12ml vials for each yeast, one is the Working Vial and the other is the Master Vial.  When it gets time to re-culture my stored yeast I will discard the Working Vial and use the Master Vial to streak a Petri Dish and then isolate a few single cell colonies and restock the Master Vial and Working Vial.  Doing it this way, I am for the most part always working with a 1st generation yeast.  Not completely true, because all of the yeast is propogated but my stored yeast has not gone through a beer brewing cycle.  Each time I culture up for brewing, I am starting with "fresh" stored yeast.

I brewed this weekend and I started the culture on Monday am (1/3) with a few drops smeared on a Slant;
on Tuesday pm I could see "impurfections" in the surface of the Slant
on Wednesday pm I could see barely visible yeast colonies;
and Thursday pm I "harvested" the yeast.
To harvest I scraped the yeast off with the innoculation loop and swirled it in 500ml of 1.040 in a Qt Mason jar.  This was fermented 24 hours on a stir plate and set in the refrigerator on Friday pm to settle out. 
On Saturday am, I prepared 1500ml 1.040 in a 2000ml Erlenmeyer.  Instead of transfering all of the yeast to the next step, I used a syringe to "suck up" a couple of squirts of yeast.  I don't have a good feel for how much yeast I transfered, maybe 0.5 to 2 B cells.  This 1500ml starter fermented on the stir plate until Sunday pm, then this when to the refrig to settle out. 

I didn't finish the boil under Monday night (yesterday) and I pitched both the first and second starters on Monday night.  Since I didn't let these ferment out and settle I don't have a good feel on the total amount of yeast, but I'm sure I'm in the 200 B range.  (The beer is 5.5 gal of American Amber, 1.053).  The fermentation temp is 65F. This morning Tues (10-12 hours) the wort is fermenting well.  When I get home tonight, I am expecting to see a roaring "boil" of CO2  :)

Here's a question; Why do I have to step up in 1 to 10 steps?  ie 10 ml to 100 ml to 1000 ml.  If I put 1 cell in 1000ml  of wort, why won't that grow to the limit of 1000 ml?  1 x 2 x 2 x 2 x 2 x 2 x 2 x 2, etc until I have used up all of the sugars?  [About 200B based on 200M/ml]  I can oxygenate the 1000ml, I can use the stir plate to agitate, replenish O2 and remove the CO2, so the yeast should just keep on growing.  This example is a bit extreme, but why not?
I need someone who knows to tell me why not.

All the best, David
 
dharalson said:
Bootleg

Are you storing the yeast on the Slant?  Do you keep the slant refrigerated?  You re-culture every six months? from a White Labs each time?

I store all my yeast on slants and yes I keep them in a refrigerator. I usually keep 2 slants of any given type of yeast. I try to re-culture every 6 months and sometimes it is directly from my second slant. Since I keep 2 slants of every stain I make starters off of one and use the other one as backup. I also occasionally re-culture directly from the back-up slant. Either way, I make sure I don't exceed 4 generations. Before getting a fresh vial of yeast to culture from.

I cant answer to the need of stepping up by 10's. I certainly do not follow that model. So far I have had no issues I can attribute to my culturing/starter techniques. My beers are always well attenuated and seem to always have enough yeast to do the job. 
 
I was going through my book on yeast by Jamil Z. and Chris White of White Labs and it seems as the reason to grow by 10's is because you get the most growth. In lab studies conducted by White Labs that yeast grew more new cells when inoculated from a smaller amount as opposed to  a larger amount of yeast. Basically the less yeast in ppm/ml the more new growth you get. Growing by 10's is recommended because of the lab studies they have done.

It works like this, If I go out and buy a vial of White Labs yeast it has approximately 100billion cells in it. If you pour the whole vial into a 1liter starter you will get much less growth than if you had just poured in a few drops because the yeast in ppm/ml are less. Yeast will grow themselves more if there are less of them. I hope this all makes sense.

 
Boot
I do understand that.  My copy is dog eared and on my desk right now as I type.  I wasn't questioning the 1:2 or 1:10 factor, I want to know if I can go 1 to 20 to 30 or even 100.  I start a culture with 100-200 cells on a slant.  Those few cells grow into colonies with about 1 MILLION Cells each!  That is much much much larger than the 2 or 3:1 growth shown in the White Labs study.  They were starting with 80-100 BILLION cells at a 50-200 Million / ml pitch rate.  I am pitch maybe 500 Million in 500 ml (1 Million / ml). 

So instead 10's I want to do 50's.  I am doing it and it is working; what I want to know is this stressful on the yeast.

Using your example of a 100B White Labs vial.  I would put 1/10 of the yeast (10M/ml) in the 1 Liter and store the remaining 90B.  The 10M/ml starter will grow to 150-200B.  Add that to the stored 90 B and now I have 240-290B instead of 150-200B that I would get by pitching all the yeast. 

Yeast are more prolific than rabbits if we give them a chance.  :)

David
 
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