Guide to Making a Frozen Yeast Bank - Page 29

**Randar** · 03-15-2010, 09:51 PM

Quote:

Originally Posted by FlyGuy

The original question that has yet to be answered is why a stepped regime of yeast growth will produce MORE yeast than one single pitch in the same (total) volume of wort?

OK, forgive me if I missed it, but a quick google search revealed nothing on that. Where is it documented that this process indeed does result in greater overall cell count?

Yeast growth curve is exponential starting at the end of the lag phase until the start of the stationary phase. I don't see how stepping the media will impact the final cell count when you insert multiple stationary phases between each step. It takes some energy for cells to go in and out of lag/stationary phase that would otherwise feed into the exponential growth phase of a single-step starter.

The only thing I can think of is that when you decant each starter once the stationary phase is entered, you are adding more media. So the total final media used is greater.

Example:

Step 1) 250 ml starter, decant 200 ml
Step 2) 1 L starter, decant 750 ml
Step 3) 4 L starter

All in all, this will provide the yeast with 5.25 L of media, and assuming you are very careful and get good flocculation before each decanting, you should end up with a yeast cake with roughly the equivalent of a 5.25L starter.

So, comparing a stepped-up-to 4L starter (5.25 L of media in our example) vs using a basic 4L starter w/ 4 L of media, you can then justify why you would find more cells in the stepped version.

Otherwise, I'm at a bit of a loss how you can expect more cells from a stepped starter vs that of a single step if you assume the same total media provided.

camiller · 03-15-2010, 11:10 PM

Quote:

Originally Posted by Randar

OK, forgive me if I missed it, but a quick google search revealed nothing on that. Where is it documented that this process indeed does result in greater overall cell count?

Yeast growth curve is exponential starting at the end of the lag phase until the start of the stationary phase. I don't see how stepping the media will impact the final cell count when you insert multiple stationary phases between each step. It takes some energy for cells to go in and out of lag/stationary phase that would otherwise feed into the exponential growth phase of a single-step starter.

The only thing I can think of is that when you decant each starter once the stationary phase is entered, you are adding more media. So the total final media used is greater.

Example:

Step 1) 250 ml starter, decant 200 ml
Step 2) 1 L starter, decant 750 ml
Step 3) 4 L starter

All in all, this will provide the yeast with 5.25 L of media, and assuming you are very careful and get good flocculation before each decanting, you should end up with a yeast cake with roughly the equivalent of a 5.25L starter.

So, comparing a stepped-up-to 4L starter (5.25 L of media in our example) vs using a basic 4L starter w/ 4 L of media, you can then justify why you would find more cells in the stepped version.

Otherwise, I'm at a bit of a loss how you can expect more cells from a stepped starter vs that of a single step if you assume the same total media provided.

At each decant you are also removing ethanol and other yeast waste products. I know I feel more like reproducing in a nice clean pool than in a septic tank....

FlyGuy · 03-16-2010, 12:36 AM

Quote:

Originally Posted by Randar

I'm at a bit of a loss how you can expect more cells from a stepped starter vs that of a single step if you assume the same total media provided.

Me too, but that's what happens. It was confirmed by the yeast manufacturers and Jamil Zainasheff in emails to me, but none could actually explain WHY it occurs.

**passedpawn** · 03-16-2010, 01:10 AM

Access to oxygen. Lots of data shows that access to oxygen makes a sizable difference in yeast cell count.

Small volumes have better access to oxygen than large volumes (in the same container). Ratio of oxygenated surface area to volume. (It would seem that stir plates make this volume irrelevant, though).

So, keeping your starter small in the beginning improves early growth.

Anyhoo, this is my guess.

**Randar** · 03-16-2010, 04:21 PM

Quote:

Originally Posted by passedpawn

Small volumes have better access to oxygen than large volumes (in the same container). Ratio of oxygenated surface area to volume. (It would seem that stir plates make this volume irrelevant, though).

Yeah, seems to make the point irrelevant that it is oxygen starvation. The only exception to this I see is that alcohol content or yeast waste products hinder oxygen absorption of the solution enough to cause premature entry into stationary phase.

Flyguy, did those guys actually say a 4L starter that was stepped up to would have more viable yeast cells than an equivalent single-step starter that had the same total media volume?

FlyGuy · 03-17-2010, 03:12 AM

Quote:

Originally Posted by Randar

Flyguy, did those guys actually say a 4L starter that was stepped up to would have more viable yeast cells than an equivalent single-step starter that had the same total media volume?

Yes, exactly. I have seen that mentioned a number of times, and sent them that exact question -- why does stepping-up a starter to a certain total volume yield more yeast than a single pitch of yeast in the same volume of wort. Everyone basically reiterated the recommended method and restated that it will produce a greater volume of healthier yeast this way. Nobody attempted to explain the mechanism why.

I emailed White Labs and Wyeast with the follow-up and got no response. I also did some digging myself through the brewing science literature and came up short. Got tired of looking and dropped it.

**Randar** · 03-17-2010, 06:36 PM

With access to spectrophotometry equipment, I am tempted to make an experiment out of this. Wish I had the time, honestly.

**passedpawn** · 03-17-2010, 06:53 PM

Quote:

Originally Posted by Randar

With access to spectrophotometry equipment, I am tempted to make an experiment out of this. Wish I had the time, honestly.

Biology microscope, light stage and haemocytometer is the way, I believe. And Trepan Blue dye to count live ones.

**Nightbiker** · 03-19-2010, 01:06 PM

I haven't caught up on reading the entire thread, yet. BUT it was enough to galvanize me into ordering a yeast bank starter kit (with 5 vials included). I'm already harvesting and saving in the fridge (mini-fridge set for 36 degrees F) but would really like to have a safe repository to build from if (or when) my actively harvested crop gets 'off'.
This thread has been a tremendous help.
I have an old microbiology scope (reaches up to 100x though for that you need the oil) I guess I really would do well to acquire slides and such to take a peek at the cells from time to time and see if I can catch them mutating before I pitch and risk ruining a batch of beer. Perhaps this summer I'll start on that phase of the project (so much to do, so little time or $$). I'm glad I kept the microscope instead of getting rid of it on Ebay. Who knew?

Redbullsushi · 03-19-2010, 09:37 PM

I have been on and off of this thread and others on this topic over the past few months. I am glad to see some info coming out on this. When I started mine it was very hard to find anybody that was promoting it. It is a great idea and I can tell you that is works with fantastic results. I currently have some 5 yeasts in my bank and with a few days of prep time you can have a strong starter for your brew.

Pulling off the alcohol is big when starting it up and moving from your first feed to the second to grow it. I also have found that keeping it a little warmer helps to push its growth. I use nothing more than a small dish towel wrapped around and under the container to keep it insulated while it grows. My next level is to turn a mini fridge into a yeast grower where I can control the temp to a "T" for its growth.