Vial=>Starter=>Multiple Vials?

I'd like to buy a vial of WLP001 for my next batch, but I'd like to stretch it out for 4 or 5 batches. Rather than top cropping or washing yeast from the first batch, can I just:

- Make a large starter with stir-plate and nutrient.
- Then maybe crash cool it for a day and decant a bit.
- Then fill 4 or 5 small mason jars with yeast slurry.
- Store them in the fridge.
- Then use each jar as it's own vial making a starter from each when I need it.

I plan to use them all within 3 or 4 months. Anybody take this approach with success or am I better off washing and reusing to keep it fresh/active from batch to batch?

I would think that is a safer and more sanitary way to culture the yeast than using it post-fermentation.

yes, this would be a great way to do it. You could even probably stretch out each of your mason jars to 1-3 starters apiece, depending on how big your initial starter was.

broadbill said:

yes, this would be a great way to do it. You could even probably stretch out each of your mason jars to 1-3 starters apiece, depending on how big your initial starter was.

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Great! I'm going to give it a try then. I wonder how long I can safely wait before I re-divide one of the jars into more again. Is a 3 month nap going to weaken the little guys too much? Does anyone have a schedule they abide by for this?

carrotmalt said:

Anybody take this approach with success or am I better off washing and reusing to keep it fresh/active from batch to batch?

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Many people use the method you have outlined with a good deal of success.

However - if you have (or can purchase) some small plastic vials or test tubes - it's easier, quicker and better to split the freshly opened packet directly into sterilized tubes and then store those.

carrotmalt said:

Is a 3 month nap going to weaken the little guys too much? Does anyone have a schedule they abide by for this?

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Yes, the 3 month nap will have some effect on the viability of the yeast. How much? The Mr. Malty calculator has a built-in viability calculator you can play with to get a ballpark. If I were to guess...I'd say you'll have a 10-25% decrease in viability over that time.

The up-side of that is you are planning to make a starter anyway, so decreased viability in your stocks isn 't that big of a deal....you'll still be able to generate a starter from them, even as they get really old (up to a year, maybe longer).

good luck!

broadbill said:

Yes, the 3 month nap will have some effect on the viability of the yeast. How much? The Mr. Malty calculator has a built-in viability calculator you can play with to get a ballpark. If I were to guess...I'd say you'll have a 10-25% decrease in viability over that time.

The up-side of that is you are planning to make a starter anyway, so decreased viability in your stocks isn 't that big of a deal....you'll still be able to generate a starter from them, even as they get really old (up to a year, maybe longer).

good luck!

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Wow, according to Mr. Malty they do take a hit over time. With today's date it uses 97% viability. I changed the production date a month at a time to see what changed:

1 month later: 75%
2 months later: 53%
3 months later: 32%

So after only 2 months, it calls for twice the starter volume. Not a big deal, but I didn't think it would be that significant.

carrotmalt said:

Wow, according to Mr. Malty they do take a hit over time.

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The MrMalty and viability date is designed for when you pitch pack(s) of yeast directly into the size starter it mentions.

While it has given you a good idea of how much and how quickly yeast viability changes over time, if you are going to split and store your yeast, and then use smaller stepped starters the calculator does not cater for that. After storing your split samples, the first (small) step required to build a starter will have many old/dead cells, but once it is stepped up (even once or twice) the vast majority of the the cells will be new, healthy and viable, the date is really only essential for the first step, or if you are pitching directly into your wort or into a single large starter.

Wolfy said:

The MrMalty and viability date is designed for when you pitch pack(s) of yeast directly into the size starter it mentions.

While it has given you a good idea of how much and how quickly yeast viability changes over time, if you are going to split and store your yeast, and then use smaller stepped starters the calculator does not cater for that. After storing your split samples, the first (small) step required to build a starter will have many old/dead cells, but once it is stepped up (even once or twice) the vast majority of the the cells will be new, healthy and viable, the date is really only essential for the first step, or if you are pitching directly into your wort or into a single large starter.

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That makes sense. I was looking at it as if I were doing what I described in my initial post and using one of the jars as a vial pitched into a quart or so of wort for the starter. I believe this is the scenario the calculator is addressing. If I take your advise and divide the initial vial up, I'd need to use multiple steps to achieve my total starter volume, but like you said, each step would be adding healthy yeast to the count. I hadn't thought about it like that before.

So if I had a vial that was 3 months old and the calculator says I now need two Liters vice one with a newer vial, would I be able to use a lesser starter volume if it were stepped up in smaller increments? I'm still trying to wrap my brain around this.

carrotmalt said:

So if I had a vial that was 3 months old and the calculator says I now need two Liters vice one with a newer vial, would I be able to use a lesser starter volume if it were stepped up in smaller increments? I'm still trying to wrap my brain around this.

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The calculator is assuming that you're pitching 2L of yeast that you grew three months ago and then threw in the refrigerator. If you take your 3-month-old jar of yeast and make a 1L starter, the age of the result is now effectively zero again.

If you split a 1L stirred starter into 5 jars, you'll have about 30B cells per jar. In three months, you'll have the equivalent of 10B cells per jar. Assuming your staters are 1.040 (~10 degrees Plato), 10B cells is a reasonably conservative pitching rate for a 1L starter, so you really shouldn't need multiple steps. Just toss one of those jars into a new 1L starter, stir, and you're ready to go.

Moreover, if you're using a stir plate you can afford to do rather large step sizes---I've pitched from the dregs of a 750mL bottle directly into a 1L starter and ended up with very healthy yeast.

Wolfy said:

The MrMalty and viability date is designed for when you pitch pack(s) of yeast directly into the size starter it mentions.

While it has given you a good idea of how much and how quickly yeast viability changes over time, if you are going to split and store your yeast, and then use smaller stepped starters the calculator does not cater for that. After storing your split samples, the first (small) step required to build a starter will have many old/dead cells, but once it is stepped up (even once or twice) the vast majority of the the cells will be new, healthy and viable, the date is really only essential for the first step, or if you are pitching directly into your wort or into a single large starter.

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Right, but I don't think the OP was planning on having to step up his starter from his stocks (nor do I think he has to). Because of this, I think Mr. Malty's number may still apply (if only roughly), only instead of using the date the smack pack was manufactured, he could plug in when he grew and split his stocks of yeast.

broadbill said:

Right, but I don't think the OP was planning on having to step up his starter from his stocks (nor do I think he has to). Because of this, I think Mr. Malty's number may still apply (if only roughly), only instead of using the date the smack pack was manufactured, he could plug in when he grew and split his stocks of yeast.

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Good point, well maybe now he'll realize the number of viable cells in his stored samples might be quite low and hence consider a stepped starter.

kwantam said:

The calculator is assuming that you're pitching 2L of yeast that you grew three months ago and then threw in the refrigerator.

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Maybe I didn't explain well, but this isn't the assumption the calculator is making. By three months old, I'm talking about the production date on the calculator, which is referring to the age of the vial according to the tool tip.

From what I'm reading here, it sounds like by stepping up my starter incrementally vs one step, my viability will be better. I have nothing against stepping up in increments, but I thought this was simply to keep other nasties from taking over before the yeast do. Adding the yeast from a slant for instance to a liter of wort could take a while before they take over the full volume, so you'd let them conquer something smaller and then step it up.

What is it about incrementally stepping it up that would change the wort volume required assuming the answer to my earlier question is yes:

carrotmalt said:

So if I had a vial that was 3 months old and the calculator says I now need two Liters vice one with a newer vial, would I be able to use a lesser starter volume if it were stepped up in smaller increments?

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Thanks for all the input by the way!

carrotmalt said:

Maybe I didn't explain well, but this isn't the assumption the calculator is making. By three months old, I'm talking about the production date on the calculator, which is referring to the age of the vial according to the tool tip.

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Once you've poured the first vial into a starter, its age is categorically irrelevant. That starter is producing new, healthy yeast, and the yeast from that starter have good healthy glycogen stores. EDIT: to be clear, as long as you let the yeast completely finish out the starter wort, the strongest factor in how many yeast cells you get from a starter is its volume. Since the yeast are approximately doubling in each generation, the final generation uses at least as much energy to multiply as the sum of all generations before (since the sum of 2^k for k from 0-(n-1) is (2^n)-1). Thus, the volume of the starter determines your final cell count to within a factor of two, which is as good an accuracy as you can expect without doing dilutions and cell counts.

Let's say that today you have a WLP vial that is 6 months old. You dump that into a 1L starter, and in a few days you have ~150B "young" cells (age=0). You split this up into five jars, J1-J5, 30B cells each. The age of these jars starts counting from today.

In one month, you decide to use J1. It's got ~23B cells in it (75%), which is on the low side for pretty much any 5gal batch, so you make a 1-2L starter (depending on gravity) and you're off.

The next month, you're busily brewing, and you make three more batches. J2-J4 all have ~15B viable cells (50%), so you make a new starter for each and pitch.

In the third month, you use your last jar, J5. This one, being three months old, has an estimated 10B viable cells (33%)---still enough to make a 1L starter according to the 1 million cells / degree plato / mL guideline.

If you had waited until the fourth month, and decided to brew a high gravity beer requiring a 2L starter, you'd be starting with only ~7B cells (25%), so instead of jumping straight to a 1L starter as your first step (which honestly would be OK), you'd make a 500mL starter and then step that up into a 2L starter before pitching.

In summary: what matters is the time since the yeast last had the ability to replenish their glycogen stores. The stuff you pull out of a starter is "new" for the purposes of the pitching rate calculator.

I understand needing a larger starter if you're starting with a smaller initial yeast count because they need more food/nutrients etc. to reproduce.

What I still don't hear is why I would prefer to step up in increments.

Wolfy said:

Good point, well maybe now he'll realize the number of viable cells in his stored samples might be quite low and hence consider a stepped starter.

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If I'm starting fewer viable cells, I need more volume according to the Mr. Malty, but does this change if I step up in increments. If so, why?

kwantam said:

If you had waited until the fourth month, and decided to brew a high gravity beer, you'd be starting with only ~7B cells (25%), so instead of jumping straight to a 1L starter (which honestly would be OK), you'd make a 500mL starter and then step that up into a 2L starter before pitching.

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Sounds like either way would work, but wouldn't I have a dramatically different cell count if I used 1 liter vs. stepping 500ml to 2L? If I stepped it from 500ml to just 1L, should I expect a similar cell count that I'd have without the 500ml step?

Sorry for so many questions after you've already done a great job at answering my initial post. I just don't get what stepping up adds to the equation.

I'll try to clarify the step up vs. no step up issue:

Whenever you pitch (be it a starter or a whole batch of beer), the initial yeast concentration is important on if you get a nice, healthy population of yeast.

Take an extreme example: Theoretically, you could pitch one yeast cell into a starter and it would be capable of fermenting it out. The resulting yeast count after fermentation would even be similar had you added more yeast from the get-go.

There are a number of problems you would run into however:
1.it would take alot longer to ferment that starter (because of the additional rounds of reproduction the yeast would go through).
2. Risk of contamination would be much greater (because the yeast would likely be out-competed by faster growing bacteria that might have gotten in there)
3. The resulting yeast population would be stressed, and the quality would be lower because of the under-pitching. That would be one tired yeast cell!

Long story short: Its not so much about yeast numbers, it is also about yeast quality. You want to avoid the the stresses of under-pitching; regardless if you are pitching for a starter vs. a 5 gallon batch of beer. This is where stepping-up comes in. Stepping-up is a way of babying the yeast culture to prevent the stresses of under-pitching. That's it.

When do you need to do it? Well, that's a tough one to answer. You can try to get an approximating of yeast count and viability from Mr. Malty, but when in doubt err on the side of over-pitching to prevent under-pitching. You might want to consider stepping-up any yeast stock that is more than 3-4 months old for example.

Hope this helps.

carrotmalt said:

If I'm starting fewer viable cells, I need more volume according to the Mr. Malty, but does this change if I step up in increments. If so, why?

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Yes.

By stepping up you do not need the larger-end volume because at your next-to-last step virtually all your yeast is new/fresh/healthy since it was only grown at the previous step.

carrotmalt said:

Sounds like either way would work, but wouldn't I have a dramatically different cell count if I used 1 liter vs. stepping 500ml to 2L? If I stepped it from 500ml to just 1L, should I expect a similar cell count that I'd have without the 500ml step?

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I'm sorry, I was unclear. In both scenarios, the final starter volume would want to be 2L (since you're pitching into a high gravity wort). You could get there either by way of 500mL or 1L as the first step, and then 2L as the second. 500mL is slightly preferable because it's gentler on the yeast.

I'll go back and edit my post to reflect this.

So the final cell count is roughly going to be the same at a given volume regardless of the number of steps, but due to under pitching stresses, they might not be of the same quality. So if the percentage of healthy yeast is lower, we need more of them, hence the larger starter as the production date gets older.

Well, I guess it takes a whole team of HBTers to straighten me out on this, but I think I get it.

I've never understood how underpitching "stresses" the yeast. Fewer yeast cells in an abundance of nutrients should make the yeasties happier, I would think. That said, I think the yeast will have to compete with whatever stray bacteria are present in the starter (or wort) and pitching a larger count would help a lot in that regard. Also, asking the yeast to reproduce fewer times (ie fewer generations) might minimize the risk of mutation/genetic drift, etc. It's for this reason that most people who bank yeast won't go much more than 4-5 generations from the initial vial.

What I do, and it's clearly a variation on the above, is a "linear" yeast bank. I make a large 2+ liter starter and before I cold crash it, I decant a well mixed pint of the starter into a sterile 1 pint mason jar. I refrigerate this and use the sludge from this jar to make my next starter. I only do this about 4-5 times before I start with a new vial/smack pack. They are easily good for 3-4 months in the fridge.

L