Can a yeast starter be too big?

[frankvw]

I'm making yeast startrs using a stir plate. My starter wort has an OG of 1.040. I'm growing up a yeast sample that has few viable cells in it (liquid yeast and fermenter sludge several months old, sediment harvested from a bottle, etc.)

What is better in this case: starting with a small starter (e.g. half a litre) and stepping that up to one and then two litres, or simply making a two litre yeast starter right off the bat? With "better" I mean what will give me the largest population of healthy cells? And, more to the point, why?

 

[InLimbo]

what will give me the largest population of healthy cells? And, more to the point, why?

The way to get the largest population of healthy cells will be to pitch a single yeast culture from a single source. Mixing at least three different yeast strains who may all have different preferred fermentation methods AND different levels of cell viability is like playing Russian roulette. I would choose one of your sources to propagate, instead of dumping all three of them together. Unless you're going to be counting cells on a slide under a microscope, there is no way of telling how much yeast you're going to be pitching into your beer.

Whatever you end up doing, I would recommend decanting most of the liquid off of your started before pitching, so I think the volume is going to be irrelevant. Yeast cell count is obviously what's important.

 

[frankvw]

The way to get the largest population of healthy cells will be to pitch a single yeast culture from a single source. Mixing at least three different yeast strains who may all have different preferred fermentation methods AND different levels of cell viability is like playing Russian roulette.

Sorry... I may have written that in a confusing manner. I'm growing up three different cultures separately, not mixing them. So I'll be doing this three times. In each case I'm not starting with a vial of liquid yeast or smack pack but with something that, given the age of the sample, has a limited number of viable cells in it.

I completely agree with what you say about the inadvisability of just dumping three different yeast sources together and relying on hope and luck from there on in.

So. With a small number of viable cells in a starter culture, what would be better: starting small and stepping up, or starting with a larger volume right away? And why?

 

[Vale71]

Starting very small. With single-cell cultures you start with a few milliliters and then step it up from there.
Too low of a pitching rate will stress out the yeast as it needs to adjust the wort to its needs, f.e. by dropping PH by as much as 1.00. Too low a pitching rate will overwork the yeast and if its viability is already marginal that might just be too much to take. The greatest risk when not working in 100% sterile conditions is of course that something unwanted might prevail and take over the starter before the stressed yeast manages to cope.

 

[frankvw]

Starting very small. With single-cell cultures you start with a few milliliters and then step it up from there.

I don't have a single cell culture (I don't have the means of isolating a single viable cell) but given the state of the starting samples, viability won't be high.

Too low of a pitching rate will stress out the yeast as it needs to adjust the wort to its needs, f.e. by dropping PH by as much as 1.00. Too low a pitching rate will overwork the yeast and if its viability is already marginal that might just be too much to take.

I see. That is exactly what I needed to know.

The greatest risk when not working in 100% sterile conditions is of course that something unwanted might prevail and take over the starter before the stressed yeast manages to cope.

Noted. Sanitary conditions will be vital since any contaminant is likely to multiply at least at the same rate as that of the yeast, possibly far greater.

Thank you!

 

[IslandLizard]

I'm growing up a yeast sample that has few viable cells in it (liquid yeast and fermenter sludge several months old

You may be surprised how vital a harvested slurry can be after a few months in the fridge, since it has 4-5x the number of cells you pitched. Decant most, mix it up, ladle or pour out a tablespoon (or 2) and make a starter.

Always use a yeast calculator such as:
BrewUnited's Yeast Calculator
I use that one most often to estimate needed cell counts and viability from fresh packs and saved out starters.
The WLP PurePitch sleeves retain as much as 90% viability over the first 3 months. 70-50% after 6 months. Tweak the appropriate cells in the calculator to reflect those numbers.

Mr. Malty
I really like the "pitch from slurry" tab to estimate cell counts from slurries. Then transfer to the one above or to the starter tab.

 

[IslandLizard]

What is better in this case: starting with a small starter (e.g. half a litre) and stepping that up to one and then two litres, or simply making a two litre yeast starter right off the bat? With "better" I mean what will give me the largest population of healthy cells? And, more to the point, why?

Just re-read your OP: Step starter or single step?

[EDITS]

It largely depends on how many cells you think you have and limits on inoculation rate.

• Inoculation rate too low (<25 million/ml), e.g., large starter volume and only a small amount of viable cells, she won't grow well.
• Inoculation rate too high (>100 million/ml), e.g., the starter is more like a vitality starter, there's not as much growth, she's being prepped for the pitch, waking up so to speak, building up sterols and her strength so she can bud.

These are general guidelines of course, target numbers.

If you harvested from a previous fermentation and you now have a pint to a quart of dense settled slurry (mixed with trub) in the fridge, estimate her age, use one or more of those yeast calcs, to see how many cells you may have, let that be your guide.

1) If it's less than 2-3 months old, you could pitch a cup or so directly. Or make a 1/2 - 1 liter vitality starter from that cup 4-6 hours ahead of pitching to wake her up, preparing her for the big job. Pitch the active starter as is, no cold crashing and decanting.

2) If it's older, say up to 6-12 months old, it may still be fine to pitch as is, just use more. Either make a vitality starter, pitch as is, or if time allows, a traditional starter, a few days to a week ahead, and overbuild it to save some out to make a starter from for a next batch.

3) If it's much older, say well over a year, and you indeed may only have a small amount of viable cells left in your harvested slurry. Making a step starter from some of it (e.g., 2 Tbsp) would be the better option, due to limits on inoculation rate. Then crash, decant and do a next step.
Before your first step you could rinse ("wash") the whole or half the slurry to help separate the live yeast from the boat load of trub and dead cells. Live cells remain in suspension while everything else settles out.
In this case use the supernatant for propagation, of course, including its volume (relatively large) in your yeast starter (total volume / gravity) calculation.

4) If it smells really bad, like marmite/vegemite, burnt rubber, or worse, don't use it, it has autolyzed. Ready for composting.

 

[bracconiere]

Noted. Sanitary conditions will be vital since any contaminant is likely to multiply at least at the same rate as that of the yeast, possibly far greater.

i'm actually not sure for yeast, but i know when i clone mushrooms, i soak them in a dilute bleach bath to sanitize them, bleach kills bacteria and spores, not fungus....so maybe a couple drops of bleach?

 

[IslandLizard]

i'm actually not sure for yeast, but i know when i clone mushrooms, i soak them in a dilute bleach bath to sanitize them, bleach kills bacteria and spores, not fungus....so maybe a couple drops of bleach?

In pro environments yeast is typically washed with a Chlorine Dioxide solution, which kills bacteria and most spores. But it also affects the yeast, so this is done right before pitching. Yeast rinsing (often, but incorrectly referred to as "washing") is what we homebrewers do to separate yeast from trub. It merely uses water.

Chlorine Dioxide is also used to chlorinate municipal drinking water.

 

[Bassman2003]

Amount of pitched cells wanders into the artistic side of brewing. Yes, pitching a decent to large amount of healthy cells is usually the best but yeast are living organisms and they can be directed or bent to act in a certain way. Underpitching does stress the yeast cells because they have to work harder with all of that food around but for certain beer styles, this stress creates certain flavors that might be desirable. On the other side, pitching a lot of yeast results in minimal stress which leads to a clean beer.

So the amount of yeast you pitch is just another tactic or tool to execute your recipe and get the beer you want.

 

[Day-Day]

I don't have a single cell culture (I don't have the means of isolating a single viable cell) but given the state of the starting samples, viability won't be high.

A single cell culture is a culture (or group) of intact single celled organisms (yeast in this case), not an actual single cell.

What he's saying is that with single cell cultures (yeast slurries, starters, etc), if the viability is low, you need to step them up... often with only a few milliliters of weak wort.

When I bottle culture I'll use maybe 75 ml of 1.020 wort per bottle (dregs of) then step it up from there.

It's not best practice to take a culture of yeast with potentially low viability and throw it into a larger ( 1 liter or larger) starter of wort, it's healthier and actually more productive for the yeast if you step it up.

I'd be shocked if anyone is isolating single cells of any kind in their home breweries.

 

[Vale71]

A single cell culture is a culture (or group) of intact single celled organisms (yeast in this case), not an actual single cell.

Sorry but a single-cell culture is actually a culture that started from a single cell isolate and not a culture of single celled organisms as opposed to multicellular organisms.

What he's saying is that with single cell cultures (yeast slurries, starters, etc), if the viability is low, you need to step them up... often with only a few milliliters of weak wort.

Nope, that is not what I meant or said. I was using the single cell example to show that starters need to be as small as necessary in order to ensure proper inoculation rates.

I'd be shocked if anyone is isolating single cells of any kind in their home breweries.

Feel free to be shocked, there are homebrewers who deep-freeze yeast and then start cultures from a single cell isolate.

 

[Day-Day]

Sorry but a single-cell culture is actually a culture that started from a single cell isolate and not a culture of single celled organisms as opposed to multicellular organisms.




Nope, that is not what I meant or said. I was using the single cell example to show that starters need to be as small as necessary in order to ensure proper inoculation rates.




Feel free to be shocked, there are homebrewers who deep-freeze yeast and then start cultures from a single cell isolate.

Sorry if I twisted your words, but I still disagree that people are isolating single cells in their homes. They may be isolating pure cultures (monoculture) similar to how hobby mycologist isolate and culture mycelium. Freezing yeast, streaking it on a plate, growing it out and isolating is not a single cell but rather a single culture. There is quite the difference.

NOW, if people really are, and they may be isolating actual single cells via centrifuge or other single cell isolation techniques (again I'm not an expert), I'd be shocked.

I'd really love to be proven wrong here... that would be inspiring if us brewers are actually going these lengths to store and propagate our cultures via single cell isolation.

I was a hobby mycologist for 10 years before I started brewing, so I'm rather familiar with fungi, and I used to have a laminar flowhood in my home to clone and isolate monocultures (pure cultures) so I'd assume that is what home brewers are doing.

More importantly, my intention was not to offend anyone or twist up words, but I feel strongly about monoculture vs single cells so please prove me wrong.

Here's a few resources

https://www.labome.com/method/Cell-Isolation.html (Single cell isolation)
https://bootlegbiology.com/diy/isolating-yeast/ (Strain isolation)
https://bootlegbiology.com/diy/banking-microbes/ ( Freezing and storing cultures)
http://braukaiser.com/wiki/index.php/Growing_Yeast_from_a_Plate (Single cell colonies.. i.e. cultures, at the home level)

 

[Vale71]

Did you actually read the last link you provided? If done right plate streaking will lead to isolating single cells and that's not that hard to do. It's single-strain multi-cell cultures that is actually very hard to do as you'll need expensive specialized equipment to select for the desired strain, without which any multi-cell culture will inevitably be multi-strain (or rather, multi-species) due to contamination of the source. What is harder in a home environment is keeping sanitary conditions, which means that the purity of subsequent large-scale cultures will be more questionable than if the same were done in a pro environment.
That said, the number of hard-core amateurs that are doing this is certainly minuscule. Most homebrewers who stock frozen yeast indeed simply grow cultures from frozen stock starting with a small sample and not with a single-cell, but that doesn't mean that there aren't a select few who go the whole way.

 

[Day-Day]

Did you actually read the last link you provided? If done right plate streaking will lead to isolating single cells and that's not that hard to do. It's single-strain multi-cell cultures that is actually very hard to do as you'll need expensive specialized equipment to select for the desired strain, without which any multi-cell culture will inevitably be multi-strain (or rather, multi-species) due to contamination of the source. What is harder in a home environment is keeping sanitary conditions, which means that the purity of subsequent large-scale cultures will be more questionable than if the same were done in a pro environment.
That said, the number of hard-core amateurs that are doing this is certainly minuscule. Most homebrewers who stock frozen yeast indeed simply grow cultures from frozen stock starting with a small sample and not with a single-cell, but that doesn't mean that there aren't a select few who go the whole way.

Taken From the Last Link

"In order to ensure that you are only using yeast colonies that have grown from a single yeast cell only consider single round colonies of yeast. Yeast colonies are off-white with dull looking surface. Some of your streaks on the plate should have produced such colonies."

Again, you're dealing with colonies on those plates.

"Using the sterile inoculation loop pick one colony at a time and place them into the wort in the vial. Repeat this until you have picked a few colonies. You could also pick only one. This would give you a pitch of yeast grown from a single cell. But I like to mix it up with a few colonies".

Again, picking one colony. They are explicitly dealing with colonies in that link.

I know they keep referring to colonies grown from a single cell, but nowhere in there does it claim that this completely isolates a single cell... maybe we're getting to deep into semantics. What i gathered from that is that you are isolating a colony (i,e. pick one) that was grown from a single cell... you are not actually isolating a single cell on those plates.

There is a difference between isolating a colony that was derived from a single cell (last link, and completely isolating a single cell and starting a new colony from there.

Again, I never said that it was impossible at the home level.

Is anyone on this board actually isolating SINGLE CELLS in their home and starting a new colony from an actual single cell?

I'd love to see it if it's being done.

Are you actually saying that strain isolation is harder than single cell isolation?

That's pretty backwards. Mushrooms are multi-cellular fungi, and home mycologist (even myself) have easily and effectively isolated single strains with nothing more than, agar plates, scaple, alcohol lamp, pressure cooker, and laminar flow hood (and a few other odds and ends).

Being sterile (not sanitary as you mentioned, because there is a clear difference) is critical, hence why some breweries out source their yeast storage and propagation to companies who have the means to deal with delicate single cells. Also why home mycologist build and utilize laminar flow hoods, as do surgeons in their operating rooms.

 

[Vale71]

We're drifting off topic here so this will be my last post on the subject.

You seem to think that to start a single-cell culture you need to physically pick up a single cell and start culturing it somewhere else. This is simply not the case. When you streak a sample you'll end up isolating a single cell from which a single colony starts to grow. You then pick up the colony, which is much easier than manipulating the single original cell, and then keep on propagating it further until your target cell count is reached. That colony is the first step in your single-cell culture. The fact that you then keep on propagating it does not negate that fact.
This is completely different from picking a sample that even if small will still contain millions of cells and starting from there, as there is no guarantee that the millions of cells are all of the same strain. With the other method you are guaranteed this but of course you cannot know whether you picked up the desired strain or some foreign contaminant, but testing for that is definitely beyond the reach of even the most dedicated amateur.

 

[Day-Day]

Here's the issue with your logic, when you streak a sample you do not end up with a single cell. Do you have any idea how small a single yeast cell is, you would not be able isolate it simply by streaking a sample onto an agar or other media based plate.

I'm not sure where you got the info that streaking plates leaves you with one single viable cell... if you feel strongly that you are correct, provide some concrete evidence, otherwise you are just assuming that is what people are doing.

The whole point of isolating on agar plates is to get your healthy yeast colony away from potentially other nasty colonies, whether it be other yeast strains, bacteria etc. You can do this because it's on a single plane and they grow away from each other unlike when it's in solution.

I never stated that the only way to start a single cell culture is to physically isolate a single cell.. although you can do that... but that's not what home brewers do.

I'm sorry man, I've worked with cultures on agar plates, I've worked with slants, never ever have I heard that streaking leads to a single cell, you isolate a pure culture. I think this is where you are confused.

I don't know what a "single culture- cell" is, I'm sure you meant single cell culture.

FYI culture/colony implies there has been growth, a single cell in it of itself is not a culture.

I'm fine with being wrong, so I'd love to see this evidence of people simply streaking agar plates and arriving at a single cell like you just stated.

 

[Vale71]

"In order to ensure that you are only using yeast colonies that have grown from a single yeast cell only consider single round colonies of yeast. Yeast colonies are off-white with dull looking surface. Some of your streaks on the plate should have produced such colonies."

Sorry, don't know how to make it more clear than that, considering it was already quite self-explanatory.

As for the term single-cell culture I'm not responsible for scientific nomenclature and really can't help you out there. FYI microwells is the original technique used to isolate single cells but as it requires a microscope it's more expensive than plate streaking.

I'm fine with being wrong, so I'd love to see this evidence of people simply streaking agar plates and arriving at a single cell like you just stated.

I'd recommend buying the book "Yeast" by White and Zainasheff which explains it all in a very clear and concise way. Anyway I'm done with this diversion as I don't want to hijack the thread.

 

[Day-Day]

Dude you completely missed the entire point of my argument

My point was not that home brewers can't isolate single celled organisms or even single cells. My point was that the technique that home brewers are using is actually isolating colonies and not reducing the colony to an actual single cell.

My argument was quite clear. That link is not saying that they are isolating single cells ( an individual cell) , they are isolating colonies of a single celled organism that ORIGINATED from single cells in the wild. They themselves are not isolating an actual single cell.

Read that sentence more carefully dude, the person utilizing that plate is isolating a colony of single celled organisms originally grew from a single cell.

[Removed - mod]