Estimating Yeast Cell Counts in Fresh Starter

[thadius856]

Understood completely. I think.

Here's where the rubber meets the road to test the viability of a yeast bank. What is the highest gravity wort that can be inoculated with a starter of no more than 3.8L (1 gal) that began with an estimated 45b cell vial (without stressing past 10:1)? This assumes that a home brewer can reasonably stir plate up 1 gallon of starter (two 2L flasks with FermCap on two plates, or one 1-gallon glass jug on a single plate).

If we assume 1.040 starter, we're at just shy of 380g DME per 3.8L batch. Starting with an estimated 45b cell, that's an estimated finish of 615b cells at 1.5b cells/g DME. Space is not the limiting factor here then, because 10:1 would be 495b finished.

10 gal of 1.060 wort would require a pitch of 417b, so that's our neighborhood. (I used Mr Malty because I'm still trying to wrap my head around inoculation rates in m/ml.) We don't pass 495b cells required until about 1.072.

So unless you're making 10g batches of Barleywine, it seems that one 2L preform tube can inoculate up just about any 10g batch with stressing the yeast past 10:1, assuming you have a minimum 45b cells viable.

Does this math check out?

 

[Kaiser]

So unless you're making 10g batches of Barleywine, it seems that one 2L preform tube can inoculate up just about any 10g batch with stressing the yeast past 10:1, assuming you have a minimum 45b cells viable.

I'd say that is correct for ales. I brew 5 gal batches and have been fine with just a 2 L flask for most of my lager yeast propagations as well.

A 5L flask is nicer b/c a 2 l starter gets a nicer vortex in the larger flask.

Kai

 

[thadius856]

I'd say that is correct for ales. I brew 5 gal batches and have been fine with just a 2 L flask for most of my lager yeast propagations as well.

A 5L flask is nicer b/c a 2 l starter gets a nicer vortex in the larger flask.

Kai

While a 5L flask would be nice, it seems a bit comically large for most users here. And also, much more expensive than two smaller flasks.

I personally use 1 gallon cider jugs for the moment being. They're a pain in the butt to get the stir bar to stick because of the concave bottom, but they work. At least, they do now that I use grade 50 neodyium magnets. No way they'd work with even entry-level neodynium magnets, let alone ceramic magnets.

 

[jww9618]

This is a great thread. I have been way over pitching.

 

[Kaiser]

I don't believe in anti foam agents, so I do like the head space that the 5 L flask gives me. Mine was only $40 at MoreBeer. The 2 L ones cost about $20 each.

Kai

 

[Coff]

Yea this has been an awesome thread to follow. I dont have anything to add but I am learning alot.

 

[thadius856]

I don't believe in anti foam agents, so I do like the head space that the 5 L flask gives me. Mine was only $40 at MoreBeer. The 2 L ones cost about $20 each.

Kai

You don't believe in them? I assure you, they exist.

I've seen the 5L flask you're referring to. NB sells it, too. Terrible reviews there of it cracking under the lightest use conditions. So I avoided it.

If I was going to buy flasks (I will, eventually), I'd go for the Pyrex or Kimax brand ones. They're the only two brands I've seen that offer the rough service flasks, usually with "heavy duty" or "thick" in the title description. They're noticeably thicker all around in the pictures.

Those are close to $25-30 for a 2L and $80-100 for a 5L.

I'll get there, eventually. Just better places to put my equipment money at the moment.

 

[Kaiser]

You don't believe in them? I assure you, they exist.

I've seen the 5L flask you're referring to. NB sells it, too. Terrible reviews there of it cracking under the lightest use conditions. So I avoided it.

I must have been lucky. The one I have seems very sturdy and I have been using it for a while.

 

[Paulgs3]

http://www.teknova.com/YPD-AGAR-PLATES-100MM-p/y1000.htm

Make serial dilutions, 1 ml of yeast into 9 ml of diluent (boiled water, uninoculated media, whatever you are using to grow your yeast...). This is a 1 to 10 dilution.

Take one ml of that and put into another 9 ml. We are now at a 1 to 100. You want to do this nine times so you have nine dilutions total in front of you. To explain what is going on, if you originally had 100 cells in 1 ml, a 1:10 dilution now gives you 10 cells per ml, a 1:100 dilution now gives you 1 cell per ml. You are shooting for 100 billion cells cells, so I would dilute out to one billion which is 9 1:10 dilutions.

Take one ml of the last dilution and pour it onto the agar plate. Its a rather large size to put on a plate but it should make spreading easy. Stick it somewhere warm for a good 24 hours.

Count the colonies, each colony will represent 1 billion cells per ml. To find out total cells, multiple the number of colonies counted by 40 ml which is the volume of your stock. That will give you an idea of where you are based on the principle of one cell = one colony.

If you count 10 cells, multiply by 40 and we get 400. You have 400 billion cells in your sample.

 

[novaris]

this has been a great thread to help get my head around storing and using yeast. Thanks for sharing the results of experiments.

 

[WoodlandBrew]

http://www.teknova.com/YPD-AGAR-PLATES-100MM-p/y1000.htm
Take one ml of the last dilution and poir it onto the agar plate. Its a rather large size to put on a plate but it should make spreading easy.

When I have pours that large of a sample onto a plate it never absorbs. So colonies don't form. The yeast just grows in the pools. I've had this happen with both agar and gelatin. Is there a trick to it?

I use one drop which is about 20ul.

Plate counts are great in that they are achievable with things that most people have, and it tells you how many cells are capable of dividing. With 9 serial dilutions I would think the errors would add up fairly high, but it would probably get you closer than estimating by volume.

 

[ScrewedBrew]

I'll add my reply to dirty up this thread and get on the subscribe list.

Kai, and Woodland, thanks for contributing your experiments to help out us novices.

My question: When stepping starters, is each propagation considered another generation? I just started a step yesterday harvested from a starter made from a smack pack (3522 Ardennes). I took about 1.5 ml of dense slurry about 3 weeks old. I'm guessing that would be about 1 B viable cells Pitched it into .27 ltrs of wort. 2nd step will be 1.2 ltrs. Third step will be 1 ltr of wort from my final batch. Using brewer's friend calculator, that will give me 103 B cells to inocculate my remaining 2.25 gallon batch.

Does this mean that I'm on fourth generation yeast by the time I pitch my batch?

 

[thadius856]

I personally only consider it a new "generation" once it's either been fermented in a hopped wort or frozen.

The reason for counting "generations" this way is because most of us don't have conicals like the pros. Thy can harvest the "cream" in the middle of the yeast cake, leaving the less and more flocculant mutants. The best we can do is get a homogeneous solution by swirling.

There's no use thinking of a generation as occuring each time yeast is multiplied. A single batch of wort could have many more than four generations in it, by that reasoning, assuming that a chart of the generations would take on a bell curve shape, with some specific standard deviation. Strictly speaking, there's no reason why the 1000th generation is any worse off than the 1st generation, assuming we started with an isolated, phenolic positive Saccharomyces culture, have kept out unwanted mutants, and have kept it free from infection of other strains/bacterium.

I'm sure Woodland and Kaiser could speak more specifically on the subjects, but hopefully that answers your question satisfactorily.

 

[Photopilot]

While a 5L flask would be nice, it seems a bit comically large for most users here. And also, much more expensive than two smaller flasks.

I think the 5L flasks are actually kind of petite so I went with this.

Not really but I needed bigger than my 2L and this was the same price as the 5L at $40 and is also made of Pyrex. As you can see I made a fairly large starter. I have saved this yeast in mason jars and will use it to brew 36 gallons before growing another batch for future beers.

 

[WoodlandBrew]

Your right that during fermentation there are several generations. A more correct term would be a passage. This is used to describe when a culture is transferred to a new medium. I keep track of passages.

 

[d_striker]

Kai,

I've read your blog at least 4 times and sort of grasp what you're saying, but I am having trouble fully understanding. I want to make sure that I am interpreting your theory/work correctly as I'm a little slower than the rest of you guys...

If (initial cells < 1.4 Billion/gram extract)
yeast growth is 1.4 Billion / gram extract
​

Assuming one uses 107g DME in 1L water to create 1.040 starter wort:

-It doesn't matter how many cells one starts with, under 149B cells (1.4*107), and the growth would always amount to 149B cells? In other words starting with 30B cells OR 80B cells (random numbers) would both grow 149B cells?

-Is this number just the new cells grown or the total number of cells after growth?


One other random question as I occasionally make 1.040 starter wort from grain and pressure can it. Does 1L of this 1.040 wort also contain 107g of sugar?

 

[thadius856]

Kai,

I've read your blog at least 4 times and sort of grasp what you're saying, but I am having trouble fully understanding. I want to make sure that I am interpreting your theory/work correctly as I'm a little slower than the rest of you guys...

If (initial cells < 1.4 Billion/gram extract)
yeast growth is 1.4 Billion / gram extract
​

Assuming one uses 107g DME in 1L water to create 1.040 starter wort:

-It doesn't matter how many cells one starts with, under 149B cells (1.4*107), and the growth would always amount to 149B cells? In other words starting with 30B cells OR 80B cells (random numbers) would both grow 149B cells?

-Is this number just the new cells grown or the total number of cells after growth?


One other random question as I occasionally make 1.040 starter wort from grain and pressure can it. Does 1L of this 1.040 wort also contain 107g of sugar?

I'm not Kai or Woodland, but I understood it a little differently.

I think what he was saying was that when your target cell count for a starter is many times the original cell count of the vial that the initial doesn't matter so much. The yeast will multiply until they run out of food, at which point the difference in the inoculation rate is very small.

In other words, the final cell count approaches a limit. Think of it as a logarithmic curve. The further x is from 0, the lower the positive slope.

What's the limit? Well, I'd have to assume the limit is the maximum inoculation rate for that strain, but honestly, I'm not sure about that. I know stepped starters hit a wall based on volume at some point, no matter how much sugar you throw at them, which is where I derived that hypothesis.

Of course, we're starting with a positive quantity, so if we let y be the cell count and x be the amount of available sugars, then the y-interval is the cell count of the starter vial or smack pack.

 

[d_striker]

I'm not Kai or Woodland, but I understood it a little differently.

I think what he was saying was that when your target cell count for a starter is many times the original cell count of the vial that the initial doesn't matter so much. The yeast will multiply until they run out of food, at which point the difference in the inoculation rate is very small.

In other words, the final cell count approaches a limit. Think of it as a logarithmic curve. The further x is from 0, the lower the positive slope.

What's the limit? Well, I'd have to assume the limit is the maximum inoculation rate for that strain, but honestly, I'm not sure about that. I know stepped starters hit a wall based on volume at some point, no matter how much sugar you throw at them, which is where I derived that hypothesis.

Of course, we're starting with a positive quantity, so if we let y be the cell count and x be the amount of available sugars, then the y-interval is the cell count of the starter vial or smack pack.

I think we're understanding it the same for the most part but you're expressing it a different way. We're both understanding Kai's model to say the final number of yeast cells is dependent on the amount of sugar in the starter wort, not the amount of starting cells.

I ran my random numbers through the BF calculator using Kai's stir plate model.

Assuming 1L of 1.040 starter wort:

#Start---------# New Cells Created--------#Total Ending
30B-----------------160B------------------------190B
80B-----------------160B------------------------240B

As you can see, starting with either 30B or 80B cells will create 160B cells according to Kai's model. This will hold true for any number of starting cells up to 159B cells (1.4*114) assuming 1L of 1.040 wort.

 

[diegobonatto]

Please let me give a small contribution to this interesting discussion. I'm working with yeast genetics and metabolism a while (more or less 15 years) and it is well understood that all microrganisms (including yeast) follow a growth curve: lag phase, exponential phase, stationary phase, and death. Of course, the growth curve for yeast is a little bit more complicated because it display a diauxic transition, a change from fermentative metabolism to respiration, where the growth rate lower during exponential.

However, if we consider the stationary phase of growth for the major yeast strains, all yeast strains reach the maximum concentration of 1-3x10^8 cellls/mL of culture medium (wort, YEPD, or other rich medium that are plenty of complex nutrients). It is possible to achieve high cell numbers (above 3x10^8 cells/mL) by using special equipments (e.g., bioreactors), where the physico-chemical parameters are better controlled (pH, O2 concentration, nutrient levels, etc...).

In our case (homebrewer's case), we always perform what is called a "batch culture", that is the use of erlenmeyers, growlers, etc. with shaking to grow yeast cells. Thus, the maximum concentrations of cells always will be in the range of 1-3x10^8 cellls/mL, even if more sugar or DME was added in the culture.

So, if you have an initial total number of 5x10^10 (50 billions) cells and added this to 1 L of wort, your final cell concentration will be 5x10^7 cell/mL. The cell will divide in 24 hours or less and reach (more or less) 2x10^8 cells/mL, giving a total of 2x10^11 cells (200 billions). I am considering 100% of viability in all cases.

If I stepped up, and inoculate this 2x10^11 cells in 4 L of wort (cell concentration in wort = 5x10^7 cells/mL), and let it grow, I will end up with more or less 8x10^11 cells (800 billions of cells). It should be noted that these numbers are theoretical, and many factors can influence the final number of cells in the wort. Viability is an important factor, as well as the initial sugar concentration in wort, dissolved O2, bacterial contamination, etc...

There are many protocol applied for yeast counting: neubauer chamber counting, colonies counting, dry mass measure, spectrophotometry. All methods give an approximation of the real number of cells in wort. The best way to get a value that is closest to the real number of cells in wort is to make, at least, three independent cell countings using serial dilution and neubauer chamber. From these raw data, the average + standard deviation can be calculated. Low standard deviation give us a good precision of cell number estimation.

Please remember that there are not a definitive method to determine cell number. Moreover, viability determination is also very important, and complements the cell counting methods.

 

[WoodlandBrew]

... all yeast strains reach the maximum concentration of 1-3x10^8 cellls/mL of culture medium.

So 100 to 300 billion cells per ml is the brick wall that a cell culture will hit?

From what Kai, and Jimal have seen growth rates seem to drop off somewhere before that. (That's what I have seen as well) Perhaps the with inoculation rates of about 200 million per ml there is a foam wall? There is growth, but it is somewhat restricted. When making starters for home brew It sounds like that brick wall is pretty far away. In the cell counts I have done the highest I have seen is 4 billion per ml.

 

[diegobonatto]

In this case, 100-300 millions of cells/mL of culture (instead of billions). Four billions of cells/mL seems to be very high in a conventional starter, even for those yeast strains that have high growth rates, like belgian strains. How did you estimate this number of cells? Just curiosity...

 

[WoodlandBrew]

In this case, 100-300 millions of cells/mL of culture (instead of billions). Four billions of cells/mL seems to be very high in a conventional starter, even for those yeast strains that have high growth rates, like belgian strains. How did you estimate this number of cells? Just curiosity...

Right 1-3E8 is 100-300 million. I shouldn't try to do even simple math in my head after a few beers. :-/

What you are saying makes much more sense now. It's good to know that we are all seeing about the same thing as someone with as much experience as you.

Cell counts were done with MB stained on a hemocytometer.
This is my procedure: http://woodlandbrew.blogspot.com/2012/11/counting-yeast-cells-to-asses-viability.html

4 billion per ml was the highest, but I have seen most of WLP566 and S-04 come in about there for the thick cell density. The actual culture density is about 100-200 million per ml. I've done a few hundred cell counts, and have pretty consistent results.

 

[diegobonatto]

Glad to know that I could help. In fact, neubauer counting + MB staining is a standard method to evaluate cell viability and number. Scientific literature reports some drawbacks by using this technique, seems that quiescent (non-dividing but viable cells) can be stained with MB and, thus, leading to an underestimation of the number of viable cells. The use of fluorescent dyes is more advisable to evaluate viability, but this is unpractical for homebrewers because epifluorescent microscopes ($$$) are needed.

 

[Kaiser]

Now I know why d_striker posted in the science forum. I neglected this thread. So here is my answer to d_striker’s question:

The lower yeast growth per sugar is something I observed in my experiments while I don't have an exact justification for the actual numbers yet I have a theory of what's going on.

Let's assume two things:
(1) - 1 gram of extract gows X Billion new cells
(2) - yeast only bud once they consumed enough resources to grow a new cell

This means that if there are more yeast cells per extract than can be grown from that extract not every cell will be able to grow a daughter cell. In an idealized culture (all cells consume nutrients at the same rate) no new cells should be able to grow since none of the cells will be able to consume enough nutrients to grow a daughter cell. But the culture is not ideal which means some cells will be able to consume enough nutrients to grow a daughter cell while others won't. The ones who don't grow buds will consume extract but don't actually contribute to cell growth (though it makes them healthier and better prepared for fermentation). This mechanism also means: the more initial cells are trying to consume the existing extract the fewer will be reach nutrient levels sufficient for budding. That's why I expect cell growth to drop with increased initial cell density.

I'm still working on solidifying or disproving this theory with additional experiments. If the theory is correct, the drop in yeast growth should be earlier and more pronounced with old cultures compared to fresh cultures since old cultures have depleted their reserves further. I have experimental data on this, but the results are not as clear as I hoped them to be. I think I have to control a few more parameters.

But I do want to hear more from diegobonatto and have a few questions. I changes the exponential notation to Billions to make it more readable. At least for me

However, if we consider the stationary phase of growth for the major yeast strains, all yeast strains reach the maximum concentration of 100-300 B cellls/L of culture medium (wort, YEPD, or other rich medium that are plenty of complex nutrients). It is possible to achieve high cell numbers (above 300 B cells/L) by using special equipments (e.g., bioreactors), where the physico-chemical parameters are better controlled (pH, O2 concentration, nutrient levels, etc...).

What is causing this limit? is it simply the available amount of nutrients or are there other mechanisms at work? I don’t have my data here at work but tonight I want to check the max culture densities that I observed.

In our case (homebrewer's case), we always perform what is called a "batch culture", that is the use of erlenmeyers, growlers, etc. with shaking to grow yeast cells. Thus, the maximum concentrations of cells always will be in the range of 100-300 B/L, even if more sugar or DME was added in the culture.

I’m wondering about that. One limiter might be the amount of alcohol that is produced when the initial sugar concentration is high.

… It should be noted that these numbers are theoretical, and many factors can influence the final number of cells in the wort. Viability is an important factor, as well as the initial sugar concentration in wort, dissolved O2, bacterial contamination, etc...

It’s exactly those factors that affect yeast growth and the final yeast cell count that I’d like to get a handle on. I think one major factor is amount of nutrients available in the wort. But I don’t think that viability plays a large role here. Especially if the amount of growth is significantly more than the initial population. My thinking is that non-viable yeast cells will not consume any of the available nutrients and leave more for the others. That means that while non viable cells may not be able to grow other cells should be able to grow a bit more since they have more nutrients available.

Kai

 

[diegobonatto]

Hello Kai!

Let me answer your questions:

Let's assume two things:
(1) - 1 gram of extract gows X Billion new cells
(2) - yeast only bud once they consumed enough resources to grow a new cell

The first proposition assumes that 1 g of yeast should have more or less some billions of new cells. However, remember that you will have a mixture of new and old cells compounding this 1 g of yeast biomass. In fact, the proportion of new/old cell depends on the viability of culture and the initial cell concentration inoculated in the wort.

This means that if there are more yeast cells per extract than can be grown from that extract not every cell will be able to grow a daughter cell. In an idealized culture (all cells consume nutrients at the same rate) no new cells should be able to grow since none of the cells will be able to consume enough nutrients to grow a daughter cell. But the culture is not ideal which means some cells will be able to consume enough nutrients to grow a daughter cell while others won't. The ones who don't grow buds will consume extract but don't actually contribute to cell growth (though it makes them healthier and better prepared for fermentation). This mechanism also means: the more initial cells are trying to consume the existing extract the fewer will be reach nutrient levels sufficient for budding. That's why I expect cell growth to drop with increased initial cell density.

This is a bit more complicated...in a batch culture, you will have non-reproducing old mother cells (which not bud anymore, but are live and nutrient-consuming cells), budding old mother cells, budding new cells, and naive new cells. All these cells consumes nutrients. Thus, if your initial cell concentration is high, the nutrients of the wort will be fast depleted and, again, the culture will reach the stationary phase of growth. In other words, the number of new cells generated will equalize the number of cells that die by aging or random mutation. After nutrient depletion, all yeast cells enter in quiescence and begins to consume its internal supply of nutrients. The concentration of intracellular nutrients depends on the age of yeast cells (new cells = more intracellular nutrients).

What is causing this limit? is it simply the available amount of nutrients or are there other mechanisms at work? I don’t have my data here at work but tonight I want to check the max culture densities that I observed.

Nutrient levels, the age of the yeast cells, genetic mechanisms, among other physiological and environmental factors regulate the upper limit of cell concentration observed in stationary phase. In the lab., we can achieve high cells numbers (>10^9 cells/mL) by using a technique called "fed-batch", which increase amount of culture media is added in a bioreactor, with O2 levels, temperature, and pH being constantly monitored and kept at specific numbers. For homebrewing batch culture, this is more complicated, seems that many environmental points are not finely regulated, influencing the final number of cells in the wort.

I’m wondering about that. One limiter might be the amount of alcohol that is produced when the initial sugar concentration is high.

Yes, and ethanol concentration above 15% is toxic for the majority of yeast strains. High sugar concentration leads to elevated ethanol levels in wort, restricting the cell cycle of viable cells

It’s exactly those factors that affect yeast growth and the final yeast cell count that I’d like to get a handle on. I think one major factor is amount of nutrients available in the wort. But I don’t think that viability plays a large role here. Especially if the amount of growth is significantly more than the initial population. My thinking is that non-viable yeast cells will not consume any of the available nutrients and leave more for the others. That means that while non viable cells may not be able to grow other cells should be able to grow a bit more since they have more nutrients available.

Viability is an important factor. If you consider that non-reproducing cells are live and consuming nutrients at levels that are more or less similar to reproducing cells, your culture will reach the stationary phase. Only dead cells will not consume nutrients. It is important to note again: the batch culture used by homebrewers will be composed by a heterogeneous population of yeast cells: senescent old cells, reproducing old cells, reproducing new cells, and naive cells. All these cells consume nutrients, depleting the wort fasting if the initial cell number inoculated is high. Is possible to get a culture only composed by new cells in a batch culture? No, because the cells will age with time, originating a heterogeneous population. Is there a method used to obtain only reproducing cells? Yes, but only with special techniques and equipments (chemostats or bioreactors) not easily available for homebrewers.

 

[Kaiser]

The first proposition assumes that 1 g of yeast should have more or less some billions of new cells. However, remember that you will have a mixture of new and old cells compounding this 1 g of yeast biomass.

I think you misread the statement. I was talking about new growth that one can expect from a gram of extract. It didn&#8217;t talk about the amount of cells in a gram of yeast.

This is a bit more complicated...in a batch culture, you will have non-reproducing old mother cells (which not bud anymore, but are live and nutrient-consuming cells), budding old mother cells, budding new cells, and naive new cells. All these cells consumes nutrients.

I would expect that the proportion of non budding old mother cells is small compared to the other cells. A given culture should contain about 50% cells with no scars, 25% with one scar, 12.5% with two scars and so forth. There should not be a significant amount of cells with more than 6 scars, for example.

In other words, the number of new cells generated will equalize the number of cells that die by aging or random mutation.

So what you are saying is that once the growth rate approaches the death rate the population of healthy cells will not grow. That makes sense and would explain an upper limit for the cell density.

Thanks,
Kai

 

[diegobonatto]

I think you misread the statement. I was talking about new growth that one can expect from a gram of extract. It didn’t talk about the amount of cells in a gram of yeast.

You are completely right! I missed the statement. Thank you for the observation.

I would expect that the proportion of non budding old mother cells is small compared to the other cells. A given culture should contain about 50% cells with no scars, 25% with one scar, 12.5% with two scars and so forth. There should not be a significant amount of cells with more than 6 scars, for example.

Depends on the aging of culture. Fresh yeast culture will have less multi-scar cells than old cultures. I like to say that fresh cultures have less senescent (non dividing) cells that old cultures. Its simplify a lot the discussion, because the number of scars is a not a reliable factor when considering the viability of culture (some yeast strains can display a large number of scar before senescence, while others stop dividing early). When and what factors that leads a yeast cell to enter in senescence is a hot topic of research and debate today in the scientific yeast literature (including my research on yeast aging).

Thanks,
Kai

[/QUOTE]

It was my pleasure!

 

[Kaiser]

Fresh yeast culture will have less multi-scar cells than old cultures. I like to say that fresh cultures have less senescent (non dividing) cells that old cultures.

I’d like to understand this aspect a bit more. How do the number of scars increase from a fresh to an old culture? If the cells aren’t dividing there should not be an increase of scars and if they are dividing there should be an increase of cells with a low scar count.

… (including my research on yeast aging)…

That’s what I like about these forums. It gets me in touch with people who may have answers for some of the questions I have.

Kai

 

[diegobonatto]

I’d like to understand this aspect a bit more. How do the number of scars increase from a fresh to an old culture? If the cells aren’t dividing there should not be an increase of scars and if they are dividing there should be an increase of cells with a low scar count.

Remember when I wrote that in stationary phase the number of new cells generated is more or less equal to the number of cells that die? By some reason that is not completely understood, the new cells (without scars) generated enters in quiescence (non-dividing state) faster than old, multi-scared cells during stationary phase. Thus, the fast explosion of new cells observed during exponential and early stationary phase is not observed during the late stationary phase. With time and presence of lower amount of nutrients, the old cells (single-scar to multi-scar cells) divide some times before senescence and death, thus increasing the number of cells with scares. But, again, I do not like to use the scares as an indicative of how old is a culture of yeast cells because the number of scares is very strain-dependent.

In scientific literature regarding yeast aging, there are two major mechanisms that describes how a yeast cell age: (i) the replicative lifespan, which corresponds to the maximum number of daughter cells generated by a mother cell (which can be determined by counting the number of scares) and (ii) chronological lifespan that is associated to the maximum time that a cell can be kept in quiescence mode before senescence and death. For my point of view, I think that chronological lifespan is very important during beer lagering of some styles (lagers). However, replicative lifespan could be important during the active phase of fermentation. Interestingly, there are some hypotheses that discuss that old cells' death release very small amount of nutrients that sustain the quiescent state of new cells until the environment becomes adequate to induces the exponential phase again.

Unfortunately, many of these studies are only performed with lab-conditioned haploid yeast strains in standard culture media. There are not any information regarding the replicative and/or chronological lifespan of brewers strains.

In conclusion, old yeast culture contain more senescent cells, with low viability and vitality, but also contain new cells in quiescent state that can be stimulate to divide when the nutrient levels of wort/beer increase again. In this sense, the starter is fundamental to restore viability and vitality of culture, especially if the original culture is very old.

That’s what I like about these forums. It gets me in touch with people who may have answers for some of the questions I have.

I have the same opinion! A learned a lot from different people with different backgrounds in this forum.

 

[tagz]

Great thread. I have a question about Woodlands latest blog post. Hope you don't mind me tacking it on here. Woodland discussed an equation showing the number of cells produced as a function of sg and fg. Seeing as most people pull their starters after 24 hours, are we not getting the optimal number of cells out of our starters? I assumed most reproduction occurred during the lag phase, but the numbers seem to indicate that reproduction chugs along until you hit fg.

Also, I've never really measured the sg of my starters. Do most starters hit fg after 24 hours?