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Old 02-07-2013, 09:10 PM   #41
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Originally Posted by Paulgs3 View Post
http://www.teknova.com/YPD-AGAR-PLAT...MM-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.
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Old 02-08-2013, 04:52 PM   #42
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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?

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Old 02-08-2013, 07:39 PM   #43
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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.

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Old 02-08-2013, 07:53 PM   #44
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Originally Posted by thadius856 View Post
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.
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Old 02-08-2013, 10:32 PM   #45
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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.

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Old 02-09-2013, 07:56 AM   #46
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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?
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Old 02-09-2013, 03:34 PM   #47
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Quote:
Originally Posted by d_striker View Post
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.
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Old 02-09-2013, 06:20 PM   #48
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Quote:
Originally Posted by thadius856 View Post
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.
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Old 02-09-2013, 08:50 PM   #49
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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.

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Old 02-10-2013, 12:08 AM   #50
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Quote:
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... 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.
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