Building a home QC lab... questions

helibrewer said:

Just saw this, sorry for the delay. I added the "T" handle back when I was experimenting with steam injection in my mash tun...Now I use it just to monitor and vent (for non liquid items like my tools and plate chiller).

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Wow, ha. Thanks for eventually answering us

Ok guys I've been messing around with this a bunch and am back for follow up Q's.

Putting cell counts and pitch rates into practice
Does this sound logical? I snagged a 7 month old Wyeast pack from the LHBS for free. Wanted to see if I could make this sucker viable for a session beer pitch in a limited amount of time. I was figuring maybe 10% viability in the pouch.

So I made a starter. 2L wort + stirplate in a 4L flask. Lots of room for gas exchange. 1.040 was the starter gravity + nutrient addition. 68F for 36 hours. Initially I was worried as there was no sign of kreusen at all, which is odd for my starters but considering the initial pitch rate, oh well. Around hour 30 it seemed to become substantially more turbid than before so I felt confident the log growth phase was progressing. Starter was cold crashed at 40F for 2 days and around 1600mL decanted and thin slurry was waiting for brew day.

MrMalty says for 5 gallons of 1.053 wort, I'll need 185 billion cells.

I remove a small sample of the 400ml thin slurry. Make a set of serial dilutions by adding 9ml of DI water to a few test tubes then 1ml of the slurry, then 1ml of the mix, etc eventually getting 1:10, 1:100 and 1:1000 samples. The 1:10 factor is what I used to count.

Using my hemocytometer, I count 170 cells in the 16 small (1/16th) squares. Phone app gives me 4.25e7 or 42.5 million cells per ml (1:10 dilution).

Then I add 1ml alkaline methyl violet solution to a 1ml sample of the 1:10 dilution creating a 1:20 dilution that I can assess for viability.

Again using the hemocytometer, I count 92 cells in 16 squares but this time with 9 staining purple (that weren't budding). Phone app gives me 2.30e7 or 23 million cells per ml (1:20 dilution). (92-9)/92x100= 90% viability.

Sooo the average pitch rate between the two counts without dilution factor is 442 million cells per ml. I have 400 ml of slurry. Multiply them together to get a pitch of 176.8 billion cells....of which 90% are viable, so 159 billion viable cells pitched, slightly under the MrMalty calculated rate.

Does this look correct? I hate math. Not a bad turn around though from a mega old yeast pack.

How are you guys decanting off starters? For some of the less flocculant strains, I don't think pouring off the top is effective. I'd like to siphon out of the flask but man that takes forever even with a 50 ml pipette, if you can even find a decent bulb for one (I can't). Is there some sort of sterile suction method/apparatus that will somewhat quickly remove large quantities of fermented starter wort from big Erlenmeyers?

Filling hemocytometers? I've been using a 100-1000ul pipetteman and make a mess every time. Gotta be a better way. Small glass pasture pipettes??

butterpants said:

I remove a small sample of the 400ml thin slurry. Make a set of serial dilutions by adding 9ml of DI water to a few test tubes then 1ml of the slurry, then 1ml of the mix, etc eventually getting 1:10, 1:100 and 1:1000 samples. The 1:10 factor is what I used to count.

Using my hemocytometer, I count 170 cells in the 16 small (1/16th) squares. Phone app gives me 4.25e7 or 42.5 million cells per ml (1:10 dilution).

Then I add 1ml alkaline methyl violet solution to a 1ml sample of the 1:10 dilution creating a 1:20 dilution that I can assess for viability.

Again using the hemocytometer, I count 92 cells in 16 squares but this time with 9 staining purple (that weren't budding). Phone app gives me 2.30e7 or 23 million cells per ml (1:20 dilution). (92-9)/92x100= 90% viability.

Sooo the average pitch rate between the two counts without dilution factor is 442 million cells per ml. I have 400 ml of slurry. Multiply them together to get a pitch of 176.8 billion cells....of which 90% are viable, so 159 billion viable cells pitched, slightly under the MrMalty calculated rate.

Does this look correct? I hate math.

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It's great that you are doing cell counts. Personally, I would have been interested in what the viability was before the starter.

It sounds like what you counted is one "box." (this area fits nicely inside the field of view at 400x on most microscopes.) Each box is 4nl. Typically five boxes are counted for statistical reasons. 170 cells / 4nl is 42.5 cells per nl or 42.5 billion cells per liter. With the 10:1 dilution that means that the original sample was 425 billion cells per liter.

92 cells / 4nl is 23 billion per liter. factor in the 20:1 dilution and you have 460 billion cells per liter. About 10% variation. That's pretty typical for what is essentially box to box variation.

Your viability assessment is correct, but as mentioned before, most people count 5 boxes.

WoodlandBrew said:

It's great that you are doing cell counts. Personally, I would have been interested in what the viability was before the starter.

It sounds like what you counted is one "box." (this area fits nicely inside the field of view at 400x on most microscopes.) Each box is 4nl. Typically five boxes are counted for statistical reasons. 170 cells / 4nl is 42.5 cells per nl or 42.5 billion cells per liter. With the 10:1 dilution that means that the original sample was 425 billion cells per liter.

92 cells / 4nl is 23 billion per liter. factor in the 20:1 dilution and you have 460 billion cells per liter. About 10% variation. That's pretty typical for what is essentially box to box variation.

Your viability assessment is correct, but as mentioned before, most people count 5 boxes.

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So my counts are off by a factor of 100?

170 cells counted in 1 small square come out of my app at 4.25e7. I would read this as 42,500,000 (42 million). Multiply by 10 for the dilution factor and you get 425 million cells per ml. You say 425 billion per ml.

If so then I pitched 159 trillion cells? That doesn't seem right. I can't imagine propagating that many in such a short time from that gimped source. Please help!

I'll attach a hemocytometer app screenshot from my phone....one sec

So I chose option #3 and enter 170 cells in 1 box.

And I was interested in the original pack viability but to be honest the starter was prepared at 2am after a long day at work and I had no desire to do lab stuff at that late hour. . A moment of weakness, HA

The number of squares you count is determined by the accuracy you want. The coefficient of variation in your estimate is 1/sqrt(n) where n is the number of cells you counted. If you count 100 cells you will have Cv = 1/sqrt(100) = 0.1 and what ever you determine that to be, dependent on how many boxes of what size it took to get 100 cells, the estimated error will be ±10%. So, suppose you go into that central area where the boxes are defined where triple lines intersect and count 19, 20, 21,18,18 and 24 cells in the top row (out of the total of 25 in that area). That's 100 cells. If the central area is 1 mm x 1 mm (typical) and the depth 0.1 mm (also typical) you would have 100 cells in 5*2E-4*2E-4*1E4) = 20*E-12 m^3 = 20*E-6 cc for a density of 100/20E-6 = 5E6 cells/mL ± 10%, If you counted two rows (10 boxes) and got a total of 200 cells your Cv would go down by a factor of sqrt(2) to 1/sqrt(200) = 0.0707 or 7%. You counted twice as many cells in twice the volume so the densty is now 200/40E-6 which is still 5E6 cells per mL but the standard error is now 7% of that or ±350,000 cells/mL. Dilution factors would, of course, need to be applied. To halve your accuracy you have to double the number of cells counted.

Thanks for the reply AJ. Yes next time, more counting, less error. My app gave me a 15% error margin so I was tracking it just didn't talk about it here. My fault.

You didn't touch on Woodlands comments and the discrepancy of 100 fold in his vs my numbers. Need to get this remedied....maybe it's just something innocuous I'm missing

I'm sure the count discrepancies are due to incorrect volume assumptions on someone's part. If the central area is 1 mm x 1mm x .1 mm then it corresponds to 1E-3*1E-3*1E-4 = 1E-10 m^3 which is 1E-4 cc. That area is subdivided into 25 boxes (each of which has 1 or 2 triple ruled lines) and they are (1E-4)/25 = (100E-6)/25 = 4E-6 cc (4 nL). Those boxes are in turn subdivided into 16 subsquares each of which contains (4E-6)/16 = (40E-7)/16 = 2.5E-7 cc (0.25 nL)

If someone else ran the numbers I would feel more confident, but it seem that you just misread the units that I wrote. We are getting the same result. 425 billion cells per liter (not billion per milliliter) is what I came up with for the first cell count, and 460 billion per liter for the second. 400ml of 425 billion per liter would be 170 billion cells. 400ml of 460 billion per liter would be 184 billion cells. At 90% viability that's 153 and 166 billion cells.

And it was that simple of a mistake on my part. Glad I'm tracking... if just not counting enough.

I actually have a pile of questions saved up but I think counting is all set