dilution for cell counting- best methods?

[Junkyard]

would anyone like to share your best practices for dilution before cell counting?

what kind of measurement containers do you use?

what do you use to transfer the yeast from container to container?

how many times do you dilute? just once to 100:1 or once to 10:1 then again to 10:1 to make it 100:1?

is having a good expensive hemocytometer important? i know the tolerances are very important for the total area of the chamber....


I've been cell counting for several batches now but i want to be able to get really accurate counts...

[SeattleBrewer]

I always use weight instead of volume because it is much more accurate of a measure of yeast. So 10g of yeast in 90g of water (which happens to be 90ml). Then mix well. Measure out 10g of that mixture, then put into 90g (ml) water. That'll get you a 1:100 dilution. Using 10g in 100g gives you more margin of error with weighing out the yeast. Plus using weight doesn't require special pipettes or measuring equipment, just a nice accurate gram scale. Once you have your 1:100 solution, add a drop or two of your methylene blue, stir it up, and I like to use a disposable Pasteur pipette to put into the hemocytometer. I'm not sure they make "bad" hemocytometer slides but they usually are pretty pricey for the standard ones.
Point to note, using grams instead of ml for measuring doesn't change the calculations from once you put it under the slide. So extrapolate like you normally would to get the number of cells per ml, and that is your number of cells per gram. Then you pitch the required number of grams or kilos for your batch. Much more accurate than volume since that can be variable. You can have a liter of yeast that contains the same amount of yeast as a 1.5 liter volume because the density of beer to yeast is different. Plus it can foam up quite a bit and throw off readings.


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[Junkyard]

A volume of yeast slurry will weigh more than the same volume of water right? Won't this throw off your dilutions?

[HorriblePunHere]

Hemocytometer cover slips can be bad if they are plastic, the osmotic pressure can flex the plastic and change the volume under the slip. If they are glass, you're fine. The price for 200 glass coverslips is something like $5. I remember reading that even if all your science, methods and equipment is perfect, you're still only going to get within 10% of the actual answer when cell counting with a hemocytometer, but I don't have a source for that, so take that with a grain of salt.(Salt not actually recommended for scientific testing.)

[SeattleBrewer]

Hey junkyard, yes the densities are different between slurry and water, so if you are going on a 1:100 dilution based on weight only (1g of yeast in 100g of water) then you don't have to deal with volumes at all. With your extrapolations for calculating up from the hemo count, just change what would be ml to grams in the notation. It just so happens that 1ml of water weighs 1g, so maybe that's where it can get confusing.


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[Bainbridge]

Correct. Typically you see cell counts volumetrically, in cells/ml. But if you go by weight, you get cells/gm of slurry. From that you can pitch by weight. So many kilos of slurry dosed in during transfer to get the right pitching rate.

[Junkyard]

Sounds good, ill try using weight and see how that works.

So two step dilutions are better than one step dilutions. 10:1 then 10:1 again


when counting up the small grids, ive seen that it is a good idea to count multiple grids. how many do you guys count?

also, say you count 5 of the small grids. do you then take the total cells counted and then divide by 5 to get the average and use the area of one small grid for your calculation? or do you calculate the entire box of 20 grids and multiply the calculation by 1/4? i hope this question makes sense.

[WoodlandBrew]

If you are just looking for cell density and can forgo viability then the cells can be unclumped by diluting with acetic acid (vinegar) instead of water. I've found this especially useful for highly flocculent strains.

You might consider using image analysis for the actual counting. It removes some of the subjectivity of the technician and, once you have you process down, is very fast. See here for details:

http://www.woodlandbrew.com/2015/02/automated-cell-counting-of-yeast-using.html

A cell density meter is another option:

http://www.woodlandbrew.com/2014/07/cell-density-meter.html

[Mike Elliott]

check out BSG lecture

Junkyard,

Couldn't paste a link, but Google "Getting the most out of your hemocytometer" for a complete procedure and explanation of logic behind the steps. BSG gave this lecture at CBC a couple years ago.

[d_striker]

I always use weight instead of volume because it is much more accurate of a measure of yeast. So 10g of yeast in 90g of water (which happens to be 90ml). Then mix well. Measure out 10g of that mixture, then put into 90g (ml) water. That'll get you a 1:100 dilution. Using 10g in 100g gives you more margin of error with weighing out the yeast. Plus using weight doesn't require special pipettes or measuring equipment, just a nice accurate gram scale. Once you have your 1:100 solution, add a drop or two of your methylene blue, stir it up, and I like to use a disposable Pasteur pipette to put into the hemocytometer. I'm not sure they make "bad" hemocytometer slides but they usually are pretty pricey for the standard ones.
Point to note, using grams instead of ml for measuring doesn't change the calculations from once you put it under the slide. So extrapolate like you normally would to get the number of cells per ml, and that is your number of cells per gram. Then you pitch the required number of grams or kilos for your batch. Much more accurate than volume since that can be variable. You can have a liter of yeast that contains the same amount of yeast as a 1.5 liter volume because the density of beer to yeast is different. Plus it can foam up quite a bit and throw off readings.


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This.

I did it by volume for quite awhile before realizing it was more accurate and easier to do it by weight.

When I did it by volume, I would let yeast settle in a graduated centrifuge tube. I would note the volume and perform the first of my serial dilutions based on settled yeast volume. Volume would be dependent on how well the yeast settled in the tube...And of course, I had to wait for it to fully settle.

[Katie Taylor]

I've found the best dilution liquid to be not-too hot wort. The cells automatically want to de-floc, and you don't have to worry about skewing your 'dead cell' count with an acid (if you leave the solution for too long the cells will weaken and start to turn blue), that is if you're looking for viability and not cell density.
Count 5 of the squares, the 4 corners and the middle-most square. I've found whichever side of the hemocytometer you load your sample from will have more cells than the opposite side, so its best to count the squares from all 4 corners.
brewingscience.com has an easy to follow protocol for cell counting including equations to calculate pitching rate.
hope this helps!