Author Topic: Hemocytometer  (Read 2898 times)

Offline lonnie mac

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Hemocytometer
« on: January 15, 2011, 05:00:18 PM »
Rather than dig up some old threads that I have read, I happened upon a nice little scope (for me anyway). A 430X. I am having a ball looking at my yeast under a slide. I think I want to give this yeast counting a go. Looking at Hemo's, I don't understand the grid patterns on some that I have seen. They seems to be a different grid count between models.

I am looking at something like this, mainly because it is a complete kit with a nice little storage case. Would this suffice for counting beer yeast?

http://www.spectrum-scientifics.com/cgi-bin/commerce.cgi?preadd=action&key=2301#

Offline tschmidlin

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Re: Hemocytometer
« Reply #1 on: January 16, 2011, 12:28:31 AM »
Yeast cells and blood cells are roughly the same size, so any hemocytometer will work.  Don't worry about the different grids between different models, just make sure you follow the directions for the one you have.  The ones I have used have 9 squares, but you only count from the corners.  The corners are further subdivided into 16 squares, and you don't need to count every square to get a good estimate of the cell count.  When it comes to ones on the line, come up with a way that works for you and stick to it.  Typically, I count cells partially in the square on 2 sides, and not on the other two sides of the square.

Your biggest problem is going to be distinguishing live from dead (Are you going to be staining?) and breaking up the clumps so you have individual cells to count.  I don't think you'll want to spring for a vortexer, but if you pipet up and down that can help break things up.  I've heard of people using "marital aids", aka personal massagers, on the bottom of a microfuge tube to break things up too. :)  Also, make sure you take accurate measurements for your dilutions, and make sure each sample is uniform before you dilute it again.

Which brings up another point, dilute your sample!  You don't need to count every cell from a thick slurry, dilute it 10-fold, look at it under the scope and see if it is too thick, if so dilute it again, repeat until it is reasonable to count.

Oh, and get yourself a clicker counter thing like they use to count people going into places, it makes the counting a lot easier and faster.

I hope that helps :)
Tom Schmidlin

Offline lonnie mac

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Re: Hemocytometer
« Reply #2 on: January 16, 2011, 09:12:46 AM »
Thanks buddy... That's kinda what I was thinking too. Now on to the math!!!! :)

The little kit in my link don't look too bad then as it comes with some graduated pipets. I think it should work well. I'll get into staining, diluting for sure. There is no way I can count that many yeast!

Offline a10t2

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Re: Hemocytometer
« Reply #3 on: January 16, 2011, 09:37:52 AM »
To give you a starting point, I've found that diluting 100:1 typically means counting about 100-200 cells.
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Offline tom

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Re: Hemocytometer
« Reply #4 on: January 16, 2011, 09:44:01 AM »
Rather than dig up some old threads that I have read, I happened upon a nice little scope (for me anyway).
Where'd ya get the 'scope?
Brew on

Offline lonnie mac

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Re: Hemocytometer
« Reply #5 on: January 16, 2011, 10:29:00 AM »
This was a good thread that I dug up when I got my scope...

http://www.homebrewersassociation.org/forum/index.php?topic=713.0

Offline Kaiser

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Re: Hemocytometer
« Reply #6 on: January 20, 2011, 11:52:31 AM »
I must have missed this thread when it was discussed, but wanted to chime in.

Tom makes a good point about breaking up yeast clumps. I found that they are best broken through reversal of the flocculation by adding the yeast to wort. Most practically this would be on a stir plate with about 1-2 qt of the wort that the yeast will be pitched in.

When I count yeast I use a clicker-counter app and this spreadsheet on an iPod. The spreadsheet allows you to enter the dilution in sample volumes. I.e. if you have a 1 ml sample and add 10 ml water you'll enter a dilution of 10. This multiplies the cell count by 11.

Tom, do the methods you described for breaking up yeast work well. I feel that the yeast's flocculation might be strong enough to re-form clumps as soon as the aggitation stops.

The only yeast I have not been able to un-flocculate well before pitching was WLP002. In this case I was also not able to count the yeast.

In yeast growth experiments, I conduct once in a while when I have time, I keep using WY2042. This is the Danish Lager yeast and it doesn't flocculate which means that clumps are easily broken up on the stir plate.

Kai

Offline lonnie mac

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Re: Hemocytometer
« Reply #7 on: January 20, 2011, 03:46:32 PM »
Thanks Kai! Great spreadsheet. Us old farts need something easy you know... Got my Hemo on the way, and some general slides for my Son to look at chicken lips or what ever he wants to look at with it. That is half the fun of having a scope now, all the other general stuff you can do with it.

Offline Kaiser

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Re: Hemocytometer
« Reply #8 on: January 20, 2011, 06:06:06 PM »
I have some general slides too, but don't use them much to loop at yeast.

Make sure you get some methylene blue as well. I used to use a 0.1% solution as part of the dilution and account for it in the calculation but now I just drop in a drop of the 1% solution and don't really account for the additional volume. If the sample volume is ~5-10 ml the error introduced by the extra volume is well within the noise.

Most of the time I don't check for viability anymore since I propagate my yeast fresh for almost every batch and was always getting viabilities in the high 90s. And you need to keep in mind that non stained yeast does not necessarily mean that it is alive. I had a 1yr old smack pack that showed less than 50% stained cells but when I plated this yeast nothing grew. I think all the cells were dead but some were still be able to metabolize the methylene blue. Maybe Tom can shed some light on this.

Kai

Offline lonnie mac

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Re: Hemocytometer
« Reply #9 on: January 20, 2011, 06:39:12 PM »
Ah yes, the methylene blue... Not quite sure where I will find that yet. It is next on the list!

Offline Kaiser

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Re: Hemocytometer
« Reply #10 on: January 20, 2011, 09:12:49 PM »
I'd check Cynmar:

http://cynmar.com/item_detail.aspx?ItemCode=CHM84184

Other useful items are 5 and 10 ml Serological pipets and a pipet pump. Those are good diluting samples

http://cynmar.com/item_detail.aspx?ItemCode=11520665

http://cynmar.com/item_detail.aspx?ItemCode=11520667

and a pipet pump

http://cynmar.com/item_detail.aspx?ItemCode=15024574

Kai