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Old 10-09-2011, 04:27 AM   #21
kcpup
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Originally Posted by passedpawn View Post
Microscope: I think 400x is the right magnification for counting. Note that the magnification is a product of the objective lens (i.e, 40x) and the eyepiece lens (10x). The objectives are the 4 silver parts on the rotatable turret in the pic. They are removable - you can add even higher magnification. There's lots to say about scopes. You can get decent cheap ones from amscope and others, but the general wisdom is get Leica, Nikon, Olympus, or Zeiss and you won't regret it later. Ebay has some decent deals. Expect this to be some painful coinage. Trinoculars are very cool and make it easy to take pics, but those heads can add some signficant cost to the scope. I wouldn't get a monocular, but if you are looking for the lowest cost scope that would be it. Also, you'll want one with an X/Y stage that allows you to easily move the counter around without fat-fisting it.

Hemocytometer: If you buy one that looks like mine, or says improved Neubaur, you're good. Available under $30 on ebay.

Stain: To determine whether the cells are actually alive or not, you'll need a stain like methylene blue or Trypan blue. Living cells can reject the stain from passing through the cell wall, but dead ones can't and turn blue when the stain is added. Look around ebay, amazon, and www.cynmar.com. I haven't done this yet, but I'll probably go with a stain kit of several different stains.

Thats about it. Go ahead and research the thing to death, that's my way too.
Thanks for the list! I'm getting excited already!

 
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Old 10-09-2011, 11:53 AM   #22
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Awesome scope. Color me jealous! SWMBO has told me to throw together a yeast lab wishlist for Christmas, but I'm not sure if I can talk her into a scope that nice. Maybe if I pitch it as a homeschooling investment for our 18month old...

 
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Old 10-09-2011, 12:20 PM   #23
COLObrewer
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Originally Posted by passedpawn View Post
. . . . Count all 16 squares, multiply x 10,000, and you have cells per ml. You also need to multiply by any dilution ratio (this was 25:1). In the example below, I counted 61 cells in the full 16 squares. 61 x 10,000 x 25 = 15.25 million cells per milliliter. Easy peezy. Now to determine how many are alive!

Dweebtastic! So splain to us the significance of the double lines on the hemocytometer (border?) How do you count cells that are split by the border? Include half, exclude all? I can't wait to see the difference between dead and live cells. Good work PP

 
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Old 10-09-2011, 12:37 PM   #24
acuenca
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NICE PICS!!! So Passedpawn if you end going with trypan stain just a warning that crap stains the hell out of everything and is tough to get out (speaking from experience).

Dude I can't believe they make an iPhone app... That's awesome.

 
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Old 10-09-2011, 01:10 PM   #25
passedpawn
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Originally Posted by COLObrewer View Post
So splain to us the significance of the double lines on the hemocytometer (border?) How do you count cells that are split by the border? Include half, exclude all? I can't wait to see the difference between dead and live cells. Good work PP
There are various boundaries in the counting chamber. The double-line forms a boundary around an area of 400 of those small squares - much more counting I needed. You might use a much larger area of the chamber for counting if your cells are much larger. Our yeast cells are small, so we only require the smallest cells.

When counting, the traditional method (as I've read) is to include the cells that touch the bottom or left lines in a cell, and exclude the ones that are touching the right or top lines.

Here's the pattern (mine is slightly different - where this diagram shows heavy dark lines, mine uses double lines).



Here is an excellent tutorial with great pics from While Labs. One one slide it says 40x is the magnification - that's incorrect, should read 400x.
http://www.whitelabs.com/beer/CellCo...&Viability.pdf

Here's the straight dope on hemocytometer counting:
http://www.ruf.rice.edu/~bioslabs/me...lcounting.html

Some interesting pics of yeast (brewer's and wild) and bacteria. Note how different they look when stained with the same stain. Thus a stain can be used not only as a tool for assessing viability, but for identifying unwanted critters in your beer.
http://www.whitelabs.com/beer/microscope.html
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Old 10-09-2011, 08:23 PM   #26
DakotaRules
 
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Pretty cool stuff. I've been thinking of counting cells but wasnt sure of the equipment and investment needed. After seeing this I think it may be more attainable then I thought.

 
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Old 10-09-2011, 10:40 PM   #27
theredben
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Just to clarify, when using a haemocytometer (I like to old spelling), you need to fill both chambers with different samples from the same source. In other words, you need to pipette a sample from the flask into the chamber, then rinse the pipette, then pipette a sample into the other chamber. Then compare the results. Haemocytometers are only as good as your QC. Also, make sure to be vigourously swirling your flask before each sample. They need to be as representative as possible. Remember that you are counting an unbelievably small section of your whole starter, so precision/repeatability is key.

 
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Old 10-09-2011, 10:49 PM   #28
passedpawn
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Thanks. I wondered why there were two chambers. (by the way, haemo is the way I thought it should be spelled also... )

My QC probably sucks, as you can imagine. My damned dog (viszla) doesn't make matters better. Yes, I swirl (except just now, since I'm looking at pond water and hoping for critters to fall to bottom).

I truly appreciate any further advice from those schooled in the art. I'm all ears.
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Old 10-09-2011, 11:02 PM   #29
acuenca
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Sure you could pipette into both chambers and you could count the 4 sets of 16 squares and then average in both chambers... but honestly since you're using it for a yeast starter where you need it to be roughly in the 2-3XX billion... if you're off by a couple million, it's not really going to make that much of a difference. Hell for some applications at work I only count a random 4 squares in a 4X4 multiply by 4 and use that number as by cells per 10(4)/mL... Comparing these numbers with machines that are automated cell counters or with other colleagues have not shown a significant difference between values and I've never had to count the same sample twice unless it's completely off from the number that I think I should have and usually the problem is that I didn't resuspend the cells well enough. The important thing is to make sure that the yeast starter solution is as homogenous as possible and then you should be good.

Obviously, it's up to you...When you have to count 30-40 samples of cell suspensions at a time, you start to look for shortcuts that won't hurt your experiments or your data and this, for me, is one of them. Hope this helps.

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Old 10-13-2011, 11:43 PM   #30
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A local brewery has a microscope; they use it to see how many generations they can use the yeast for. I always wanted one, post a pic up if you ever get an infected batch!

 
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