Help selecting a hemocytometer and microscope

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WoodlandBrew

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For counting yeast cell density and viability what would be the most appropriate tools?

With a few searches on this site it seems that the $20 Hemocytometer, and an inexpensive microscope with 400x magnification are adequate, but I would like to hear about peoples experience with cell counting. What tools and techniques work, and which ones just don't cut the mustard.

Hemocytometers seem to range from $20 to over $200! For homebrewing I would imagine counts that are within a factor of two would be adequate. Would there be any need in a brew lab for the expensive hemocytometer?

The two microscope I have my eye on now are both from Amscope. One is about $80, monocular, fixed stage, up to 1000x, The second is $165, binocular, mechanical stage, up to 2000x.

$80 microscope
$165 microscope

$20 Hemacytometer
$200 hemocytometer
 
I have that binocular Amscope, it works great for cell counting and is a very reasonable price. Mine goes up to 1000X, I can't imagine trying to use it at 2000X. Be very careful when installing the eye pieces that you don't get any lint or dust in. It is impossible to get out once any gets in, at least in my experience. My hemocytometer was $30 shipped I think, works fine.
There are some pics in my gallery taken from an iphone at one of the eye pieces. What you see in the scope is much clearer than what the pics show but it gives you an idea.
 
That's good to know. It's nice to hear from someone that has real experience with very similar, if not the same, items. Thanks!

A picture speaks 1000 words. I know what you mean about the image not being quite representative with a cell phone. I took some pictures through my brother's Olympus scope and it just doesn't do it justice. It looks like the yeast pictures were taken at about 400x (10x eyepiece with a 40x objective) is that right?

One of the reasons that I am getting a microscope is to control my pitch rate from slurries. I noticed some other pictures in your gallery labeled "Re-used Slurry Viability" "Commercial Yeast Viability" and "Inoculation Rate Vs. Doublings"

Where did you get those graphs?

My experience is very limited, but here is what I have found so far:
http://woodlandbrew.blogspot.com/2012/11/counting-cells.html
 
Yes, they were taken at 400X. 400X shows a grid full view that you would count. 100X shows full view all 25 grids to see if you have even distribution.

I made those graphs with data from Jamil's calculator and info from the Yeast book.

More often than not, when I make a stir-plate starter using Jamil's calculator, I get ~75% of the cells the calculator predicts. So, I've been making bigger starters than it recommends, then do a cell count prior to pitching and use only as many cells (by volume) as the pitching rate I'm using dictates.

The hardest part about cell counting is figuring out what dilution to use and then measuring it accurately. I use 2 mL and 10 mL pipettes for starters and a 100 mL burette for slurries. The 1 mL pipette I got for taking a 1 mL yeast sample couldn't suck up starter slurry, the opening in the tip is too small so I have to use the 2 mL.
 
dstar,
That's some good information. I have had a hunch that the calculators may have some inaccuracy, but wasn't sure how much. The 75% discrepancy you see is good to know. Have you seen a deviation between yeast strains and the accuracy of the calculated starters?

passedpawn,
You started one epic thread! I spent about 30 minutes reading it, but didn't get to the end. It is actually one of the sources I used to come to the equipment that I am considering.
 
Have you seen a deviation between yeast strains and the accuracy of the calculated starters?

I've observed WLP001 multiple times and that is where I can confidently say 75%. I've used the WLP029, WLP380, WLP838 and Wyeast 1007 strains once each and seen ~75%

Under the same conditions, Wyeast 1098 and Wyeast 1728 grew to what Jamil expected. I thought starter size may have something to do with it since the 1098 was the first time I've seen Jamil numbers and it was a 2L starter (all others were 5L). Alas, the 1728 was also a 5L so the starter size theory can't be confirmed.

I've been playing with the Brett Trois strain from White Labs (WLP644) lately and it grows like crazy. 2.5 times the doublings of what Jamil predicts, after repeated starters.
 
$165 for a binocular microscope that does 2000X sounds too good to be true. The 2000X is what we call 'empty magnification'. IOW instead of having a small blurry yeast cell you have a big blurry yeast cell. 1000X is more than you can get in an optical scope without oil immersion (is the 100X objective oil?). Empty magnification has sold lots of microscopes and telescopes to the unsuspecting.

Having said that you don't need anything like 1000x to count yeast cells. 100X is fine. At the price, if the thing performs decently at 100X it may be a good buy.

I usually advise people to try to find a good used microscope such as the ones used by medical students. These are somewhat over $1000 new and you ought to be able to find one in good shape for a couple of hundred. If you want to impress your friends with empty magnifications you can always buy a 20X eyepiece.
 
Thanks AJ,

That's what I was afraid of. The 2000x did sound to good to be true. The 100x is an oil immersion objective. The most attractive things with the more expensive scope are that it is binocular and has a mechanical table.

The reviews are good on amazon. I'm hoping the 400x is good enough for counting cells and checking viability with methylene blue or trypan blue. If the higher powers aren't to blurry maybe I'll be able to see bacteria, however I hear plating is a better test for bacteria.

The 2000x is actually from a 20x eye piece and the 100x objective... maybe I need a 25x eyepiece to really impress my friends! ha ha.
 
Yes, I saw those Amazon reviews. I'm still scratching my head. An oil immersion capability in a binocular scope for $165 is really unbelievable. You'll see what you'll see when you get it but you should be able to use it for counting in any case.
 
Yes, I saw those Amazon reviews. I'm still scratching my head. An oil immersion capability in a binocular scope for $165 is really unbelievable. You'll see what you'll see when you get it but you should be able to use it for counting in any case.

Yeah, anyone can sell you a lens and tell you to use immersion oil with it. The tricky part is actually producing a lens that will be diffraction limited when used with immersion oil and a coverslip. I'd pretty much guarantee that at this price the higher magnifications will have some massive aberrations (see the amazon review about 'Focus Issues' - sounds like significant spherical aberration). The fact that the listing won't tell you any of the lens specifications (other than magnification) is a bit worrisome too, but if all you want is cell counts, fuzzy blobs should be ok.
 
Thanks for the input bdh

Do you have an idea what the markings on the objective may mean? This is what is written on them:
Plan 4/0.1 160/0.17
Plan 10/0.25 160/0.17
Plan 40/0.65 160/0.17
Plan 100/1.25 oil 160/0.17
 
The first two numbers are, respectively, the magnification and the numerical aperture (n*sin(acceptance angle)) where n is the index of refraction of the air or oil. I think the second two may be, respectively, the focus distance and cover slip thickness. Not so sure here and don't know what the working distance is measured from or what units it's in.
 
The first two numbers are, respectively, the magnification and the numerical aperture (n*sin(acceptance angle) where n is the index of refraction of the air or oil. I think the second two may be, respectively, the focus distance and cover slip thickness. Not so sure here and don't know what the working distance is measured from or what units it's in.

the last number is indeed the cover slip thickness.
 
Yeah, the values are:

Magnification, numerical aperture, tube length, and coverslip thickness. Of all of these, numerical aperture is probably the most important, since it directly limits the maximum possible resolution of the system.

The tube length describes how far behind the back aperture of the objective the image will be focused. Most modern microscopes use infinity corrected objectives (i.e. when you place the specimen at the working distance of the objective all of the light rays coming out the back aperture of the objective are parallel) since you can place other optics (such as color filters, polarizers, dichromatic mirrors, etc...) in the light path without changing the focal length of the system. The 160mm listed on these objectives was the 'old' standard that was used on microscopes up until the 1980s or so. The actual value isn't that important as long as you use the objective with a compatible microscope and don't try to add any intermediate optics to the system.

The 'Plan' listed on these objectives refers to a 'Planar' corrected objective, which means the objective has been corrected to have a flat field of focus across the entire plane in the field of view.

If these objectives really are what they're listed as, then this is a great deal. If you were to call up Olympia/Nikon/Zeiss and ask for their cheapest Planar 4X 0.1NA objective lens you'd probably be looking at a few hundred dollars already (for just the lens). To get the higher magnification and higher NA lenses the price would probably go into the thousands. So, that being said, I'm still pretty skeptical of these lenses, but I suppose it might be possible that this company can get objectives with a 160mm tube length for cheap since they're not used much anymore....
 
Thank you everyone for your valuable information. I ended up getting the Binocular Amscope. Like everyone said, it's not an Olympus, but fine for counting cells. So far, I am pleasantly surprised by its capability. It has already saved a batch of beer! When doing cell counts on my S-03 slurry I saw an enormous number of small moving partials. They are approximately 1 micron in size which is about the limit of the resolution at 400x. The concentration of them is about twice that of the viable yeast cells. On other slurrys I didn't see that type of bacteria, so the S-03 is going down the sink instead of into my next brew.

For several pictures and more information about this scope see this blog post:
http://woodlandbrew.blogspot.com/2012/11/amscope-binocular-compound-microscope.html
 
You might have been a bit hasty. If they are irregular at all in shape then they are probably protein globules which are quite harmless. If they are uniformly shaped and in particular if they are paired they are the dreaded pediococcus and you did the right thing.
 
Thanks AJ, would protein globules be moving around? Brownian motion? They were very small, so I really couldn't make out the shape. Less than 1/10th of the width of a yeast cell. They were not visible in my control or any of the other slurries. Before putting it down the drain I made up another slide being very careful about procedure and cleanliness.
 
Yes, they are in continuous (Brownian) motion and about 1/10th the size of a yeast cell is about the right size but the real key that they aren't bacteria is that they have no symmetry i.e. they are not rods, spheres, cubes etc - just globs of stuff seemingly randomly joined together.
 
It was hard to tell the shape of these objects because they were all very small. There were a variety of gobs larger than the yeast cells, but the moving ones all seemed to be about the same size. There were no structures that were irregular and the size of yeast, just these ones smaller than yeast.

What I saw looked very similar to this:
https://www.youtube.com/watch?v=8FZwKFC75XI
 
Just a couple updates if anyone is interested.

AJ, yes I probably did act hastily discarding the slurry that I thought was infected. I should have tried acid washing the yeast, or killing everything with alcohol to see if the amount of movement changed, or plated it on yeast inhibiting medium. … or a number of other test.

$200 vs $20 hemocytometers:
Having the opportunity to use both I can say there is a very distinct difference between the two. The Brite-Line is much nicer a cost of ten times the price. The lines are much more clear and well... brighter as the name sugests. The edges of the lines are also less jagged. The tan background makes both the cells and lines easier to distinguish. However the $20 hemocytometer does the job just fine. The resulting counts of cells are the same although it may take small amount more time to count because it is simply not as crisp of a grid.

I can't say that there is any benefit to spending 10x the money on the more expensive one.
 
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