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-   -   Recommendations for Microscope / Hemacytometer? (http://www.homebrewtalk.com/f128/recommendations-microscope-hemacytometer-125499/)

stoutaholic 06-25-2009 08:22 PM

Recommendations for Microscope / Hemacytometer?
 
So I want to perform some yeast starter experiments and am trying to figure out the cheapest way to perform yeast counts. According to the White Labs website, I basically need a hemacytometer, a 400X microscope, and methylene blue solution. Has anyone used this equipment before? Can I buy just any microscope with a 400X magnification, or do only certain types of microscopes support the use of a hemacytometer? Also, when I google for hemacytometers, they come up in a wide variety of price ranges, from $30 to $340. Are there any particular characteristics of the hemacytometer that I need to look for? Is a hemacytometer made for counting red blood cells applicable for yeast cell counting? Would this one work: Neubauer Hemacytometer Blood Count for Microscope - eBay (item 380089722252 end time Jul-11-09 01:03:06 PDT)
And how about this microscope: LiveScience Store: Ultimate 400x Digital Microscope
I thought this one would be cool because I think it would allow me to just do the counting on my computer screen, rather than staring through a microscope.

By the way, for the experiment, I don't really care about actual cell counts. Those are apparently pretty difficult to extrapolate from a hemacytometer count. I will approximately know the beginning cell count based upon the number of fresh liquid yeast packs that I will empty into the starter. What I am trying to determine is the percentage increase in yeast cells over time. So I will just be comparing my later counts to my initial count, and so as long as my counting procedure is consistent, I should be able to determine the percentage increase from time 1 to time 2. One of the objectives is to determine how long the lag phase lasts, given certain dissolved oxygen concentrations (which I will measure with a DO meter).

Thanks for any feedback!

menschmaschine 06-25-2009 09:02 PM

I think 400X is a bit weak. You'll be able to see the yeast, but they'll be small. I'd try to get one that has 800-1000X. This will help you better see yeast health and bacterial contamination.

sw341034 07-01-2009 05:41 AM

I work doing microbiology research with a university winemaking program and we use this stuff all the time. Obviously the better the microscope the easier counting is going to be. We use phase-contrast scopes which eliminates the need for your staining solution but these can be very pricey. Most compound microscopes should suffice. As for the wide price range of hemacytometers, again its mostly a quality issue. The more expensive ones are more heavy duty and meant for long term repeated use while the cheaper ones can often be made of plastic and are more "disposable" however if you treat it nice the cheap ones should last you a good while. They all essentially are the same thing however. Also most hemacytometers are labeled for red blood cell count but there is no difference (their original use was for counting blood cells but are now used for virtually all cell counting). The hemacytometer you linked to should work great. Can't speak on that particular microscope as I have no experience with it. Also you might consider buying a pipette of some sort to transfer your suspension to your hemacytometer. Also some slide wipes might be a wise investment if you would like to keep you hemacytometer nice. Finally you mention "I don't really care about actual cell counts. Those are apparently pretty difficult to extrapolate from a hemacytometer count."....It's really not all that hard and is straight forward calculations. If you have taken the time to buy all the equipment, stain your samples, spend the time counting under the scope, you have already done 99.9% of the work. Simple multiplication and your there!

Poindexter 07-01-2009 06:41 AM

The only thing I can really contribute is about optical glass. I got some rifle scopes, some binoculars, several camera lenses. Been wearing glasses for 30 some years.

Good quality optical glass is not cheap. For what you are descrbing I would rather have a binocular vision Nikon microscope at 400x than any higher powered monocular scope at whatever magnification from some maker I never heard of. Among microscopes I am sure there are a few companies in Switzerland I have never heard of...

stoutaholic 07-01-2009 03:04 PM

sw341034, do you guys ever you CellProfiler? It appears that I should be able to take a picture of my cell grid and then feed it into the software, which will do the cell counting for me.There are various "pipelines" that you can use for specific applications with this software (e.g. one pipeline for fruit fly cells, another for human cells, etc.). If you guys have ever used this, I was wondering if you might be able to recommend the best pipeline for yeast cell counting, and how difficult it was for you to set this up. Also, what magnification do you guys use when counting?

Thanks for all your info about the microscope and hemyocytometer. My main concern about estimating actual cell counts is getting the dilution calculation right.

Gremlyn 07-01-2009 03:41 PM

Counting is really quick and easy, using software like that is by far over kill. I agree that the haemocytometer will work just fine, they are only called that because they originally were used for counting blood cells. But cells are cells. I disagree that 400x will not be enough to count. Often you want to be able to see the whole field (usually 1 sq mm) to get a count and yeast cells are pretty big compared to some of the stuff I've had to count ;) On average, a yeast cell you'll see will be 5-10 um, which under 400x will appear as a 2 mm cell, plenty big to see. If you use the stain, then only the viable (live) cells will absorb the die and you can see how healthy your yeast are, which can be quite helpful.

If you want to spend a ton of money and have stupid accurate counts, get a FACS machine................. but seriously, don't (unless you have thousands of dollars burning a hole in your pocket).

Look around for biotech surplus sales, you can often get decent equipment for a great price. Some of them are auctions so you may have to be a little patient, but it pays off. I wish I had the room to accumulate some equipment. I'd want a spectrophotometer as well to get better SRM values :)

Edcculus 07-01-2009 09:01 PM

Quote:

Originally Posted by Gremlyn1 (Post 1409702)
I wish I had the room to accumulate some equipment. I'd want a spectrophotometer as well to get better SRM values :)

Sorry to hijack the thread...

I have an extra spectrophotometer laying around (I work at a printing company). I've been proposing using one to take color readings of beer, but am kind of unsure how to proceed on the software side. Nothing we use in the printing industry would really translate to beer. Any ideas?

Gremlyn 07-01-2009 09:13 PM

Quote:

Originally Posted by Edcculus (Post 1410559)
Sorry to hijack the thread...

I have an extra spectrophotometer laying around (I work at a printing company). I've been proposing using one to take color readings of beer, but am kind of unsure how to proceed on the software side. Nothing we use in the printing industry would really translate to beer. Any ideas?

I suggest a calculator :) Calculating SRM from a sample measured in a 1 cm cuvette is done at 430 nm, and you multiply the absorbance by 10. Obviously this only gets you up to 20 SRM, but you can do dilutions and get pretty close, though the dilution of beer is NOT linear with regards to absorbance reading. You can do a few similar commercial beers with known SRMs to try to accurately predict the curve of the line and get your final SRM.

Either PM me or we can start a new thread so we don't hijack this one any more :)

/hijack off

sw341034 07-02-2009 08:37 AM

We don't use cellprofiler for the majority of the work since as gremlyn1 mentioned counting yourself is usually much faster and less of a hassle than running through image analysis however, we do on occasion use similar programs since the goal of a lot of what we do is to get papers published and such, and that being the case the consistency that you can get with those types of software is bonus, but again overkill for your everyday cell counting. Let me know when you get your equipment and I can go over some calculations with you. There are actually a few methods you can use but all are equally easy. If you can multiply by ten then you can do this.


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