Author Topic: Microscope and yeast counting  (Read 14817 times)

Offline Thirsty_Monk

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Re: Microscope and yeast counting
« Reply #30 on: January 09, 2010, 09:22:57 PM »
Would it have enough magnification?
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Offline Kaiser

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Re: Microscope and yeast counting
« Reply #31 on: January 10, 2010, 06:16:41 AM »
I count cells at 400x (40x objective and 10x eyepiece) but can already see individual cells at 100x. My advice is that if you have a 100x microscope you may try it and buy a hemavytometer. If you buy one don't buy anything below 400x. Otherwise you'll catch the bug, become unhappy with it and buy a better one anyway.

If you are going for a good price, find a monocular version. That saves about $100+ and the image quality is the same. 

Kai

Offline karlh

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Re: Microscope and yeast counting
« Reply #32 on: January 11, 2010, 07:38:17 AM »
Wow, you actually broke through the whole thing.

Kai

I am used to leaving things, and returning to find them in the same condition I left them at work.  Unfortunately with kids around the house, that is almost never the case.  I broke the whole thing.  That said, 37 bucks wasn't bad (I have seen hemocytometers for more than 200 at various scientific supply houses). 

Drew, I would advise against purchasing a stereomicroscope for cell counting.  Like Kai says, you will have a lower net magnification and probably end up spending more in the long run buying a good optical microscope.  I would check Craiglist and ebay for higher quality (Nikon, Zeiss, Leitz, Leica, etc.) used microscopes over new "off-brand" microscopes from cynmar, optics planet, etc..  I watched craigslist for a while and ended up with a research grade Leitz microscope that has outstanding optics for $100.  Brand aside, a key requirement for me is having an adjustable condenser below the stage in order to obtain a Kohler alignment.  This focuses the light source from below at the sample plane while the objective is focused at the same point from above, and effectively optimizes imaging.   
Karl
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Offline karlh

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Re: Microscope and yeast counting
« Reply #33 on: January 20, 2010, 04:12:53 PM »


This is a (B&W) photo of an actively fermenting wort on the hemocytometer grid square.  As you can see, I need to dilute the sample to facilitiate counting.
Karl
Mundelein, IL  USA

Offline karlh

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Re: Microscope and yeast counting
« Reply #34 on: January 20, 2010, 05:01:10 PM »
OK,  I didn't have the patience to count all the cells manually, but did a quick filter, threshold, and count on 5 squares of the 25 square grid.  One square looked like this after filtering:

This counted as 477 cells (477+460+417+440+444)

The rough count of cells in five squares is 2238 (477+460+417+440+444) (x5 for the uncounted grid squares) (x104) = 1,163,760 cells/ml at day 3 of the ferment, so not too bad, and my filter was pretty sloppy so it could be higher.  I'll dilute 10 ml into 90 ml H2O the next time I attempt this.
Karl
Mundelein, IL  USA

Offline Kaiser

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Re: Microscope and yeast counting
« Reply #35 on: January 20, 2010, 05:03:03 PM »
Karl, that is a nice pic. How did you take it? 

Another thing I found important for flocculant strains is the need to un-flocculate them in glucose solution or wort. Otherwise they can form rather large clumps.

Kai

Offline karlh

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Re: Microscope and yeast counting
« Reply #36 on: January 20, 2010, 07:03:48 PM »
Its a really nice microscope.  I have an unfair advantage when it comes to microscopes. 





I have tried to point out that you can find a lot better optics if you look for a used microscope that is research grade.  My home microscope is an "Ernst Leitz Wetzlar" medical microscope from about 1950-1952,  I purchased it on Craiglist for $100 from a doctor closing down his office.  Unfortunately I had to watch the Chicago craiglist for about a year before I found a really high quality microscope at a low price that didn't appear to be stolen.  I actually brought a slide with some yeast on it to check out the scope before I bought it. 

There are a few really good names in optical microscopes and you can search ebay or craiglist for Carl Zeiss, Leitz, Leica, Nikon, Olympus.  The latter two have made good units for the last 20 years, and the others for the last 50 or 60 years.  I would rather take my chances on a 100 year old Carl Zeiss microscope than a no name"student" microscope.  The one inexpensive model that seems to be very high quality is "LOMO", a research grade microscope out of Russia that is surprisingly low cost available from optics planet. 
Karl
Mundelein, IL  USA

Offline bluesman

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Re: Microscope and yeast counting
« Reply #37 on: January 20, 2010, 09:27:47 PM »
Karl, that is a nice pic. How did you take it? 

Another thing I found important for flocculant strains is the need to un-flocculate them in glucose solution or wort. Otherwise they can form rather large clumps.

Kai

+1

This is a really cool thread. Keep up the great work guys!
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Offline roffenburger

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Re: Microscope and yeast counting
« Reply #38 on: January 21, 2010, 08:06:02 AM »
Its a really nice microscope.  I have an unfair advantage when it comes to microscopes. 


Yes, yes you do. I wish I had acces to an SEM.... ;) Did you take those pics?

I totally agree with your statements about getting a quality microscope. It would be well worth patiently searching for a used one.
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Offline Kaiser

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Re: Microscope and yeast counting
« Reply #39 on: January 21, 2010, 08:27:08 AM »
Its a really nice microscope.  I have an unfair advantage when it comes to microscopes. 

I’m actually happy with the image quality I get from my microscope. The only thing I’m not happy with at this point is the inability to shoot pics that match the quality that I get when I look though the scope. I’ll have to buy a camera adaptor for that. Right now I just use a PS camera and it works well enough for sharing pics.

I also started taking pics to count cells later on the computer. But since I have trub and other particles in the samples I have to determine which cells are and which are not. Is there any simple software out there I could use for counting objects in an image. I’m thinking of a use model where I click an object to mark it. the software then counts the marked objects. Right now I just use a tally counter.

Kai

Offline karlh

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Re: Microscope and yeast counting
« Reply #40 on: January 21, 2010, 11:40:11 AM »
Kai,

Try finding Uthsca Image Tool.  Its freeware, has some basic imaging filters and thresholding tools.  You should be able to
1: determine size of grid square on hemocytometer in pixels (use a photo and the calibrate function)
2: take photos on hemocytometer (outside grid square for minimum noise, but having same volume as grid squares) showing cells
3: Crop images or create region of interest same size as grid square
4: measure cells using combination of filtering and thresholding. 
5: export count data to txt or csv format
6: count and do math in excel.  In theory, you could take a much lower mag photo and count in this manner automatically for better statistics.  Scale as you see fit.
Karl
Mundelein, IL  USA

Offline Tristan

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Re: Microscope and yeast counting
« Reply #41 on: March 12, 2011, 04:42:22 PM »
Thought I would resurrect this old thread.  I recently was given a Leitz microscope, circa mid 80s, that was "retired" and no longer used.  I purchased a Hemacytometer (http://cgi.ebay.com/ws/eBayISAPI.dll?ViewItem&item=180512548686&ssPageName=STRK:MEWNX:IT#ht_4590wt_1117)  a few weeks ago and have done one count on it thus far.  I was lucky enough to get a .3ml micropipet with a good supply of tips to fill the chamber.

I am eager to apply this process to improve my yeast handling and propagation.    In retrospect for those gentlemen who've gone down this path; how long did it take you to become comfortable with the cell counting process?  Would you say you had it down after a few tries or did it take a significant effort?  At what point did you use it to improve your propagation process?

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Offline Kaiser

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Re: Microscope and yeast counting
« Reply #42 on: March 14, 2011, 07:56:08 PM »
I am eager to apply this process to improve my yeast handling and propagation.    In retrospect for those gentlemen who've gone down this path; how long did it take you to become comfortable with the cell counting process?  Would you say you had it down after a few tries or did it take a significant effort?  At what point did you use it to improve your propagation process?

I'm mainly using the microscope to keep track of my yeast propagation and run a few experiments. In the end I want to be able to have good guidelines that allow me to estimate cell count from the weight of a yeast slurry. I'm starting to get there.

Counting cells is pretty easy once you have a "clicker-counter", but it takes time and having the ability to do that definitely added time to my brew day.

Karl, I haven't seen your reply until now. I'll have to check that out.

Kai