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General Category => Yeast and Fermentation => Topic started by: karlh on December 15, 2009, 03:21:07 PM

Title: Microscope and yeast counting
Post by: karlh on December 15, 2009, 03:21:07 PM
Does anyone out there use a microscope and counting chamber for yeast counting?  I am wondering what the basic equipment and procedures are, what common dilution levels are, and whether it is worth it.  I have a decent microscope, and could very likely be yeast counting tomorrow with cookbook type instructions, but am uncertain whether you collect a sample from the yeast cake and dilute or shake up a starter and sample the liquid slurry.  If you were to do the latter, would you then pitch the entire volume?  Is anyone doing any yeast counting?
Title: Re: Microscope and yeast counting
Post by: a10t2 on December 15, 2009, 04:02:05 PM
http://www.cascadebio.com/uploads/File/pdf/hemat.pdf
Title: Re: Microscope and yeast counting
Post by: Kaiser on December 15, 2009, 04:03:23 PM
I'm getting one and a counting chamer for christmas. I'm looking forward to that and playing around with it. But at this point I only have a vague idea about what to do exactly.

I believe you want to get a hemacytometer and methylene blue stain. The stain is not necessary for counting but can give you a rough idea which cells are dead and which are alive. 

Kai
Title: Re: Microscope and yeast counting
Post by: tom on December 15, 2009, 05:20:22 PM
Page 7 of http://www.brewingscience.com/PDF/BSI_brewers_lab_handbook.pdf
Title: Re: Microscope and yeast counting
Post by: Kaiser on December 15, 2009, 05:22:57 PM
Page 7 of http://www.brewingscience.com/PDF/BSI_brewers_lab_handbook.pdf

That's a very nice find. Thanks.

Kai
Title: Re: Microscope and yeast counting
Post by: tom on December 15, 2009, 07:45:18 PM
Their whole website is a great source. Too bad they don't sell yeast to homebrewers.
Title: Re: Microscope and yeast counting
Post by: philsner on December 16, 2009, 03:03:21 PM
Thanks, good info on the proceedure. I have a 1000x scope, so where is the best place to get a hemacytometer and methylene blue stain?

Philsner The Great
Title: Re: Microscope and yeast counting
Post by: narvin on December 16, 2009, 03:57:16 PM
I purchased this one -- http://www.cynmar.com/item_detail.aspx?ItemCode=01200123.  Kai recommended cynmar at one point, and I was happy with their service.

It's definitely interesting, although I can't see myself doing it on every batch.  I'm actually more interested in using the microscope for wild Belgians.
Title: Re: Microscope and yeast counting
Post by: Kaiser on December 16, 2009, 04:17:09 PM
I love shopping and browsing at Cynmar. At this point I’m ordering more there than I do at home brew stores. I have their catalog as bathroom reading material.

I got the methylene blue as dry power and plan to make small batches of it for staining. I figure that I won’t need much.

I’m with Narvin in that the excitement over being able to count cells will wear off at some point. But it’s still nice to be able to check one more intermediate point in the process.

And my daughter (5) seems to be very interested in looking at stuff under the microscope. So getting a good one wasn’t a hard sell to the wife if it helps with the education of the kids.

Kai

Title: Re: Microscope and yeast counting
Post by: denny on December 16, 2009, 04:56:23 PM
And my daughter (5) seems to be very interested in looking at stuff under the microscope. So getting a good one wasn’t a hard sell to the wife if it helps with the education of the kids

When I was a kid, my parents bought us chemistry sets, microscopes, electronics construction kits, all kinds of science stuff.  I think that's one of the main reasons I turned into a geek!
Title: Re: Microscope and yeast counting
Post by: bluesman on December 16, 2009, 05:35:52 PM
I believe one would be really enlightened and suprised at the behavior and viability of yeast under a microscope. I have never examined yeast under a scope but sure would like to give it a try sometime. My wish list is pretty long and getting longer.  ;D
Title: Re: Microscope and yeast counting
Post by: karlh on December 16, 2009, 06:57:14 PM
I believe one would be really enlightened and suprised at the behavior and viability of yeast under a microscope. I have never examined yeast under a scope but sure would like to give it a try sometime. My wish list is pretty long and getting longer.  ;D

I have a microscope and counting chamber, and started the thread to get an idea if and how folks are using these.  What I can say from my very limited experience is that the quality of the microscope is important.  New student and lab microscopes are very expensive, costing hundreds if not thousands of dollars. Toy microscopes will not give you a quality approaching that of a 100 year old lab microscope.  They have plastic optics, and really don't work well.  I found a high quality (Leitz) microscope from around 1950 on craigslist for $100, and have been very pleased with it.  If you can find a decent quality lab microscope it will be of a far greater value than a new, inexpensive, microscope.

I have followed the procedures Kai linked to, which are similar to those on the Brewing Techniques site and elsewhere, and the cells are easy to see and count.  I have not worked on the staining yet, but will note that you can also find Meth blue in the pet supplies for aquarium use, its inexpensive, and would work well.  The yeast does not really "behave"... it just sits there, and using a 40-50x objective they just look like little spheres in the counting squares.  The math is tougher than the microscope. 
Title: Re: Microscope and yeast counting
Post by: Thirsty_Monk on December 16, 2009, 08:13:28 PM
I have their catalog as bathroom reading material.
This is pretty hard core :)

Kai
What kind of microscope did you buy?

By the way this is pretty interesting subject.
Title: Re: Microscope and yeast counting
Post by: Kaiser on December 17, 2009, 04:20:56 AM
What kind of microscope did you buy?

I think it was this: http://cynmar.com/item_detail.aspx?ItemCode=CSH08017

Kai
Title: Re: Microscope and yeast counting
Post by: Thirsty_Monk on December 17, 2009, 03:24:25 PM
Thank you :)
Title: Re: Microscope and yeast counting
Post by: Kaiser on December 17, 2009, 03:36:06 PM
What kind of microscope did you buy?

I think it was this: http://cynmar.com/item_detail.aspx?ItemCode=CSH08017

Kai

I just checked and they don’t have the one that I bough (753 SRS) anymore. It was about the same price and has very similar specs. I don’t think it matters much and I didn’t really do a lot of research either. I went for the monocular version which will be a pain but I didn’t feel like spending an additional $100. I’ll just buy and eye patch ;)

Kai
Title: Re: Microscope and yeast counting
Post by: Kaiser on December 26, 2009, 10:28:30 PM
So I finally got to play around with the microscope and the hemacytometer.

So cool to see the yeast and with methylene blue stain it is easy to tell the difference between viable and non-viable cells. 400x seems to work best for counting for me. But counting  cells is a lot of prep work and some clean-up.

Kai
Title: Re: Microscope and yeast counting
Post by: karlh on December 31, 2009, 05:26:43 AM
I was messing around with mine, and one of the kids must have been turning knobs.  I tried to focus the microscope and "crack"... there went my counting chamber.  I focused right through the slide (and they aren't cheap slides).  I thought that I might mention that (at least if you have kids around) you should start at the lowest magnification, find the area you are looking for, and then increase the magnification one objective at a time until you are up at 400X. 

I work with microscopes every day, and never thought I would make this mistake at home, but I'm back shopping for a new counting chamber. 
Title: Re: Microscope and yeast counting
Post by: Kaiser on December 31, 2009, 02:47:41 PM
Karl,

That sucks. But can't you just use a normal cover slide? The grid is on the bottom of the chamber which is hard to break. Or get a replacement for the slides. I think that this must happen quite often and mine came with a spare cover slide.

Kai
Title: Re: Microscope and yeast counting
Post by: tubercle on December 31, 2009, 06:17:41 PM
 Crack? :D :D :D

  I'm not laughing at you, I'm laughing with you. Hope 2010 is a little better for ya!

  Reminds me of a guy who once tried to get a "running go" with his malt crusher and flipped it across the floor.

  It sucks to be human.
 
Title: Re: Microscope and yeast counting
Post by: Kaiser on December 31, 2009, 07:02:40 PM
looks like someone is making a Cynmar order: http://www.cynmar.com/productdetails/01200122.aspx

Kai
Title: Re: Microscope and yeast counting
Post by: Thirsty_Monk on December 31, 2009, 08:52:54 PM
And you get 10 cover glass with it :)
Title: Re: Microscope and yeast counting
Post by: roffenburger on January 05, 2010, 06:05:14 PM
I'm pretty spoiled and get to use a $5-6K Ziess scope at work. I've also used an inverted scope for a few months while doing research on cardiac myocytes that ran around 80K :o Cool stuff!

I want to get one for home use, but don't really see the need at this point. I would LOVE to have one though!
Title: Re: Microscope and yeast counting
Post by: Kaiser on January 05, 2010, 06:56:14 PM
Having a microscope certainly lengthened my brewday ;). At some point the frequent use is likely to wear off though.

To make determining cell densities and cell counts easier I wrote a simple spread sheet that I keep on my ipod and together with a tally counter app it’s pretty simple to use. I thought I may share it here: http://braukaiser.com/documents/cell_count.xls

Instead of giving dilutions you specify how many additional volumes of the sample volume you added for methylene blue dye and/or additional water. This seems to work better for me.

The current setting of depth and width (0.1 mm x 0.05 mm) is for one of the small squares in an improved Neubauer counting chamber.

Kai


Title: Re: Microscope and yeast counting
Post by: roffenburger on January 05, 2010, 07:46:00 PM
Its definitely a nice tools to have around--even if the usage reduces.
Title: Re: Microscope and yeast counting
Post by: karlh on January 09, 2010, 04:23:13 PM
I was messing around with mine, and one of the kids must have been turning knobs.  I tried to focus the microscope and "crack"... there went my counting chamber.  I focused right through the slide (and they aren't cheap slides).  I thought that I might mention that (at least if you have kids around) you should start at the lowest magnification, find the area you are looking for, and then increase the magnification one objective at a time until you are up at 400X. 

I work with microscopes every day, and never thought I would make this mistake at home, but I'm back shopping for a new counting chamber. 
I ordered a replacement counting chamber, through amazon of all places, as that was the best deal I could find with same day shipping (about $37 vs $50 on cynmar).  Hopefully I will be back in the cell counting business soon.  I have been looking at yeast on plain glass slides with Methylene blue, and Kai is right about the dramatic difference between the viable and dead cells.  If I can get pictures I will try to put some online (I might have to bring some yeast to work for this).
Title: Re: Microscope and yeast counting
Post by: Kaiser on January 09, 2010, 11:29:23 PM
Wow, you actually broke through the whole thing.

Kai
Title: Re: Microscope and yeast counting
Post by: dbeechum on January 10, 2010, 12:08:03 AM
I'm noticing cynmar's having a sale on some simple stereo micros.. may have to break down and get one.
Title: Re: Microscope and yeast counting
Post by: Thirsty_Monk on January 10, 2010, 02:24:08 AM
I'm noticing cynmar's having a sale on some simple stereo micros.. may have to break down and get one.
Do you have a link?
Title: Re: Microscope and yeast counting
Post by: dbeechum on January 10, 2010, 03:27:35 AM
http://www.cynmar.com/item_detail.aspx?ItemCode=CSS08409
Title: Re: Microscope and yeast counting
Post by: Thirsty_Monk on January 10, 2010, 04:22:57 AM
Would it have enough magnification?
Title: Re: Microscope and yeast counting
Post by: Kaiser on January 10, 2010, 01:16:41 PM
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
Title: Re: Microscope and yeast counting
Post by: karlh on January 11, 2010, 02:38:17 PM
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.   
Title: Re: Microscope and yeast counting
Post by: karlh on January 20, 2010, 11:12:53 PM
(http://api.ning.com/files/BAeAaxXrS191thQQdFsZDRA3Rc73aMUmVD4TvQCn4C9TuYAeAmVxII8C14*fuGkpavcGp3833KBEUEf-IuyplRt62Yh-D5Td/yeastcounting.JPG)

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.
Title: Re: Microscope and yeast counting
Post by: karlh on January 21, 2010, 12:01:10 AM
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:
(http://api.ning.com/files/OW11qYPBPs1MiNy8VfA3Obek6LNaizScW9cfxx2HHcvwEgqz76jSwW7zHpu97P1tLHB9t47NSDl-n8SSINoatOcJ2AlniuEC/yeastcountingb.JPG)
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.
Title: Re: Microscope and yeast counting
Post by: Kaiser on January 21, 2010, 12:03:03 AM
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
Title: Re: Microscope and yeast counting
Post by: karlh on January 21, 2010, 02:03:48 AM
Its a really nice microscope.  I have an unfair advantage when it comes to microscopes. 

(http://api.ning.com/files/Az-oNHrgWMfO3RuvkJW86NlmfR-*Ab7TJj78t-jq46se6FV32KngK1o56p5UM0yYo3P4Tf8PBn2F7650J*0mdRSWoZ76SDoX/yeast05.jpg)

(http://api.ning.com/files/Az-oNHrgWMfSUiiN3OeVwXSaejrAaRhBaSSdvK58iv6q2Oaib2vcKbyl1Gz9N4Rtpeg24NgACKrq255cnh8aqUdyenoIMmyR/yeast01.jpg)

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. 
Title: Re: Microscope and yeast counting
Post by: bluesman on January 21, 2010, 04:27:47 AM
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!
Title: Re: Microscope and yeast counting
Post by: roffenburger on January 21, 2010, 03:06:02 PM
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.
Title: Re: Microscope and yeast counting
Post by: Kaiser on January 21, 2010, 03:27:08 PM
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
Title: Re: Microscope and yeast counting
Post by: karlh on January 21, 2010, 06:40:11 PM
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.
Title: Re: Microscope and yeast counting
Post by: Tristan on March 12, 2011, 11:42:22 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?

Title: Re: Microscope and yeast counting
Post by: Kaiser on March 15, 2011, 02:56:08 AM
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