Pics of Yeast under my new microscope

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passedpawn

passedpawn

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Just bought a used scope and a hemocytometer for doing yeast counts and viability measurements. Man is this cool. (eek, I think I see a bacterium... yet another thing to worry about!)

I still have to get some stains to spot the dead ones. I can see the cells easily under 400x. I took this pic with a camera pressed up to the eyepiece. Looks much better with the eyes. I don't know why the lines of the hemocytometer don't show up in the pic... they are plain as day when viewed directly.

These cells are from some very dirty BoPils yeast cake, diluted 10:1. Wyeast 2001.

IMG_4674.JPG


2011-10-08_at_16_11_22.jpg
 
Is that set up in your home?! I'm a lab rat and am so freaking jealous right now!!! I've thought about ordering a "spare" hemocytometer for the lab for yeast use. As I'm doing starters, I'm thinking man... a nice set of pipets and an easypet.

Where did you get the supplies? Fisher?
 
acuenca said:
Is that set up in your home?! I'm a lab rat and am so freaking jealous right now!!! I've thought about ordering a "spare" hemocytometer for the lab for yeast use. As I'm doing starters, I'm thinking man... a nice set of pipets and an easypet.

Where did you get the supplies? Fisher?
Click to expand...

I bought the hemocytometer off ebay. Improved Neubaur type, looks great, came with a couple of cover slips. Got scope there too.

Yea, that's my den in my house. Not a lab at all, sort of a mess of odds and ends equipment of various types. I have lots of equipment in my closet that I don't keep out all the time. The counter you see there is something I built onto a built-in hutch bookshelf thing... I've got a pic, maybe I'll post that instead of rambling here.

This scope is a lot of fun to play with. Of course, I'm trying to put bugs under it right now.
 
OK, I'm getting a better idea how to feed sample into the hemocytometer (I've been spelling that wrong for about 2 years!).

The coverslip goes on first, then use pipette to drop sample just outside the slip, and the sample capillaries into the 0.1mm channel between the cover slip and the grid. Here, I took another pic at 400x where you can see some of the grid lines. Each of these small boxes is 0.05mm across (0.0025mm^2). 16 of them make up the actual area you have to count (i.e., in the picture below you see half of the squares I counted).

Count all 16 squares, multiply x 250,000, 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 250,000 x 25 = 381.25 million cells per milliliter. Easy peezy. Now to determine how many are alive!

A lot of the tutorials have you continue counting blocks, essentially getting the count in the larger 25 blocks in the middle. Then they multiply by 10,000 instead of 250,000. As long as you understand the dimensions of each block, and know that there are 1000 cubic mm in 1 ml, you'll figure out the math.

IMG_4675.JPG
 
Yooper said:
That's awesome! I'm jealous. You geek!
Click to expand...

I'm having fun with this thing.

Turns out there is a Hemocytometer Counter app for the iPhone. Crazy. It's only $2, but I'll probably skip it since the math is simple. Still, pretty nifty for you app-crazed iPhone users. Just replace "red cells" with 'yeast cells".

[edit] turns out I was screwing up the math repeatedly. Maybe I'll get the app afterall :eek:

iphone_app.jpg
 
What kind of scope is that? My dad used to work for leica and I think he had two scopes. Not sure if they go to 400x.
 
passedpawn said:
The coverslip goes on first, then use pipette to drop sample just outside the slip, and the sample capillaries into the 0.1mm channel between the cover slip and the grid.
Click to expand...

Bingo. Doing it that way makes me more confident that I'm looking at a 100 micron gap.
 
Ok, I'm not a microbiologist or anything, but that is seriously cool. What exactly would I need to do that type of yeast cell count stuff?

I need a microscope that goes to what magnification?
What else exactly? If there is a place where I can research the needs list, I'm happy to do the work. Does Jamil's new book have the supply list? I'm out of town and the book is back home...

Thank you for sharing your setup. It's inspiring!
 
I've got a cheap child's microscope that works fine to count cells on the cytometer at 100x (10x objective with 10x eyepiece). You can't make out any bacteria or do viability staining, but the counting is quite visible. The hardcore stuff is what the scope in the lab is for.
 
PoppinCaps said:
I've got a cheap child's microscope that works fine to count cells on the cytometer at 100x (10x objective with 10x eyepiece). You can't make out any bacteria or do viability staining, but the counting is quite visible. The hardcore stuff is what the scope in the lab is for.
Click to expand...

That's encouraging. Maybe I could do this...
 
You could easily do it for under the price of a brew. There are tons of online science shops that will have everything, including a POS scope, hemocytometer (they can come cheap too), agar, petri dishes, pipettes, etc for keeping strains, the sky's the limit. At the very least, you'll finally know how many cells you're pitching.
 
kcpup said:
Ok, I'm not a microbiologist or anything, but that is seriously cool. What exactly would I need to do that type of yeast cell count stuff?

I need a microscope that goes to what magnification?
What else exactly? If there is a place where I can research the needs list, I'm happy to do the work. Does Jamil's new book have the supply list? I'm out of town and the book is back home...

Thank you for sharing your setup. It's inspiring!
Click to expand...

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.
 
PoppinCaps said:
You could easily do it for under the price of a brew. There are tons of online science shops that will have everything, including a POS scope, hemocytometer (they can come cheap too), agar, petri dishes, pipettes, etc for keeping strains, the sky's the limit. At the very least, you'll finally know how many cells you're pitching.
Click to expand...

PoppinCaps - thanks for that list and advice. Perhaps Santa will bring me this...better make sure I'm on the nice list. :mug:
 
passedpawn said:
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.
Click to expand...

Thanks for the list! I'm getting excited already!
 
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...
 
passedpawn said:
. . . . 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!

IMG_4675.JPG
Click to expand...

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:mug:
 
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.
 
COLObrewer said:
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:mug:
Click to expand...

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).

counting_chamber.jpg


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/CellCounting&Viability.pdf

Here's the straight dope on hemocytometer counting:
http://www.ruf.rice.edu/~bioslabs/methods/microscopy/cellcounting.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
 
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.
 
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.
 
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.
 
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.
 
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!
 
GodsStepBrother said:
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!
Click to expand...

I've got batches intentionally infected - with lacto (flanders red & berliner weisse). I will post up a pic of bacteria this wkend for comparison here. Hopefully I can get both them and the yeast in the same pic. Also, gonna work on getting my webcam into the eyepiece tube for some microscope video. Would be good to show a sped-up video of sacc budding.
 
This picture is so interesting. From Brewing Science and Practice,

“The Mean Cell size increases with generational age. For any given strain there is a rough correlation between number of bud scars and mean cell size. Using a diploid lager yeast strain Barker and Smart (1996) reported that the mean cell volume of virgin daughter cells was approximately 150 um3. Cells, which had undergone around 20 rounds of budding and where approaching the end of their life spans, had mean cell volumes of approximately 850 um3. “

Could be the picture below. Hmmm. :rolleyes:

cells.png
 
Kcpup, that's a good price for a semi-serious scope for brewing. Maybe it doesn't have the optical superiority as an Olympus or Leica, but you don't need publication worthy images (other than for HBT posts, that is...). For an inverted, binocular scope ( that works, make sure), solid find. You can get fancy and add a camera and such later via one of the eye pieces.
 
PoppinCaps said:
Kcpup, that's a good price for a semi-serious scope for brewing. Maybe it doesn't have the optical superiority as an Olympus or Leica, but you don't need publication worthy images (other than for HBT posts, that is...). For an inverted, binocular scope ( that works, make sure), solid find. You can get fancy and add a camera and such later via one of the eye pieces.
Click to expand...

PoppinCaps, thank you for the feedback!
 
kcpup said:
http://kansascity.craigslist.org/sys/2575287392.html

Trying to figure out what to target when looking at craigslist.

Thanks in advance for any feedback.
Click to expand...

I'd jump on that. Looks almost like mine. Optics are everything of course. Just buy it and hope for the best.

I think I see an Abbe condensor, X/Y stage controls, slide clip, that thing is an Olympus CH clone. The 100x is probably an oil immersion lens, which uses oil on the slide. You can find more info on that by searching. Make sure to as the seller if there is a 40x objective.
 
Here's a quick video that shows 40x, 200x, and 400x. I can see a lot more in the eyepiece than my photos and video show... I'm not getting the field of view for some reason.

I had to take apart my webcam tonight and remove the lens to get this to work... check it out. I will get better at this. I am going to make a PVC tube to extend the distance of the webcam from the objective and see if that helps.

 
Last edited by a moderator:
FYI, here's a pic of my webcam taped to the eyepiece tube (eyepiece optics removed, and also the webcam lens removed (had to desolder a couple of pads there - probably to the zoom lens inside it). This only took a few minutes to do and worked surprisingly well. Very happy with quick result.

2011-10-14_at_20_09_00.jpg
 
I love the beautiful juxtaposition of the great, shiney technology coupled with the jerry-rigged webcam. :D

Cool video though.
 

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