School Science Project - Counting yeast cells

[jasonclick]

So my son (6th grade) brought home his homework and has to do a science project. He already thought of a few things... fruit batteries and the like. I said "how about we do an experiment about growing yeast cells... maybe compare using a stir plate and straight oxygen injected into the wort?". I've always wanted to get my boys interested in the process. Well, me thinking he would have just ignored my ideas like usual, went to school and got the project approved. Now I've got to help him with the project but I need equipment. I've watched some YouTube videos on yeast cell counting and have read Jamil's yeast book so I've got a basic understanding of the procedure. What I'm wondering is what equipment is needed and how is the cheapest way of buying it?

I've got a 2 liter flask, stir plate and oxygen stone already. I assume I'll need a microscope and hemocytometer. Any suggestions on those? Where to get a good deal and the basic specs on what would be needed? Any other equipment? Thanks.

 

[Homercidal]

Those and you may want to get some stain so you can count living cells vs dead cells.

 

[trentm]

What a great project for you and your son!

Yes a microscope and hemocytometer are needed as well as some way to make accurate dilutions. In order to count cells from a starter I use a 20:1 dilution as it is impossible to make good counts at the cell concentration of a starter. While I use micro pippetors and a 1ml centrifuge tube, there are cheaper materials available to make the dilutions. One method would be to use a 20ml graduated cylinder and a 1ml disposable pipette. Simply add 1ml of the yeast starter to 19 ml of water. Always make sure the yeast are in suspension, they settle quickly. It is very easy to make errors in this type of counting so be sure your dilutions are accurate and follow an exact counting method. An error of 5 cells per square in your count can mean a difference of 25 million cells per ml. That's a 50 billion cell error in your 2 liter starter.

Below is a list of equipment I use along with price and Cynmar number (http://cynmar.com/). The microscope was purchased from AmScope (http://www.amscope.com/). For your microscope, be sure to get one that has a mechanical stage and at least a 40X objective with a 10X eyepiece. For the hemocytometer, I tried the cheaper versions with much regret and ended up getting the one described below. The more expensive versions are probably even better but this works fine.

There is a nice free app (Hemocytap) for iPhones that helps with counting and calculations.

If you want to do viability staining, you can get methylene blue at you local aquarium store or from Cynmar.

 

[ajdelange]

There are some notes on how to use a hemocytometer at
http://wetnewf.org/pdfs/hemocytometer.html
There are lots of other places to find instructions as well.

On the microscope: You only need perhaps 50 - 100x (10x eyepiece, 5 - 10x objective) to count yeast (as the photomicrograph in the web article makes clear). If you have the opportunity to obtain a good used low power microscope this is the better option as most of the toys that sell for a couple of hundred dollars and which advertise 400 and sometimes 1000x are playing a marketing numbers game and selling 'empty resolution'. Unfortunately, you are probably stuck with one of these unless you can find something on e-bay, have a friend with one he wants to sell...

 

[jasonclick]

thanks for the info... looks like this will be an expensive science project... it may be cheaper to just pay the teacher off

 

[trentm]

There are some notes on how to use a hemocytometer at
http://wetnewf.org/pdfs/hemocytometer.html
There are lots of other places to find instructions as well.

On the microscope: You only need perhaps 50 - 100x (10x eyepiece, 5 - 10x objective) to count yeast (as the photomicrograph in the web article makes clear). If you have the opportunity to obtain a good used low power microscope this is the better option as most of the toys that sell for a couple of hundred dollars and which advertise 400 and sometimes 1000x are playing a marketing numbers game and selling 'empty resolution'. Unfortunately, you are probably stuck with one of these unless you can find something on e-bay, have a friend with one he wants to sell...

Yes this is true. I once had access to a really good Olympus microscope (~$20,000). Counting yeast cells could easily be done with a 5 or 10X objective. It was actually easier than with the 40X objective in my "toy" microscope. Unfortunately, as A. J. points out the high quality optics may not be affordable for most of us. Even the high quality low power microscopes on eBay sell for several hundred to over a thousand dollars and are typically sold "as is". Minor problems, say with fine focus or the stage mechanics can make a great microscope unusable and repairs can be very expensive. On the other hand if you can find a used Olympus, Nikon, Lecia, Zeiss, etc. that is in good working condition for a reasonable price, by all means that's your best option. That said the "toy" microscopes will do the job.

Does your son's school have microscopes available?

 

[Kingbogart]

Maybe a different type of "counting" would reduce the cost but still lead to a result. Could you get by with say, a measurement of yeast material after the starter was done? For a stagnant starter you might be able to graph the volume of trub that's accumulating in the flask day to day, or hourly blocks. For the stir plate version, that doesn't work for obvious reasons, but you could compare a completely stagnant sample to one that is shaken on occasion. Oxygen/no oxygen, shaken/not shaken gives a couple variables to play with. Not sure of the specifics of the project, but it seems more in line complication wise and cost wise.

 

[jasonclick]

good idea... it's a 6th grade project so I don't believe they are asking for in depth analysis. But if I do want to go the scope way, I'm assuming the minimum I would need would be a microscope and a hemocytometer?

 

[jasonclick]

just to give an update... our experiment is over... we used a microscope and hemocytometer. Here are the results after 24 hours:

starting cell count per mL in each flask: 21,208,000
ending count with no stir plate: 52,924,000
ending count with a stir plate: 158,772,000

what a difference a stir plate makes!

 

[trentm]

just to give an update... our experiment is over... we used a microscope and hemocytometer. Here are the results after 24 hours:

starting cell count per mL in each flask: 21,208,000
ending count with no stir plate: 52,924,000
ending count with a stir plate: 158,772,000

what a difference a stir plate makes!

Thanks. Great info for us and I bet you have stimulated an interest in science for your son.

 

[Black Island Brewer]

just to give an update... our experiment is over... we used a microscope and hemocytometer. Here are the results after 24 hours:

starting cell count per mL in each flask: 21,208,000
ending count with no stir plate: 52,924,000
ending count with a stir plate: 158,772,000

what a difference a stir plate makes!

Where did you end up getting your microscope and hemocytometer?

 

[jasonclick]

Where did you end up getting your microscope and hemocytometer?

I got the microscope on Amazon:
http://www.amazon.com/dp/B004UMDLAA

I initially purchased the Hemocytometer on Amazon as well but returned it after I couldn't find any lines on it. I ended up getting this one on Cynmar:

http://www.cynmar.com/ProductDetail/01200123_Darklined-Hemacytometer-Specialty-Cell-Counter

I tell you going down one of the off shoots of brewing is leading to things I wouldn't have been interested in before. I also got vials and plates to start culturing and storing yeast. Lab equipment is as cool as some of the brewing equipment I have.

 

[Black Island Brewer]

I got the microscope on Amazon:
http://www.amazon.com/dp/B004UMDLAA/?tag=skimlinks_replacement-20

I initially purchased the Hemocytometer on Amazon as well but returned it after I couldn't find any lines on it. I ended up getting this one on Cynmar:

http://www.cynmar.com/ProductDetail/01200123_Darklined-Hemacytometer-Specialty-Cell-Counter

I tell you going down one of the off shoots of brewing is leading to things I wouldn't have been interested in before. I also got vials and plates to start culturing and storing yeast. Lab equipment is as cool as some of the brewing equipment I have.

Awesome, thanks! In guessing you were happy with the gear? I'm wanting to experiment with using one yeast strain for all of 2015, and want to observe it for viability and mutation, growing extras starter and using the extra for the next starter. Don't know yet if I will, but I'm exploring the possibility.

 

[jasonclick]

yes it all seems to work as it's supposed to. The one thing that surprised me was how hard it is to find the lines on the hemocytometer. The one I got on Amazon might have been good if I had setup the microscope properly to find the lines. I thought the brighter the better on the microscope but actually I had to turn down the light to it's lowest point to see the lines. Once I did that, it works as it supposed to.

 

[gmonken]

Jasonclick,
I'm very interested in your yeast cell counting. In fact I got a microscope and hemocytometer for Christmas. What type of process or steps did you use in counting yeast cells? I've never done this before but have much chemistry lab experience. How did you do your dilutions and what equipment did you use? Where did you start? I am more interested in making sure I have the right amount of yeast for my fermentations. I brew 10 gal. batches and am never really certain if I'm pitching the right amount of yeast or not. I've never made a starter for this very reason because I can never be sure of the results of my starter. So I just buy extra vials of White Labs yeast and pitch them. By counting I can get into the right park. I've also read and downloaded a lot of info on counting yeast cells but never pulled the trigger. Could you fill me in on some of your procedures and processes?

 

[GilaMinumBeer]

Now THIS is one helluva way to get brewery tech through funding.

 

[jasonclick]

Jasonclick,
I'm very interested in your yeast cell counting. In fact I got a microscope and hemocytometer for Christmas. What type of process or steps did you use in counting yeast cells? I've never done this before but have much chemistry lab experience. How did you do your dilutions and what equipment did you use? Where did you start? I am more interested in making sure I have the right amount of yeast for my fermentations. I brew 10 gal. batches and am never really certain if I'm pitching the right amount of yeast or not. I've never made a starter for this very reason because I can never be sure of the results of my starter. So I just buy extra vials of White Labs yeast and pitch them. By counting I can get into the right park. I've also read and downloaded a lot of info on counting yeast cells but never pulled the trigger. Could you fill me in on some of your procedures and processes?

gmonken,
I basically started with the tips in this thread from others and then watched some youtube videos. Here's a link to the playlist:
https://www.youtube.com/playlist?list=PLuj0sGVhDPwM6J7RDhkLbjPk34R0R3Wzv

When we did the experiment for school, we didn't pay too much attention to sanitation since I wasn't going to use the yeast afterwards. The following is what I did to count the yeast.

1- we started with a slurry from a previous batch of beer but I'm sure you could start with a white labs tube and just dilute it down

2- I measured out two 20mL vials with 9mL of distilled water each

3- I transferred 1mL of the slurry to one of the vials. This is the first dilution. 1:10

4- I then took 1mL of the above and added to the 2nd vial. This was the 1:100 dilution.

5- I added a drop of that solution to the hemacytometer.

6- I used an app for my iPhone... HemocyTap to keep track of the counts. It also calculates the cell count if you plug in your starting volume.

7- when you are counting, my dilutions were 15-20 cells per square which are pretty easy to count. When I was doing counts after growth it was 80-100 cells per square which was a real chore to count. If there are too many cells per square, it's easier to dilute again.

8- cell counts can be done before and after using a stir plate to see the percentage growth or just afterwards to see how many cells you are starting with.

The most difficult part for me was setting the light properly to see the lines on the hemcytometer. Once I got the hang of it, it was no problem.

These are my basic procedures. I'm sure others with more experience can chime in.

 

[IslandLizard]

Awesome! ^ ^^ ^^^ ^^^^^ ...

Don't forget this is for a 6th grader who should do this all by himself only with as little assistance from his dad as possible. It's your son's project, right? Or not...

Your son maybe really smart and very interested (looking cool among his peers and with dad), but I bet the dilution/reduction schedule is already challenging. I'd stick to something way way waaay simpler. More qualitative too.
How about growing some yeast? Volume indicates growth.

I remember the school "science" projects from my son during those fruitful years.
Involvement: 90% dad, 9% mom, 1% son. Mom and dad switched roles for non-scientific projects. Son's involvement remained the same. Thank goodness he's 23 now with a BA in business. As far away from science and rhetorical theory as he can get. Science the most.

:smack:

 

[gmonken]

Jasonclick,
Thank you so much for the insights into your process of yeast cell counting. It surely helps to kick me off into counting my own yeast cells.
Another question: Have you ever done any viability counts? And if so, what procedures did you use? Dye and dilution of dye and yeast?
Thank you again jasonclick for all your information.

 

[jasonclick]

I bought the stain but haven't used it yet. That's my next thing to try.

 

[WoodlandBrew]

What an awesome science experiment! I was going to suggest an optical cell density meter instead of a microscope, but it looks like I'm a little late to this thread.

Stir plates do increase the growth rate, but the yield is about the same if you let them both run to completion.