Yeast basics: Counting yeast cells

Eureka, this post starts the yeast basic series. Today, we are talking about a basic technique how you count yeast cells to determine the cell concentration in a yeast starter or calculate the amount of yeasts you have in total. This method is only for those of you having access to a microscope. However, there is another easier technique to get an idea about the yeast concentration. Will cover this technique in a future post. Lets begin with the microscope based technique.

Why count the yeast cells?

Counting cells is a technique to determine the cell concentration in a liquid. Imagine you want to make a 5 gal (18.9 L) batch of beer with an original gravity of 1.048 (12°). How much yeast do you need to get a healthy fermentation? This can be calculated by different yeast calculators such as the one from J. Zainasheff (mrmalty.com). The right amount of yeast would be 170E9 cells for this example. Lets assume you do not have the right amount of yeast available and you need to propagate the yeast with starter(s). You could even harvest yeast from a previous batch to ferment another batch. The question now arises, how can you be certain to have the exact number of yeast cells after the propagation step(s) or how much of the yeast cake do you need to harvest to get the right amount of yeast cells? This question can be answered with a cell count.

What do I need to count yeast cells?

First of all, you need a counting chamber. A Neubauer-improved counting chamber works very well for yeast cells. More about the counting chamber later on. Second, you need a microscope with a magnification greater than 400x. Third, equipment to dilute the yeast samples. Pipettes and graduated cylinders work very well.

What is a counting chamber?

A counting chamber is a special glass slide (the size of a normal microscope slide) with engraved squares and a cover slip (not shown). The squares are located in the middle of the chamber (indicated with the circle in Fig 1).

Fig 1: Neubauer-improved counting chamber

Fig 2: Haemocytometer grid [from Wikipedia]

Now about the squares of the counting device. There are nine big squares (red). In the red square are again 16 squares (green). There are yet smaller squares (yellow and blue ones). The counting chamber can be used for different cell sizes. If you have bigger cells such as leukocytes, you could count them in the red and green squares. In case of yeast cells, you can use the yellow squares because these cells are smaller than leukocytes.

When you prepare the chamber, you cover the squares with a cover slip. The gap between the squares and the cover slip is exactly 0.1 mm. The area of the red square equals 1 mm2, a yellow square equals 0.040 mm2 and therefore equals 4 nL (= 4 nano liters) of volume with the cover slip mounted on the counting chamber. The squares therefore represent a very small volume. Counting the cells in these squares can give you information about the cell concentration in the end.

How do you prepare and use the counting chamber?

Please consult White Labs homepage and have a look at the following video. Please notice the particles shown in the video are about 100 times bigger than yeast cells. For these particles the red squares are the most useful ones. For yeast cells on the other hand, use the yellow squares.

Two more things. First concerning the cover slip. In the video the cover slip is just laid on the counting chamber (synonym for hemacytometer). I breathe on the cover slip first and then lay it on the counting chamber. If you do it right you should see some Newton’s rings. If these rings can be observed the gap between the counting chamber and cover slip is tight.

Second concerning the counting rules. As mentioned there are several accepted ways to do so. I learned to count the cells touching the tripled line on top and the right side. Cells touching the lines on the bottom and the left side are not counted. More about that later on. So far about the principles.

Lets make an example

You made a starter (1’000 mL) and want to know how many yeast cells you have in there and check if you have enough yeast cells to ferment your batch.

1. You first dilute 1 mL of the yeast starter with 99 mL water or isotonic sodium chloride solution (9 g sodium chloride dissolved in 1 L of water). This is a 1:100 dilution step. If you expect a low yeast concentration, a 1:10 dilution could work as well or even load the chamber with undiluted starter.

2. You then load the chamber and it looks like the following picture:

Fig 3: Yeast cells in counting chamber

What you can see here are mostly yeast cells. Don’t bother about the blue color. This is how your yeast sample in the counting chamber could look like. Now, please notice the triple lines on the left side, top and right side of the picture. The bottom one is not visible in the picture. But there are some of these lines at the bottom as well. If you compare it with the lines in Fig 2, you can see that you are looking at a yellow square with the 4 x 4 blue squares. The triple lines are the border lines of the yellow squares. Next thing to do is count all the cells in the yellow square (between the triple lines). Please have a look at the video mentioned above how you count the cells. As mentioned above, there are several rules about how to count the cells. My technique is to count only the cells touching the lines on top and the right side. Lets have a look at the picture above. In the second square on the top left, there is a cell touching the tripled line (marked as 1). This cell is included in the count. The cell marked as 2 and 3 are included as well. The two cells in proximity of 4 are included as well. The cell below the 4 is still in the square. The cells above the 4 are not included since they are not in the square. Next dark blue cell 5. If you look at the cell it is just at the border. I guess here you can decide whether you include the cell or not. And to finish, cell number 6 is not included since it touches the lines on the left side. I hope this helped to understand how to count the cells.

Lets say you counted a total of 65 cells. Next you count another four of these squares. In total five yellow squares. Lets say you counted 60, 70, 62 and 55 in the next yellow squares.

3. Add all the numbers together: 65+60+70+62+55 = 312 cells. You therefore have 312 cells in 20 nL (since one yellow square equals 4 nL and you counted five in total).

4. Multiply 312 by 5 to get the concentration in 100 nL. In this case you have 1560 cells in 100 nL. Or 15600 cells in 1 µL.

5. Then multiply the 1560 cells with 10000 to get the concentration per mL (conversion from 100 nL to mL). In this case 1.56E7 cells in 1 mL.

6. Last thing to do is to include the dilution of 1:100 from the beginning. Multiply the 1.56E7 cells per mL with 100 to get the final yeast concentration of 1.56E9 cells per mL. This is the yeast concentration you have in your 1000 mL starter. In total, you now have 1.56E12 yeast cells in the yeast starter (1.56E9 cells per mL x 1000 mL = 1.56E12).

Coming back to the 5 gal (18.9 L) batch of beer with an original gravity of 1.048 (12°). We previously calculated that you need 170E9 yeast cells for this batch. Your starter has a cell concentration of 1.56E9 cells per mL. 170E9 cells divided by 1.56E9 cells per mL gives you a volume of approximately 109 mL. You therefore need to pitch 109 mL of the yeast starter to have the right amount of yeast cells. In fact, your yeast starter would have enough yeast cells to ferment approximately 170 L (45 gal) of a 1.048 (12°P) wort.

To summarize, the best way to get an idea about the yeast concentration of a starter or a yeast slurry is to use a counting chamber and count five yellow squares. Add all the numbers together and multiply it by 50000 to get to the concentration in cells per mL. And don’t forget the dilution factor. Simple as that.

In my opinion, if you count yeast from a starter of a fresh yeast slurry, you do not have to worry about the yeast health. Just count the yeast cells and get an idea about the yeast concentration. On the other hand, if you count yeasts which are not the healthiest anymore, it seems advisable to determine the viability of the yeast as well. This means, get an idea about how many yeast cells are dead. More about the viability test in a future post. Stay tuned for future posts and please let me know if something is not clear.

Brettanomyces lambicus microscopy pictures

Eureka, its time for yet another post about Brettanomyces. And today, we are looking at some Brettanomyces lambicus cells. You might remember, that I did some plating of the two Brettanomyces strains from Wyeast: Brettanomyces bruxellensis and Brettanomyces lambicus on Sabouraud agar. And I already posted the results from the microscopy observation of a B. bruxellensis sample. Now is the time to talk about the microscopy observation of the B. lambicus cells from Wyeast.

Lets begin with a few pictures now to get an overview. I will get into further details in the pictures below.

Fig 1: Wyeast's Brettanomyces lambicus (low magnification)

Fig 2: Wyeast's Brettanomyces lambicus (low magnification)

Fig 3: Wyeast's Brettanomyces lambicus (low magnification)

My first impression is that B. lambicus, much like B. bruxellensis, appears in very different shapes. There are boat-shaped cells, elongated cells, and even cells that attach to each other. But very few of the cells are circular like normal brewers yeast. Maybe the circular cells here might be brewers yeast impurities in the Brettanomyces sample. To summarize, B. lambicus seems to look quite different from brewers yeast (Saccharomyces cerevisiae). Lets go into more details at a higher magnification.

Fig 4: Wyeast's Brettanomyces lambicus (high magnification)

The cells seem to have a kind of vacuole (Fig 4). The vacuole can be observed as a kind of compartment in the cell itself. It is much easier to observe in the next picture (Fig 5).

Fig 5: Wyeast's Brettanomyces lambicus (high magnification)

Interestingly, there is something circular in the vacuole. I will come back to this observation later on.

Fig 6: Wyeast's Brettanomyces lambicus (high magnification)

It can be observed now, that the cells have again, like B. bruxellensis, one or two dark spots (Fig 6). I am still figuring out what these dark spots might be. But now back to the most interesting observation from my point of view. I already mentioned, that I could observe some kind of circular structure in the cells vacuole (see Fig 5). One disadvantage of pictures is their snapshot nature. The pictures just represent a short state of a cell. Just have a look the following video I took to see what the cells really looked like for real. Observe what happens inside the vacuole. I have to apologize for the bad quality of the video.

Have you noticed that the circular structure moved in the cells vacuole? Well, I have never observed something like that before. In my opinion there are several possible explanations for such a movement.

– One would be that there is a kind of dynamic movement in the vacuole itself. Much like mixing the vacuoles content in some way. And the circular structure then could be maybe a vesicle with some sort of nutrients in it and swirl around due to the dynamic movement in the vacuole.

– Another explanation, there is a kind of other cell in there. But this seems to be very unlikely to me. I have several reasons to come to such a conclusion. One would be how these cells would have gotten there in the first place.

– Sporulation. Could these structures be spores?

This is very exciting in my opinion. A lot of questions. I have several further investigations in mind to get further information about that circular structure in there. If someone out there has any idea what these structures could be, please comment below. Please comment as well if you have observed such a movement yourself.

What have I learned from the microscopy observations so far?

The observations helped me to understand the different morphologies of the Brettanomyces yeast cells. This can be observed if you look at different microscopy pictures as well. But looking at them yourself is very different. And this helped me to identify several yeasts I harvested from different sources to be Brettanomyces. On the other hand, colonies on the agar plates of the two strains looked very similar and I could not distinct between them. I addition, the colonies of Saccharomyces cerevisiae looked very similar to the ones from the Brettanomyces as well. I could not even distinct between Saccharomyces and Brettanomyces by just looking at the colonies on the Sabouraud agar. Only a microscopy observation can help to make such a distinction. It is therefore inevitable to look at the cells to get an idea what kind of yeast you have.

That’s all so far about the two Brettanomyces strains from Wyeast. Unfortunately, I can’t get my hands on the White Labs strains here. So no further pure strain observations will follow. What I have planned is to write some posts about Brettanomyces in general. And the first post about “Brettology” will be a post about the taxonomy of Brettanomyces. So stay tuned and thank you for your comments!

Brettanomyces bruxellensis microscopy pictures

Eureka, today’s post is all about Brettanomyces bruxellensis from Wyeast. One reason why I started posting about beer and souring bugs in the first place was because of the very limited available microscopy pictures of Brettanomyces. Sure there are some great sources like the Brettanomyces project page or Jason’s pictures to have a look at some Brettanomyces cells. But since I own a microscope myself, why not have a look at the cells myself. So I started with a pure culture of Brettanomyces bruxellensis from Wyeast and took some pictures while looking at them. Pictures of agar plates with the Brettanomyces will follow as well. Well, this post is more or less a picture library from my point of view. Lets begin.

Size of Brettanomyces bruxellensis

First about the size of B. bruxellensis. I loaded a Neubauer counting chamber with some B. bruxellensis cells (Fig 1). The width of one square is 50 µm. This information can now be used to determine the size of a cell. A B. bruxellensis yeast cell has a size of approximately 5 – 7 µm. Lets compare this size to other microorganisms: A typical yeast cell is around 5 µm. The Brettanomyces therefore has a very similar size like a normal yeast cell. This is no surprise since Brettanomyces are yeasts as well.

Fig 1: Brettanomyces bruxellensis in Neubauer counting chamber (width of square = 50 µm)

Morphology of Brettanomyces bruxellensis

Maybe you already observed that the cell morphology are very inhomogeneous (Fig 1). The following pictures are all Brettanomyces bruxellensis with all sort of morphological appearances. I have to apologize for the bad quality of the pictures. But I do not own the appropriate objectives for taking pictures. I hope to take some pictures in the lab with a far better microscope in the future.

Fig 2: Brettanomyces bruxellensis low magnification

Fig 3: Brettanomyces bruxellensis low magnification

Fig 4: Brettanomyces bruxellensis low magnification

So far for the lower magnification. In my opinion there are at least two different morphologies. There are circular cells with a dark spot, or two. And there are the slender cells like the ones in the upper left corner in Fig 4 that stick to other cells. The next pictures are taken at a higher magnification than the ones above.

Fig 5: Brettanomyces bruxellensis high magnification

Some cells look like small lemons. Or at least not perfectly circular (Fig 5). And these cells have a dark spot and some kind of vacuole. These cells look way different from normal Saccharomyces cerevisiae (brewer’s yeast). I have never observed a dark spot in S. cerevisiae before. Maybe this spot is something common in Brettanomyces? We will see about that as we look at Brettanomyces lambicus in a future post. And I have no idea what this dark spot could be.

Fig 6: Brettanomyces bruxellensis high magnification

It gets even more interesting. Lets have a look at the cell in the middle in Fig 6. The one connected to another cell. The lower cell, with the dark spot, looks like every other cell with a spot. But this one seems to be elongated. I assume the elongated cells are just elongated circular cells. This would make sense since there is only one strain of Brettanomyces in there (I hope). But some cells look like S. cerevisiae. Maybe there are some S. cerevisiae in Wyeast’s Activator package?

Fig 7: Brettanomyces bruxellensis high magnification

Here is another elongated cell in the lower left corner (Fig 7). And the typical dark spot can be observed again.

Fig 8: Brettanomyces bruxellensis high magnification

This pictures makes it easy to observe the kind of vacuole (Fig 8). And right in the middle of the picture is a cell with two dark spots.

Fig 9: Brettanomyces bruxellensis high magnification

And here is another shape to observe (Fig 9). This Brettanomyces strain tends to adhere to other cells. What you get is a kind of network of different cells sticking togheter. And the size of the elongated cells is way bigger than all the other cells around them. I have some sort of hypothesis here. I could imagine that two Brettanomyces cells somehow elongate to form bigger cells?

Maybe some of you remember the pictures I took from a beer I fermented with a kind of souring bug. The first one can be found here, the second one here. Now I am pretty sure that Brettanomyces are in the dregs of the BFM La Torpille. The cells in the pictures there look very similar to the ones in Fig 9 and Fig 10.

Fig 10: Brettanomyces bruxellensis high magnification

Fig 11: Brettanomyces bruxellensis high magnification

And there is yet another cell that looks like two cells kind of fused (Fig 11).

I have additional pictures but nothing interesting to see there that can’t be observed in any other pictures above. I am very happy about the pictures because there are some structures which I could not observe in other published pictures of Brettanomyces. One in particular are the adhering cells. Quite amazing stuff in my opinion.

I end this post with a preview about stuff I publish in the future. First, there will be a post about microscopy pictures of Brettanomyces lambicus from Wyeast. Second, two posts about the agar plates with the two Brett strains. And there are some further recipes to talk about as well. So stay tuned. Some really interesting stuff is coming up soon.