The New Starter: A novel method for ensuring yeast vitality

A 2-L flask sealed with a sterile filter in the bung allows gases to flow both ways from a flask without compromising the sterile nature of the closed envrionment.

After spending 15 years as a pro brewer I am back doing small-batch trials. One of the most annoying transitions has been yeast quality. A professional brewery has access to very fresh yeast on demand. A brewery can have a full pitch of yeast overnighted from the lab the day of release but more often there are many fermentations in varying stages of completion a brewer can choose from. In the homebrewing environment cell count, yeast viability, and vitality are something that requires much more attention if your goal is to maintain quality and consistency. Good measures of quality and consistency are having consistent attenuations, consistent ester qualities, and consistent fermentation times. I propose a novel method adapted from Coors of England to achieve these goals that has been shown by homebrewers to also be effective on a smaller scale.

Cell count is simply the number of yeast cells available for fermentative purposes. In practice, obtaining a cell count is not very difficult or time-consuming but there is a big hurdle in acquiring equipment and setting a procedure in place to get consistent and reliable results.

The equipment needed is a 1,000x microscope, a hemocytometer, and some glassware. A camera is cheaper to produce than a good eyepiece so the cheapest microscopes now are hooked up to a computer and a usable one can be purchased for under $150 (USD). A nice stereo eyepiece model can approach $1,000 or more. Stereo is very nice if you are going to be using it for hours at a time. A hemocytometer is a special microscope slide (see page 92) that holds a very precise volume of liquid and has a grid in order to facilitate the counting of cells. Hemocytometers can be found from $15 to $100. Useful glassware is a 10 mL pipette, a two-liter flask, and a 100 mL flask. A stir plate will also be required if you plan on using this technique.

Collecting a sample to count is not a trivial task. In a homebrew size, it involves putting the pitch into a flask and stirring it to make sure it is homogenous. Use a micropipette to remove 1 mL of yeast and put it in a small flask. Then 99 mL of distilled water is added to dilute the sample to a countable number. It is important the number of cells to count is between 100 to 300 cells per µL to make counting easy and consistent and you may need a different dilution rate than the 99/1 described above. Once this is complete a small amount is removed with an eyedropper or micropipette and placed into the input port on the hemocytometer that has been fitted with a cover slip.

The hemocytometer will have some markings on it that allow you to know how to do the math. (It is really easy to be off by a factor of 10 so make sure to keep track of all the zeros.) A commonly used hemocytometer is marked: 0.1 mm and 0.0025 mm2. The 0.1 mm tells you how high the cover slip sits off the grid. The 0.0025 mm2 tells you the size of the smallest square. A large square is typically 20×20 small squares or 1 mm2. The area over a large square is given by area x depth = volume or in the case of this hemocytometer: 1 mm2 x 0.1 = 1 µL. Then the number of cells counted x dilution (100 in our example) x pitch size in mL x 1000 µL per mL = the total cell count. Again, make sure to keep track of all the zeros.

Some important cell counting tips: Only count every other small square in a checkerboard pattern and multiply by two. Count all cells that touch the top or left lines but none of the cells touching the right or bottom lines as an example.

Once you have the equipment to count cells, yeast viability is an easy to measure parameter of how many live vs. dead cells are in a pitch of yeast. The yeast slurry is mixed and then a sample is removed and precisely diluted. The diluted sample is placed on a hemocytometer and the cells are counted. To perform a viability check a small amount of methylene blue or methylene violet (methylene violet is preferred as it makes it easier to differentiate weak cells from healthy cells) is added to the sample and the count is performed again. Any cells that turn blue are considered dead and the ratio of live to dead cells is your viability. If the measured vitality is over 90% you can use the pitch with a correction factor to get your desired pitch rate. If it is under 90% it is recommended to discard the pitch.

Image of a 0.2 mm2 in a hemocytometer with yeast at 400x (small division visible are 0.5 mm2). Image courtesy of White Labs.

Your pitching rate for an ale (1,000,000 cells/mL/˚P) will be: (pitch rate x OG in Plato x volume of wort in mL)/(yeast count x viability).

Vitality or more precisely “predictive fermentation test” on the other hand is very difficult to measure but extremely important to understand. Vitality is a measure of the yeast’s ability to create a vigorous and rapid fermentation. Many methods have been proposed but a simple test that not only tells us the average vitality of the pitch and also the deviation from ideal has been elusive. An accepted method would be to use fluorescence spectroscopy to compare the relative concentrations of NAD+/NADH but this equipment is beyond the budget of most breweries much less homebrewers. Another method used is to take a yeast sample and measure the specific rate of oxygen uptake. This is basically preformed with a dissolved oxygen (DO) meter set up to chart the O2 levels of the sample. This correlates very accurately with yeast sterol reserves. Getting yeast sterol reserves is very important to consistent fermentation performance and many of the things we do are designed to do just that.

Beer is one of the only food products that is serially pitched. Most other food products use a mother. In a brewer’s world, yeast is required to come from a resting state, uptake oxygen during a respiration phase, reproduce, then enter a fermentation stage where glycolysis turns sugars to alcohol and CO2. When its work is completed we expect the yeast to flocculate to the bottom without leaving too many of the intermediate chemicals we call off-flavors.

Advanced homebrewers usually have a starter process that looks something like this: Take a pitch of liquid yeast purchased from a store, add it to 1 L of sterile wort and ferment it out. If it has flocculated, pour off the barm beer (the decanted portion of a starter) and pitch the yeast into 5 gallons (19 L) of aerated wort. This is a good strategy and results in decent beers but it works best if the purchased yeast is fresh and the oxygen level can be carefully controlled. Most homebrewers prefer oxygen to sterile air but it is my opinion that it is much safer to use sterile air. I have been making this argument for 20 years. So far, the foaming logistics of air have outweighed the dangers of over oxygenating to most homebrewers.

It is very difficult to over oxygenate with sterile air although it’s effectively limited to beers with starting gravities under 1.080. At 1.080 and 68 °F (20 °C) the solubility limit is 7 mg/L with air and 32 mg/L with pure O2, 7 mg/L is not sufficient enough to maintain yeast health through several reproduction cycles. For worts 1.060 and under it is much more common for large breweries to use sterile air. I have seen sterile air systems in use at Sierra Nevada and Anheuser-Busch although both also have oxygenation abilities for higher gravity products.

In 2004 I was given a scholarship from the Master Brewers Association to help me learn about yeast performance. I purchased several books that would have been unavailable on a brewer’s salary. In exchange, 9 months later I was asked to give a talk about what I had learned. These books (one referenced at the end) influenced my thinking about yeast and I highly recommend for advanced homebrewers.

One of the things I read about during that time was a method piloted at Coors England by David Quain. I found the method fascinating and, while my budget never allowed me to set up the equipment to work with the 3 gallons (11 L) of yeast that I needed to pitch, I had lectured about it to many homebrewers over the years. I am very excited about the reported results. Homebrewers have reported shorter lag times, quicker fermentations, and both lower and more consistent final gravities.

Here is the outline of the method I adapted from Quain: Make a starter as normal to get your yeast cell count to the correct level using your preferred method. On brew day, while you are heating your strike water, make up a liter of 1.040 wort in a flask (about 115 grams of DME and 1 L of distilled water boiled for 15 minutes). Seal with a sterile filter that allows air to flow both ways. Cool. Pour the barm beer off your starter and add the fresh wort. Spin this pitch on the stirplate with a sterile filter until your brew day is ready for the yeast pitch. The spinning will keep adding oxygen to the wort. Ideally this should be 4 to 6 hours. DO NOT oxygenate the wort from the brew and pitch as normal. This is the magic part of the process.

What we are hoping to do is to keep the yeast in its aerobic fermentation (more precisely respiro-fermentation) phase until it has maxed its sterol reserves. It is good we elucidate this often-misunderstood point about yeast.
Saccharomyces has three general pathways of metabolism and, like herding cats, not all cells will be on the same page so we have to talk about what the majority of cells are doing at a given stage. Yeast prefers to ferment. If there is enough food, more than 0.4% glucose as an example, yeast will ferment to alcohol even in the presence of oxygen. This is called the Crabtree Effect.

If there is no oxygen present we call this anaerobic fermentation and this is the primary task we are asking our yeast to do. This is the most important part of fermentation, however, its fascinating details are beyond the scope of this discussion. Below 0.4% glucose and without other easily assimilable sugars present yeast will, in the presence of oxygen, respire. Respiration makes by far more energy for the yeast cell but, this pathway is not important to brewers because by the time the glucose level is this low, no oxygen remains for the yeast to utilize.

Yeast has a third metabolic pathway that is very important to this discussion. Since yeast does not respire in a normal fermentation what is happening? Yeast will consume all available oxygen whether it is respiring or not. This pathway is called respiro-fermentation. It does not have the energy advantages of respiration but it does have some anabolic advantages. (Anabolism is the synthesis of needed molecules.) Some of the most important things yeast can synthesize only in the presence of oxygen are the lipids needed for the cell wall membrane.

This is the reason we oxygenate the wort. Unless we supply a source of lipids in the wort, yeast will be required to make sterols in order to keep the yeast walls healthy. Once these walls get weak the yeast can become less alcohol-tolerant, die, and/or create off-flavors. One of the things we did in our yeast count was look for leaky walls with the staining. Storage time will naturally deplete sterol reserves.

Now that we have supplied the starter with 4 to 6 hours of oxygen there is no need to oxygenate the wort. For a larger pitch, bubbling sterile air or pure O2 while mixing is required. This maxes the vitality of the yeast without oxidizing the trub or adding excess O2 to the wort at all.

In Quain’s studies he was able to get much more consistent fermentations. This is exactly what homebrewing needs. A package of yeast can be months old before it is used and its vitality and viability should be very much in question. Homebrewers using this method have reported to me lower and more consistent final gravities as well as less fermentation derived off-flavors.

Further Reading:

Boulton, Christopher, and David Quain. Brewing Yeast and Fermentation, 1st Edition. Wiley, 2001.

Aquilla, Tracy. “The Biochemistry of Yeast.” Brewing Techniques, (Volume 5, Number 2).

Issue: March-April 2019