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Count on Your HEMACYTOMETER For Consistent Pitching Rates

11/30/-1

by David Sohigian (Brewing Techniques)

 

 

 

Variations in pitching rate can drastically alter your beer’s character. Assessing cell counts and viability on a regular basis takes just a few minutes out of your day and will pay off in consistent fermentations.

 


 

One of the most important factors that brewers can control is the population of yeast at the beginning of fermentation, known as pitch rate. The pitch rate will have a major influence on the performance of the yeast during fermentation. Underpinning will spur yeast growth, resulting in more production of the by-products of amino acid synthesis. It will lead to the production of more higher alcohols (fusel oils), esters, and diacetyl, but lower alcohol and acetaldehyde (green apple character) production. Furthermore, because of poor yeast health, many sugars may not be metabolized, resulting in lower attenuation. Underpitching also makes the wort more susceptible to infection by opportunistic bacteria or wild yeast.

Overpitching means that the yeast does not have to vigorously increase its population, and therefore more of the sugars will be converted into alcohol rather than yeast mass. In general, fewer fermentation characters will develop. Low yeast growth produces beers that lack in many fermentation qualities, such as higher alcohols and diacetyl, but increases the “green beer” character from acetaldehyde. Low growth may also result in excessive yeast autolysis, leading to astringency and yeast “bite.”

By pitching at the proper rate, you can ensure not only consistent fermentation performance in the beer being pitched, but also consistent performance of the yeast over multiple generations. Although pitch rate does have a direct effect on fermentation performance, it has an even bigger effect on the overall health of the yeast over time. For example, if yeast is overpitched over multiple generations, the resulting low growth will lead to an aging yeast population that lacks the vigor to properly ferment the beer.

The simplest and cheapest means of accurately determining cell concentration is the use of a hemacytometer and a microscope. A hemacytometer is a specialized slide that has a counting chamber of known volume. By placing a slurry of diluted pitching yeast on the slide and counting the cells in the chamber, you can ascertain the concentration of cells per milliliter in the pitching yeast. The viability of the pitching yeast can be estimated by staining the slurry with methylene blue before counting. The membranes of healthy cells are less permeable to methylene blue than those of dead or dying cells, and when it does enter, healthy cells can more readily metabolize it than unhealthy cells can. Viable cells therefore will not stain blue, whereas dead cells will take up the stain and turn blue.

Methylene blue staining may not give accurate results on dehydrated dry yeast because drying disrupts the cell walls and membranes of the yeast. In addition, it is difficult to get the cells to deflocculate.

The first choice you must make is whether you will measure the yeast concentration by weight or volume. You should base your decision on how you plan to measure the yeast when you actually pitch. If you pitch by weight, you should measure the concentration by weight as well. Measuring the yeast concentration by weight will give you more accurate results, but these results will be of little use if you pitch by volume.Dilution

The first step in measuring the concentration of your pitching yeast is to get an accurate sample. The sample should be representative of the population you will be pitching, so take the sample from the same area that you will be pitching from, whether it is from the bottom of the fermentor or from a yeast brink. Take a fairly large sample, 100 mL or so, and make sure that the sample is well mixed before measuring out the quantity that you will dilute. The dilution procedure you will use depends on whether you will count by volume or weight.

Counting by volume: Mix your sample thoroughly, and pipette out 10 mL of yeast. Try to avoid getting bubbles or large pieces of trub into the pipette. Dispense the sample into the 1-L graduated cylinder, and then wash any remaining yeast out of the pipette into the cylinder using tap water. Fill the cylinder up to the 1-L mark with tap water, and mix thoroughly. You will take your sample for counting directly from the cylinder. Doing the dilution in this fashion will give you a 100:1 dilution rate. For a 200:1 dilution rate (required for very thick slurries), pipette 5 mL of yeast into the 1-L graduated cylinder. The dilution rate will be factored into the pitching rate equation that follows, expressed as milliliters of liquid per milliliter of yeast. If you choose a different amount of yeast to sample, just divide the amount of liquid in milliliters by the amount of yeast. For example, if you used 12 mL of yeast and topped it up with 1 L of water, you would have a ratio of 1000 mL of liquid per 12 mL of yeast, which equals 83 mL of liquid per 1 mL of yeast.

Counting by weight: Place your 1-L graduated cylinder on a scale that can handle up to 2 kg of weight and is accurate to at least 1 g. Hit the “tare” button on the scale so that it reads “zero” with the empty cylinder on it. Mix your sample thoroughly and pipette out around 10 mL of yeast. Dispense the sample into the 1-L graduated cylinder, but don’t worry about washing out any yeast that remains in the pipette. Write down the reading from the scale, which should be close to 10 g. Remove the cylinder from the scale, fill it up to the 1-L mark with tap water, and mix thoroughly. You will take your sample for counting directly from the cylinder. The above quantities will give you a dilution of 1 g of yeast per 100 mL. To get a 1 g to 200 mL dilution, pipette about 5 g of yeast into the 1-L cylinder. The dilution rate will be factored into the pitching rate equation that follows, expressed as milliliters of liquid per milliliter of yeast. If you choose a different amount of yeast to sample, just divide the amount of liquid in milliliters by the amount of yeast. For example, if you put in 8 g of yeast and topped it up to 1 L, you would have 1000 mL of liquid per 8 g of yeast, which is equal to 125 mL of liquid per 1 g of yeast.

 

Testing for Viability: Methylene Blue

 

The American Society of Brewing Chemists (ASBC) outlines several methods for preparing a methylene blue solution to assess the viability of your yeast. (Viability is factored into the pitching rate equation explained later.) You can purchase powdered methylene blue and then add either water or a buffered (at pH 4.6) solution to make up your methylene blue solution. The buffered solution will give more consistent results, and the methods for making it up are outlined in the ASBC Methods of Analysis, as well as the recently released version for craft brewers. To produce the aqueous solution (unbuffered), dissolve the powder in distilled water to give a concentration of 0.01 g per 100 mL (put 0.1 g into 1 L of water). Stored at refrigerator temperatures, the solution will last for several months. The ASBC recommends that you use this methylene blue solution in place of your dilution water when you dilute the yeast down to the proper concentration so that you can exclude the cells that aren’t viable from your count.

You might consider instead doing the methylene blue count separately, either in the second chamber of the hemacytometer or on a standard slide. It is often easier to use a plain slide than to risk making mistakes in your cell counts on the hemacytometer. Methylene blue stain results must be read 1–5 minutes after staining for accurate results, and this time period may be difficult to adhere to if you have to fool with the hemacytometer. However, if you are comfortable using the second chamber of the hemacytometer, your results will probably be more accurate.

Remember that you don’t need a precise dilution rate to determine viability, because it is a percentage of cells, not a concentration. The easiest method for determining viability is to take a drop of your diluted sample and place it on an ordinary slide. Add to that a drop of methylene blue solution, and place a cover slip on top. View with 400X magnification and count all of the cells in your field of vision. Then go through and count all of the cells that have stained dark blue (those that are stained light blue are considered viable). Use the following equation to determine your viability:

Many brewers swear by methylene blue stain (in combination with a cell count) for determining their pitching rate. Unfortunately most research concludes that the stain is accurate only at viabilities above 85%, and recent research has suggested that it is accurate only above 95%. This means that if methylene blue shows a viability of 70%, you really have no idea of the actual percentage of viable cells in your slurry, and should probably consider throwing away your yeast rather than upping your pitching rate. For this reason, I suggest that you use methylene blue as a quick and dirty method of determining yeast health, but use it in conjunction with careful yeast tracking to determine your planned pitching rate. If you found a low viability with your first test, and your yeast tracking records showed that the yeast should be in good health, consider pulling a sample from elsewhere in the harvest and testing again.

 

Using a Hemacytometer for Counting

 

Setting up your sample: Clean the hemacytometer with tap water, and dab it dry with an ordinary paper towel. To remove any dust or other particles, wipe the surface of the hemacytometer firmly with lens paper, or some other paper that will not scratch the counting area. Place the cover slip carefully on top of the counting chamber, making sure that both sides are resting on the raised area. You must use the cover slips provided with the hemacytometer, because they are thicker than normal cover slips and will not deform when the sample is added to the chamber.

It is easiest to load the hemacytometer on the table, then move it to the stage of the microscope. Mix your diluted yeast thoroughly, draw a homogenous sample of it into a Pasteur pipette, and place the tip on the filling notch of the hemacytometer. Allow the counting chamber to fill, avoiding spillover into the overflow area. If the slurry overflows onto the raised area, it will raise the cover glass, changing the volume of the counting chamber and making the count inaccurate (see Figure 1 on page 56).

Counting cells: First, focus the microscope at 100X and make sure that you have the chamber well situated in the center of the field of vision and that there are no bubbles or large pieces of trub in the chamber. If there are, you might want to try another sample. Then move up to 400X and move the slide so that you are focusing on the upper left cell in the counting grid, as shown in the diagram. You should see somewhere between 5 and 75 yeast cells. If the count is outside of this range, you must change your dilution rate because it will be difficult to get an accurate count.

Follow these rules when you count:

  • If a cell is touching the upper or left triple lines, do not count it, but if it is touching the lower or right triple lines, do count it (see example above).
  • If a cell is budding, count the daughter cell only if it is more than half the size of the mother cell.
  • Use the 4 X 4 grid within the counting area as a frame of reference as you count, but do not bother to note the exact number of cells in each of the 16 little boxes.
  • Use a standardized counting order. For example, start in the upper left corner, work your way across, move down a row, and repeat.

You must decide up front how many of the counting areas you will count, since you can count as few as five and take an average of those five. I would suggest that you count all of the areas to obtain a more accurate count. You will find that the time commitment involved is very small once you get some practice; the real work is in setting up the slide and dilution.

Using a hand-held tally counter will help you keep your eyes focused on the counting. Write down your count on a grid laid out like the counting chamber to allow for easier interpretation (see Figure 2, above). Come up with a total cell count for all the areas you counted, and note the number of areas counted (from 5 to 25).

 

Calculating the Pitching Quantity

 

Once you have a count of the number of cells in the chamber, you must determine the concentration of cells in the slurry and then the amount of pitching yeast you will require. The equation takes into account the volume of the counting chamber, the desired pitching rate, batch size, and target gravity. The first two factors can be combined into a constant (shown left), but if you do not agree with my assumptions, you will have to arrive at your own constant.

Chamber volume: The counting chamber has dimensions of 1 mm x 1 mm x 0.1 mm. Once you’ve converted to centimeters and calculated the volume, you can easily convert to mL (1 mL is equal to 1 cubic centimeter).

0.1 cm x 0.1 cm x 0.01 cm = 0.0001 cm3 (or 1 x 10-4mL)

Pitching rate: The other assumption is a standard pitching rate of 1 million cells per mL per °Plato (1 x 106 cells/mL/°P).

Dilution rate: Whether or not your dilution rate was calculated as a volume-to-volume ratio or a weight-to-volume ratio, the dilution rate used in the equation is expressed as whole number (for example, 1 mL in 100 mL is a 100:1 dilution rate, so you should put “100” into the equation).

Viability: The equation below assumes you have factored in your viability figure to the cell count. If you used methylene blue to dilute your sample, simply subtract the number of dead cells from the total count. If you tested for viability as a separate step, you need to take the total cells counted and multiply by the viability percentage to give the number of viable cells.

Pitching amount: Once you have all the numbers in hand, you can determine the total amount of amount to pitch using the formulas shown in Figure 2 (opposite).

For a beer of average gravity (say 12 °P), a ballpark figure of a normal pitching rate (with a slurry of average thickness) is 1 lb of yeast per bbl of wort or 1 gallon of yeast per 8 bbl of wort.

 

Verifying Pitching Rate

 

After you have pitched your yeast, you may want to verify whether or not you got the pitching rate correct. You can do this by taking a sample of the pitched wort (as long as the yeast mixed well with the wort in the tank) and dropping it directly into the counting chamber. If you had a fairly high pitching rate (for a strong beer, for example) you may want to do a 2:1 dilution first (add 10 mL of yeast to 10 mL of water). Do your count using the method outlined above, and use the following equation to determine your actual pitching rate:

where

TVC = total viable cell count from the hemacytometer

AC = areas counted on the hemacytometer

Dil = dilution rate

 

Consistency You Can Count On

 

Using the methods outlined above you should be able to control not only your pitching rate, but also the fermentation performance and flavor generation that are determined largely by pitching rate. Once you get practiced at these techniques, they should take up less than 10 minutes of your brew day, and I recommend that you determine the cell concentration with every brew.

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