Cell Count and Viability Testing

Controlling and managing yeast pitch rates is a critical parameter in maintaining beer quality and consistency as well as fermentation performance and yeast health.  

Cell counts and viability tests are the main tools used for assessing how much yeast (by weight) should be pitched.  When these tools are used, brewers can expect to achieve consistent and controlled pitch rates every batch every time.  

Below is a guide with helpful tips for cell counting and viability analysis.

Sampling 

Accurate cell counts and viability analysis begins with proper sampling.  

• Yeast samples are commonly collected from tanks, brinks, and/or containers into non-breakable, sealable 500mL Nalgene type bottles
• The sampled slurry should be homogenous and representative of the yeast to be repitched
• Samples should be refrigerated and tested as soon as possible following collection
•  Samples are typically highly concentrated and will require a dilution step prior to counting and viability testing

Dilutions 

• A common serial dilution scheme used in cell counting is 1:1,000
• Methylene blue stain is added during the final dilution step to combine viability assessments with cell counting
• To perform a serial dilution and cell count you will need the following equipment:
• Scale (2,000 g, 0.01g)
• Stir plate with magnetic bar
• 500mL beaker
• 10 mL serological pipette and bulb (or handheld pump)
• 1-1,000uL adjustable micropipette with disposable 1000uL (1mL) tips
• 2 - Clean test tubes
• Test tube rack
• KimWipes
• Hand tally counter
• 0.01% Methylene blue stock solution
• 1L Tap water (not distilled or deionized)
• 200-300mL of freshly collected homogenized yeast slurry

1:1,000 Dilution

Steps for performing a 1:1,000 dilution with 0.01% Methylene Blue are as follows:

• Prepare Dilution Blanks
• 8 ml. Water blank (by volume)
• Use a new serological pipette
• See below section on using pipettes
• Slowly draw 8 mL of tap water to the bottom of the meniscus
• Dispense 8 mL of tap water into a new clean test tube
• 1 ml 0.01% Methylene Blue blank (by volume)
•  Use a 1,000uL micropipette and a clean 1,000uL tip
• See below section on using pipettes
• Add 1 mL 0.01% Methylene Blue to a new test tube
• 495g Water Blank (by weight)
• On the scale, tare a 500 mL beaker to zero
• Add 495g of tap water
• Record water weight
• Perform yeast dilutions
• Prepare the 1:100 dilution
•  Using the scale, add 5g of yeast slurry to the 495 g Water Blank
• Record yeast weight
• Place on a stir plate and stir gently
• Prepare the 1:500 dilution
•  Use a 1,000uL micropipette and a clean 1,000uL tip
• See below section on using pipettes
• Collect and dispense twice 1 mL (TOTAL 2 ml) of the stirred 1:100 dilution into the 8 mL water blank
• Gently agitate tube to mix
• Prepare the 1:1,000 dilution
•  Use a 1,000uL micropipette and a clean 1,000uL tip 
• See below section on using pipettes
• Add 1 mL of the mixed 1:500 dilution to the Methylene Blue test tube
• Gently agitate tube to mix

Using pipettes

• Serological pipettes
• Holding upright, insert the tip into the sample
• Draw liquid slowly and steadily using a handheld pump or bulb
• Measure from the bottom of the meniscus 
• If using a filtered pipette, avoid allowing liquid to contact the cotton plug
• Dispense liquid fully
• Micropipette
• Depress the plunger to the first stop
• Holding upright, insert the pipette tip into the sample
• Slowly release the plunger to draw liquid into the tip
• Wipe the outside of the tip with a KimWipe - Avoid contact with the bottom of the tip
• In a test tube, depress the plunger to the last stop to expel all of the liquid
• Insert the tip into the test tube liquid, rinse the tip three times by depressing and releasing the plunger fully - Discard the tip

Using a microscope 

• Place the slide on the stage
• Align the slide using the stage clips
• Turn on the light and adjust the brightness if necessary
• Rotate the 40x objective in place.
• Use the coarse focus knob to bring the stage up to the objective lens (without touching)
• Look through the eyepiece
• Use the fine focus knob to bring the slide into focus
• The 10x objective may be used if a larger field of view is needed

Using a hemocytometer 

• Hemocytometers are specialized slides with two counting chambers etched with 25 square grids
• A specialized thick cover slip is used with the slide to allow a consistent volume of 0.0001mL in the chambers 
• To load a hemocytometer: 
• Place the coverslip over both chambers
• Pipette the 1:1000 dilution into the chamber area by placing the pipette tip at the edge where the coverslip covers the slide
• Add the liquid slowly until the chamber appears shiny and is full, but not too full
• If too full, use a Kimwipe to absorb excess liquid

Hemocytometer grids

• Each counting chamber contains 25 square grids
• Within each grid, there are 4x4 counting squares
• Each counting square is outlined by a thick or double exterior grid line

 Counting cells in the 4x4 grids

• In each 25 square grid chambers, begin in the most upper left corner, count down the first column and up the second column, repeating until all columns are counted
• Using the hand tally counter, count the cells using the below rules:

• Do not count 

• Cells that are touching any bottom and right exterior grid lines  
• Cells that are outside of any exterior grid lines

• Count 

• Cells that are touching any top and left exterior grid lines
• Cells within the grid lines
• Budding cells that are ≥50% of the mother cell are counted as two cells
• Record the number from each count
• Perform the count twice
• If the difference in cell count between the two chambers is greater than 10%, perform a new 1:1000 dilution and cell count 
• A range of 80-150 cells should be in each 25 square counting chambers
• The dilution scheme should be adjusted If the count is out of range

Assessing Viability 

• Methylene Blue stained cells will appear:
• Clear/Light Blue - Alive
• Dark blue - Dead
• Each brewery must decide how blue is blue
• Use a hand tally clicker to count:
• Total cells (Clear and Dark blue)
• Dead cells (Dark Blue)
• Record the number from each count

Calculations

• Cell Count
• Enter total counts into the equation below to calculate slurry concentration (Cells/mL)
• Total cell count • 1/0.0001mL • Dilution Factor = Cells/mL
• Ex: 120 • 1/0.0001mL • 1000 = Cells/mL = 1.20E+09 Cells/mL

• Viability
• Enter viable counts into the equation below to calculate % Viability
• (Total cells - Dead cells) / Total cells = % Viability
• Ex: (120 - 10) / 120 = 92% Viability
• Viable Cell Count
• Enter Cell Count and Viability into the equation below to calculate Viable Cells/mL
• Cells/mL • % Viability = Viable Cells Per mL
• Ex. 1.29E+09 • 0.92 = 1.10E+09 Viable Cells Per mL

Imperial Yeast Repitching Calculator 

• To calculate the pitch weight input the following information:
• Dilution factors 
• Living cell count
• Dead cell count
• Number of grids counted
• Batch size
• Original gravity
• Desired pitch rate 

Resources

For more information, please see our Imperial Yeast Cell Counting & Viability Testing video, or contact us at techsupport@imperialyeast.com.