Materials: (White Labs product number for reference)
Methylene blue solution (MA1420) 
Hemocytometer (MA1410)
Fine tip glass pipets (LW2600)
Hand held counter (MA1470)
Gloves (optional) (LM 4710-LM4730)
Transfer pipets (LW2800)
Kimwipes (MA1460)
You will need to provide: Microscope with 400X capability

1. Make certain hemocytometer is clean and dry before use. Hemocytometer can be easily cleaned with water. Chamber and cover slip may be scrubbed gently using a lint free towelette (kimwipe).
2. Position cover slip so that glass covers both counting areas equally.
3. Preparing sample:
   This is the most critical step of the protocol. A highly concentrated sample may be too difficult to count and a very dilute sample may give erroneous results. It is best to have 100 or fewer yeast cells per microscope field at 400X. Sample can be diluted with distilled water or with 0.5% H2SO4 if cells clump excessively. Make sure you note your dilution factor.
   It is important that the sample contain as little air bubbles as possible. Degas if possible. Lastly, it is imperative that your sample is well mixed (without introducing bubbles). Once you have prepared a correct dilution, mix the sample by inverting and/or shaking for several minutes. You may have to vent sample to prevent pressure build-up.
   If you are combining cell count with yeast cell viability, you will need to perform one final sample preparation. Mix 9ml of your diluted yeast sample with 1ml of White Labs methylene blue solution. Mix and let sit for approximately 1 minute before filling hemocytometer. Again, make sure sample is well mixed.
4. Filling the hemocytometer chamber
5. Take a portion of your sample by placing the glass pipet tip into the liquid mixture and letting it fill via capillary action (draw upwards automatically.) Blot a small portion on a paper towel prior to filling hemacytometer. Fill chamber by gently setting pipet tip on edge of chamber at etched cut. Be careful not to overfill; avoid getting sample into moat.
6. Carefully place hemocytometer on microscope stage. As you focus at each objective leading up to the 400X lens, note the distribution of yeast cells. If cells are well distributed then you can use the short cell count method. If cells are grouped or clumped together, you may need to prepare another sample or use the long cell count method.
7. Cell counting: You will be counting squares within the 1-mm2 ruled area centrally located on the chamber (see Figure). It is helpful to establish a counting protocol for all cell counts. For example, cells touching or lying on the top and right boundary lines are not counted, whereas cells touching or lying on the bottom or left boundary lines are counted (see example below). Yeast cell buds emerging from mother cells are counted as a separate cell if the bud is at least one-half the size of the mother cell.
   
   If performing viability counts, dead cells will stain dark blue. Non-viable cells do not have the metabolic capability to expel the intruding dye. Do not count cells that are pale blue in color as dead. Some budding cells will also stain blue, do not count these cells as non-viable. If you are performing a cell count and a viability count simultaneously, it is best to count all cells on the hand held counter and record noted dead cells on a written tally.  The methylene blue stain procedure is not accurate below 95% viability.
   

Short method: For an evenly distributed sample.

• You will be counting cells within the 5 numbered squares (see figure above).
• # of cells counted in 5 numbered squares X 5= Number of cells in total grid (estimate)
• Yeast cells/ml= Number of cells in total grid x dilution factor x 10^4 (or 10,000)*
   * This factor incorporates the volume of fluid in counting chamber.
• Note: 10^6 are millions and 10^9 are billions.

Long method: For clumped or unevenly distributed cells

• Count all squares in the counting grid. (25 total).
• Yeast cells/ml= Total cells in grid x dilution factor x 10^4 (or 10,000)*
  * This factor incorporates the volume of fluid in counting chamber.
• Note: 10^6 are millions and 10^9 are billions.

EXAMPLE
A. 220 cells counted within the 5 numbered squares.
Dilution factor of 1:100. (1 ml of yeast slurry + 99 ml of water)
Yeast cells/ml= Total cells in grid x dilution factor x 10^4

= 220 x 5 x 100 x 10^4
= 1100000000
= 1.1 x 10^9 or 1.1 billion cells/ ml

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