Nexcelom Bioscience

978-327-5340

  • Cellometer

Counting Yeast Cells Using a Hemacytometer
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Introduction

The hemacytometer has been an essential tool for hematologists, medical practitioners, biologist and now brewers and ethanol production researchers. Yeasts are an economically important organism used for ethanol production, in the beverage and alternative fuels industries as well as a leavening agent in the baking industry. Concentration and viability determinations are routinely performed for quality control purposes in yeast production, fermentation processes, and fungicides research to monitor proliferation of pathogenic yeasts.

Manual Cell Counting with Hemacytometer

Step 1. Prepare the Hemacytometer

parts of a hemacytometer

Clean the hemacytometer and glass cover slip with 70% EtOH.

Step 2. Prep Sample & Load

preparing a hemacytomter

Place the glass cover slip over the counting chambers.

 

loading sample into a hemacytomter

Pipette 10 microliters of cell sample into the hemacytometer.

Step 3. Manually Count Cells in Sample

loaded hemacytometer

Place the hemacytometer under a microscope with a typical magnification of 100.

 

hemacytometer cell counting grid

Focus both onto the grid pattern and the cell particles, and count the total number of cells found in 4 large corner squares.

If cells are touching the 4 perimeter sides of a corner square, only count cells on 2 sides, either the 2 outer sides or 2 inner sides.

Step 4. Cell Calculations & Disposal of Hemacytometer

Multiply the dilution factor by the total number of cells, divide by the # of corner squares counted, and multiply by 104 to obtain cell concentration (cells/ml).

cell concentration equation

Clean hemacytometer and glass cover slip with 70% EtOH.

Manual Cell Viability Measurement with Hemacytometer

Using Methylene Blue to Measure Yeast Cell Viability

In general, methylene blue is used to measure yeast viability/vitality. Methylene blue is a metachromatic stain that has a molecular mass of 319.85 g/mol. Metabolically active viable/vital yeast cells with dehydrogenase activity can convert the methylene blue to a colorless substance, while the dead cells retain the blue color of the stain. Therefore, live and dead yeast cells can be manually counted using the hemacytometer to determine yeast cell concentration and viability. For more information on yeast analysis, please visit the "Yeast Concentration & Viability" webpage.

Methylene Blue Protocol

Methylene Blue Preparation

Step 1. Dissolve methylene blue in sodium citrate solution (2% w/v) to a final concentration 0.01% (w/v)

Step 2. Filter the methylene blue with 0.2 micron filter

Hemacytometer Preparation

Step 3. Clean the hemacytometer and glass cover slip with 70% EtOH

Step 4. Place the glass cover slip over the counting chambers

cell counting steps with a hemacytometer

Cell Counting Procedure

Step 5. Vortex the target yeast cell suspension and mix 1:1 with 0.01% methylene blue

Step 6. Pipette 10 microliters of cell sample into the hemacytometer

Step 7. Wait 60 seconds for the cells to settle

Manually Count Cells in Sample

Step 8. Place the hemacytometer under a microscope with a typical magnification of 100

Step 9. Focus both onto the grid pattern and the cell particles, and count the total number of cells found in 4 large corner squares. If cells are touching the 4 perimeter sides of a corner square, only count cells on 2 sides, either the 2 outer sides or 2 inner sides

Step 10. Count the live yeast cells (without methylene blue) and dead yeast cells (with methylene blue)

Cell Calculations & Disposal of Hemacytometer

Step 11. Multiply the dilution factor by the total number of cells, divide by the # of corner squares counted, and multiply by 104 to obtain cell concentration (cells/ml)

live yeast cell concentration equation
yeast viability equation

Step 12. Clean hemacytometer and glass cover slip with 70% EtOH

Crystal Violet Protocol for Total Nuclei Counting

Crystal Violet Protocol

Crystal Violet Preparation

Step 1. Prepare 0.1 M citric acid by dissolving 1.9212 g in 100 mL distilled water

Step 2. Prepare 0.1 M citric acid containing 0.01% (w/v) crystal violet by dissolving 0.005 g crystal violet (also known as basic violet 3 or gentian violet; C.I. 42555) in 50 mL of the 0.1 M citric acid prepared in Step 1

Hemacytometer Preparation

Step 3. Clean the hemacytometer and glass cover slip with 70% EtOH

Step 4. Place the glass cover slip over the counting chambers

cell counting steps with a hemacytometer

Cell Counting Procedure

Step 5. Centrifuge target cell suspension at 500 ± 50 g for 5 to 10 minutes.

Step 6. Decant supernatant. Add 1.0 mL 0.1M citric acid solution to the cell pellet. Mix well and incubate at 35° C for 1 to 2 hours

Step 7. Separate nuclei by violent shaking followed by centrifugation at 1000 ± 100 g for 20 to 25 minutes

Step 8. Discard supernatant. Re-suspend the cell pellet in 0.5 to 1.0 mL citric acid-crystal violet solution

Step 9. Pipette 10 microliters of cell sample into the hemacytometer

Step 10. Wait 60 seconds for the cells to settle

Manually Count Nuclei in Sample

Step 11. Place the hemacytometer under a microscope with a typical magnification of 100

Step 12. Focus both onto the grid pattern and the cell particles, and count the total number of nuclei found in 4 large corner squares

Step 13. If nuclei are touching the 4 perimeter sides of a corner square, only count cells on 2 sides, either the 2 outer sides or 2 inner sides

Step 14. Count the live nuclei (with crystal violet)

Nuclei Calculations & Cleaning of Hemacytometer

Step 15. Multiply the dilution factor by the total number of nuclei, divide by the # of corner squares counted, and multiply by 104 to obtain cell concentration (cells/ml)

nuclei cell concentration calculation

Step 16. Clean hemacytometer and glass cover slip with 70% EtOH

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