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Hemacytometer Cellometer Applications

Manual Cell Counting and Viability Using a Hemacytometer

Introduction

The hemacytometer has been an essential tool for hematologists, medical practitioners, and biologists for over a century. Depending on where it is being used, the word has multiple spellings such as hemacytometer, hemocytometer, haemacytometer, or haemocytometer, but for consistency purposes the word "hemacytometer" will be used in this review. The prefix "hema", "hemo", "haema", or "haemo" means blood, while "cytometer" meant a device to measure cells. The device was initially used by medical practitioners to analyze patient blood samples, which was the initial spark that created the field of hematology. The hemacytometer has gone through a series of major development in the 1800s and early 1900s. The modern-day hemacytometer utilizes a double-chamber format and counting grids developed by O. Neubauer. This format allows the user to perform two cell counts for each sample without having to clean the hemacytometer. For a complete hemacytometer historical review, please download our white paper.

The hemacytometer has been used to count cells ranging from algae, yeast, cancer cells, stem cells, blood cells, even parasites and spores. Although a variety of automated cell counting instruments have been developed, the current golden standard that researchers fall back on is still manual counting with hemacytometer.

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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 Hemacytometer

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.

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Manual Cell Viability Measurement with Hemacytometer

Using Trypan Blue to Measure Mammalian Cell Viability

One of the earliest and most common methods for measuring cell viability is the trypan blue (TB) exclusion assay. Trypan blue is a ~960 Daltons molecule that is cell membrane impermeable and therefore only enters cells with compromised membranes. Upon entry into the cell, trypan blue binds to intracellular proteins thereby rendering the cells a bluish color. The trypan blue exclusion assay allows for a direct identification and enumeration of live (unstained) and dead (blue) cells in a given population. For more information on Trypan blue viability analysis, please visit the "Using Trypan Blue and Acridine Orange/Propidium Iodide to Measure Cell Viability" webpage.

Trypan Blue Protocol

Trypan Blue Preparation

Step 1. Dilute the stock (0.4%) Trypan Blue with PBS to 0.2%

Step 2. Filter the trypan blue with 0.2 micron filter

Hemacytometer Preparation

Step 3. Prepare the Hemacytometer

Clean the hemacytometer and glass cover slip with 70% EtOH

Step 4. Prepare Sample & Load Hemacytometer

Place the glass cover slip over the counting chambers

cell counting steps with a hemacytometer

Cell Counting Procedure

Step 5.

Vortex the target cell suspension and mix 1:1 with 0.2% trypan blue

Pipette 10 microliters of cell sample into the hemacytometer

Wait 30 seconds for the cells to settle

Step 6. Manually Count Cells in Sample

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

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

Count the live cells (without trypan blue) and dead cells (with trypan blue)

Step 7. 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)

live cell concentration equation
cell viability concentration equation

Clean hemacytometer and glass cover slip with 70% EtOH

Similar counting procedures can also be performed using the Cellometer disposable hemacytometer. For more information, please visit the product page "Cellometer Disposable 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. Prepare the Hemacytometer

Clean the hemacytometer and glass cover slip with 70% EtOH

Step 4. Prepare Sample & Load Hemacytometer

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

Pipette 10 microliters of cell sample into the hemacytometer

Wait 60 seconds for the cells to settle

Step 6. Manually Count Cells in Sample

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

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

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

Step 7. 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)

live yeast cell concentration equation
yeast viability equation

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 1.9212g in 100 mL distilled water

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

Hemacytometer Preparation

Step 3. Prepare the Hemacytometer

Clean the hemacytometer and glass cover slip with 70% EtOH

Step 4. Prepare Sample & Load Hemacytometer

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 ± 50g for 5 to 10 minutes.

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

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

Discard supernatant. Resuspend the cell pellet in 0.5 to 1.0mL citric acid-crystal violet solution

Pipette 10 microliters of cell sample into the hemacytometer

Wait 60 seconds for the cells to settle

Step 6. Manually Count Nuclei in Sample

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

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

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

Count the live nuclei (with crystal violet)

Step 7. Nuclei Calculations & Cleaning of Hemacytometer

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

Clean hemacytometer and glass cover slip with 70% EtOH

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Cellometer Disposable Hemacytometer

Disposable Hemacytometer

Nexcelom Bioscience's Disposable Hemacytometer is an enclosed chamber with two ports for sample introduction. The chamber contains precisely spaced lines in a grid pattern. There are two counting chambers per slide for replicates.

Improved Neubauer, as in a common Hemacytometer

The counting grid pattern is the Improved Neubauer, as in a common Hemacytometer. The consistent pattern design allows the standard cell counting procedure to be followed.

Learn more about the Cellometer Disposable Hemacytometer »

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