Accurately determining cell number is a fundamental technique in various life science disciplines. Whether you’re a researcher studying cell proliferation, a technician analyzing blood samples, or a doctor monitoring cell cultures, the **hemocytometer (also spelled hemocytometer)** emerges as an essential tool.

**What is a Hemocytometer?**

A hemocytometer is a specialized microscope slide with a etched grid. The grid design creates a defined counting area, allowing you to calculate the number of cells within a specific volume of your cell suspension. Hemocytometers typically have a double counting chamber, each engraved with a specific grid pattern.

#### Structure of a Hemocytometer

**Counting Chamber**: The chamber has a grid etched into its surface, typically divided into nine large squares, each with a known area.**Cover Slip**: A special thick cover slip is placed over the chamber to create a precise volume.**Grid Dimensions**: The grid lines define areas of 1 mm², and the depth of the chamber is usually 0.1 mm, creating a volume of 0.1 mm³ (1 µL) for each square.

#### Applications of Hemocytometry

**Cell Culture**: Estimating cell concentrations in culture.**Blood Cell Counting**: Determining red and white blood cell counts.**Microbiology**: Counting bacteria or yeast cells.**Environmental Science**: Assessing microbial content in water samples.

#### How to Use a Hemocytometer

**Materials Needed**:

- Hemocytometer
- Cover slip
- Pipette
- Cell suspension
- Staining solution (e.g., Trypan Blue for viability staining)
- Microscope

**Procedure**:

**Preparation**:- Clean the hemocytometer and cover slip with 70% ethanol and dry them.
- Mix the cell suspension thoroughly to ensure even distribution.

**Loading the Sample**:- Pipette a small volume (10-20 µL) of the cell suspension.
- Carefully place the cover slip over the counting chamber.
- Gently load the cell suspension into the chamber by touching the pipette tip to the edge of the cover slip. Capillary action will draw the liquid into the chamber.

**Counting Cells**:- Place the hemocytometer on the microscope stage and focus on the grid.
- Count the cells in the designated squares (usually the four corner squares and the center square of the grid).

**How Does it Work?**

The hemocytometer counting process involves these key steps:

**Sample Preparation:**Dilute your cell suspension to an appropriate concentration that yields a countable number of cells within the hemocytometer grid.**Filling the Counting Chamber:**Using a micropipette, carefully introduce a small volume of the diluted cell suspension onto the hemocytometer platform. Capillary action draws the suspension into the counting chamber.**Cell Visualization:**Place the hemocytometer on a microscope stage and adjust the magnification to visualize the cells clearly within the grid.**Cell Counting:**Systematically count the cells within the defined squares of the grid pattern. Depending on the hemocytometer design, specific counting rules apply (explained in detail below).

**Counting Rules and Calculations:**

Hemocytometers typically have a central counting area with a grid pattern of 9 large squares, each further subdivided into 16 smaller squares.

Here’s how to calculate cell concentration:

**Choose Counting Squares:**Select a defined number of large squares (usually 4 or 5) for counting. Ensure even distribution of cells across the chosen squares.**Count Cells:**Methodically count the number of cells within the small squares of your chosen large squares.**Calculation:**

The following formula allows you to calculate the cell concentration (cells/mL) in your original, undiluted sample:

ell concentration (cells/mL) = (Total Counted Cells) / (Area of Counted Squares x Dilution Factor x Volume Loaded)

**xample Calculation:**

Let’s assume you counted a total of 80 cells in 4 large squares (each with 16 small squares) of your hemocytometer. You diluted your cell suspension 1:100 before loading 10 μL (0.01 mL) onto the hemocytometer.

**Area of Counted Squares:** Since each large square has 16 small squares and you counted 4 large squares, the total area of counted squares is 4 squares * 16 squares/square = 64 squares.

**Calculation:**

Cell concentration (cells/mL) = (80 cells) / (64 squares * 100 dilution factor * 0.01 mL) = 1.25 x 10^4 cells/mL

Therefore, your original, undiluted cell suspension has a concentration of 1.25 x 10^4 cells/mL.

**Cell Viability Measurement using Hemocytometer**

Trypan blue is a vital stain used to selectively color dead tissues or cells blue. It is a diazo dye. Live cells or tissues with intact cell membranes are not colored Since cells are very selective in the compounds that pass through the membrane, in a viable cell Trypan blue is not absorbed; however, it traverses the membrane in a dead cell. Hence, dead cells are shown as a distinctive blue color under a microscope. Since live cells are excluded from staining, this staining method is also described as a Dye Exclusion Method.

**Procedure for calculating cell count and % viability of BHK-21 cell line**

- 100uL of the BHK-21 cell suspension was aseptically transferred into an eppendorf tube inside laminar air flow unit.
- Mix the cell suspension with 100 uL of Trypan Blue.
- Fill the haemocytometer chamber with the cell suspension and observe under phase contrast microscope in 10X magnification.
- Count the cells in each of the four grids bordered by triple lines on any two side of the square.
- the percentage of vialbility can be calculated using the formula

**% viability = (No of viable cells / Total no of cells) * 100**

**Cell Concentration **

**Number of cells/ml = Average number of cells/grid x Dilution factor x 10^4.**

=24.75 * 2 * 10^4

= 4.9*10^5

% viability = (No of viable cells / Total no of cells) * 100

% viability = (24.75/ 27.75) * 100 = **89.18%**

#### Tips for Accurate Counting

**Homogenize Sample**: Ensure the cell suspension is well-mixed to avoid clumping or uneven distribution.**Avoid Overfilling**: Do not overfill the chamber, as this can lead to inaccurate counts.**Count Multiple Squares**: Increase accuracy by counting cells in multiple squares and averaging the results.**Stain Cells**: Use viability stains like Trypan Blue to differentiate between live and dead cells.

#### Commercial and Clinical Relevance

**Research Laboratories**:**Cell Culture Monitoring**: Ensuring optimal cell density for experiments.**Tissue Engineering**: Accurate cell counts are vital for scaffold seeding and growth studies.

**Clinical Diagnostics**:**Hematology**: Counting blood cells for diagnosing conditions like anemia or infections.**Cancer Research**: Assessing tumor cell concentrations in samples.

**Biotechnology**:**Bioprocessing**: Monitoring microbial populations in fermentation processes.**Pharmaceuticals**: Ensuring cell line consistency for drug production.