ZEISS How to Use a Hemocytometer User Guide

Step-by-Step Guide

How to Use a Hemocytometer

Author: Dr. Benjamin-Maximilian Schwarz
Carl Zeiss Microscopy GmbH, Germany
Date: June 2025

Understanding the Hemocytometer

A hemocytometer is a precision-engineered microscope slide originally developed in 1874 by Louis-Charles Malassez for counting blood cells. Today, this simple yet versatile device has become indispensable in cell biology labs for accurately counting cells from various sources, including mammalian cell cultures, yeast, algae, and bacterial cultures.

The hemocytometer consists of a thick microscope glass slide with a rectangular indentation, engraved with a precise laser-etched grid of perpendicular lines and a specialized coverslip. The area bounded by the lines is known, and the depth of the chamber is also known. By observing a defined area of the grid, it is therefore possible to count the number of cells in a specific volume of fluid, and thereby calculate the concentration of cells in the fluid overall. In conclusion, properly loading the hemocytometer is crucial, as inaccurate loading can lead to errors in counting and, consequently, incorrect experimental results.

This tutorial provides step-by step instructions along with practical tips and tricks to ensure accurate, consistent, and reliable cell counting results every time.

How to Load and Use a Hemocytometer Correctly

Materials Needed:

• Hemocytometer (clean and dry)
• Specialized hemocytometer coverslip (thicker than standard coverslips)
• Pipette and sterile pipette tips
• Cell suspension prepared at an appropriate dilution
• Microscope (preferably an inverted or standard light microscope)

Step 1
Preparation of Hemocytometer and coverslip
Cleanliness Matters: Always ensure the hemocytometer and coverslip are meticulously clean and free of dust, debris, or fingerprints, as these can lead to counting errors.
Clean the hemocytometer chamber and coverslip thoroughly with ethanol or isopropanol. Allow both the hemocytometer and coverslip to air dry or carefully dry them with lens paper to prevent scratching.

Step 2
Attaching the coverslip with proper adhesion (“Breathing Technique”)
Proper attachment of the coverslip to the hemocytometer slide is crucial to ensure optimal chamber volume. Carefully place the coverslip centrally over the hemocytometer counting grid area: Exhale gently onto the slide surface as you would when fogging up a mirror to create a slight moisture film. This thin film helps the coverslip adhere uniformly, reducing the risk of air bubbles or slippage. Press gently but firmly on the edges of the coverslip. The coverslip should remain firmly attached when you lightly tilt the hemocytometer. When the two glass surfaces are in proper contact Newton’s rings can be observed.

Step 3
Loading the cell suspension
Ensure the cell suspension is well-mixed and diluted appropriately (recommended dilution typically between 1:2 to 1:10, depending on cell density). Using a micropipette, carefully aspirate approximately 10 µL of the diluted cell suspension. Touch the pipette tip gently to the edge of the coverslip at the chamber entry notch, allowing capillary action to draw the cell suspension under the coverslip and into the counting chamber.
Do not inject or forcefully dispense the sample, as this can cause bubbles or uneven cell distribution. The fluid should uniformly fill the chamber. If air bubbles appear or the filling is uneven, thoroughly clean and reload the chamber.Step 4
Settling of Cells
Allow the cells to settle briefly (about 1–2 minutes). Waiting allows cells to stabilize and settle evenly onto the counting grid, ensuring a more accurate count.
Step 5
Counting Cells under the Microscope
Use the microscope at a suitable magnification (typically 15× or 10× objectives). Count the cells in the four outer corner squares or the central large square depending on your protocol. Employ consistent counting rules, such as counting cells touching the top and left borders but not those touching the bottom and right borders, to avoid double-counting.

Step 6
Calculating Cell Density
Use the following formula to determine the cell concentration:

Neubauer Hemocytometer Calculation

Note: 0.1 mm depth is constant in Neubauer Hemocytometer
Example from Neubauer Hemocytometer
The gridded area of the Improved Neubauer ruled hemocytometer consists of nine
1 × 1 mm (1 mm2) squares. These are subdivided in three directions; 0.25 × 0.25 mm (0.0625 mm2 ), 0.25 × 0.20 mm (0.05 mm2) and 0.20 × 0.20 mm (0.04 mm2). The central square is further subdivided into 0.05 × 0.05 mm (0.0025 mm2 ) squares. The raised edges of the hemocytometer hold the coverslip 0.1 mm off the marked grid, giving each square a defined volume         Image courtesy of:
By Zephyris at the English-language Wikipedia, CC BY-SA 3.0
https://commons.wikimedia.org/w/index.php?curid=10553213

Practical Tips and Tricks:

Tip 1: Coverslip Thickness
Always use the specialized coverslip provided for the hemocytometer. Standard coverslips are typically thinner and may alter the chamber’s internal volume, leading to incorrect cell counts.
Tip 2: Avoid Air Bubbles
If bubbles occur when loading the chamber, completely rinse and dry the chamber and reload the sample. Bubbles severely distort cell counts and accuracy.
Tip 3: Correct Chamber Filling
Ensure capillary action fills the chamber evenly. If the sample does not flow evenly, slightly adjust the angle of the pipette or check for proper coverslip attachment.
Tip 4: Maintain Even Distribution
Thoroughly mix your cell suspension immediately before loading the hemocytometer, as cells tend to settle quickly.
Tip 5: Always Double-Check Counts
Perform counting on both chambers and take the average for improved accuracy and reliability.

Conclusion
Mastering hemocytometer loading and counting ensures reliable and reproducible results critical for downstream applications in cell culture and experimental biology. With careful attention to these detailed steps and practical tips, you can achieve consistently accurate and trustworthy results in your daily laboratory practice.

Not for therapeutic use, treatment or medical diagnostic evidence. Not all products are available in every country. Contact your local ZEISS representative for more information.
EN_41_013_347 | Rel. 1.0 | CZ 06-2025 | Design, scope of delivery and technical progress subject to change without notice. | © Carl Zeiss Microscopy GmbH

Carl Zeiss Microscopy GmbH
07745 Jena, Germany
microscopy@zeiss.com
www.zeiss.com/microscopyhttp://www.zeiss.com/microscopy
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