How to Use a Hemocytometer
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Specifications
- Cover glass support
- Cover glass
- Counting chamber 0.1 mm depth
Product Usage Instructions
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: Loading the Hemocytometer
Place the cleaned coverslip on the hemocytometer chamber
carefully to avoid trapping air bubbles. Ensure a tight seal
between the coverslip and the chamber to prevent leakage.
Step 3: Adding the Sample
Load your cell suspension into the chamber using a pipette,
allowing capillary action to evenly fill the chamber. Avoid
creating air bubbles during this process.
Step 4: Microscopy and Counting
Place the hemocytometer under a microscope and focus on the grid
lines. Count the cells within the specified grid areas, following
the guidelines provided with the hemocytometer.
Step 5: Calculating Cell Density
Use the provided formula to determine the cell concentration
based on the grid areas counted and the volume at 0.1 mm depth.
FAQ
Q: What should I do if air bubbles occur during loading?
A: If bubbles occur when loading the chamber,
completely rinse and dry the chamber and reload the sample to
ensure accurate cell counts.
Q: Why is it important to use a specialized coverslip?
A: Standard coverslips may alter the chamber’s
internal volume, leading to incorrect cell counts. Using the
provided specialized coverslip ensures accuracy.
Q: How can I improve counting accuracy?
A: Thoroughly mix your cell suspension before
loading, count cells in both chambers, and take the average for
improved accuracy and reliability.
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How to Use a Hemocytometer
Step-by-Step Guide
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.
Cover glass support
Cover glass Counting chamber 0.1 mm depth
The parts of the hemocytometer
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This tutorial provides step-bystep 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.
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Step 4 Settling of Cells Allow the cells to settle briefly (about 12 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.
Image courtesy of: By Zephyris at the English-language Wikipedia, CC BY-SA 3.0 https://commons.wikimedia.org/w/index. php?curid=10553213
Step 6 Calculating Cell Density Use the following formula to determine the cell concentration:
Neubauer Hemocytometer Calculation Cells × dilution = cells/µL squares × 0.1
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
Dimensions 1 × 1 mm 0.25 × 0.25 mm 0.25 × 0.20 mm 0.20 × 0.20 mm 0.05 × 0.05 mm
Area 1 mm2 0.0625 mm2 0.05 mm2 0.04 mm2 0.0025 mm2
Volume at 0.1 mm depth 100 nL 6.25 nL 5 nL 4 nL 0.25 nL
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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.
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.
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.
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Carl Zeiss Microscopy GmbH 07745 Jena, Germany microscopy@zeiss.com www.zeiss.com/microscopy
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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
Documents / Resources
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ZEISS How to Use a Hemocytometer [pdf] User Guide How to Use a Hemocytometer, Use a Hemocytometer, Hemocytometer |