Cell Counting Chamber, Haemocytometer

The Improved Neubauer Counting Chamber is a precision-engineered laboratory device used for counting cells and particles in a specific volume of fluid under a microscope. It is widely utilized in hematology, microbiology, cell culture, and other biological sciences to determine cell concentration in a liquid sample accurately.

Design and Construction

• Material: Made from high-quality, optically clear glass to ensure accurate microscopic observations.
• Dimensions:
  • Chamber Depth: Precisely set at 0.100 mm (100 µm) by the thickness of the special cover glass placed over the counting grid.
  • Overall Size: Fits standard microscope stages for ease of use.
• Counting Grid Layout:
  • Central Counting Area:
    • Divided into nine large squares, each measuring 1 mm × 1 mm.
    • The total area of the grid is 9 mm².
  • Subdivision of Squares:
    • The central large square is further subdivided into 25 groups of small squares.
    • Each group contains 16 smallest squares, making a total of 400 smallest squares in the central square.
    • The sides of the smallest squares measure 0.05 mm (50 µm).

Features

1. Precision Engineering:
   • Uniform Depth: The exact chamber depth ensures consistent volume measurements across the grid.
   • Accurate Grid Dimensions: Etched grids with precise dimensions allow for reliable counting.
2. Double Neubauer Ruling:
   • Dual Counting Areas: Some chambers feature two identical counting grids, enabling duplicate counts without relocating the slide.
3. Enhanced Visibility:
   • Etched and Coated Lines: Grid lines are engraved and may be coated with a metallic layer to improve contrast under the microscope.
4. Compatibility with Specialized Cover Glass:
   • Defined Thickness: Uses specific cover slips (No. 4 cover glass) with a thickness of 0.4 mm to establish the correct chamber depth.

Usage Procedure

1. Preparation:
   • Cleaning: Ensure the chamber and cover glass are clean and free from residues or debris.
   • Placing the Cover Glass: Moisten the edges slightly and place the cover glass over the counting area. The cover glass should adhere by surface tension, producing Newton's rings (colorful interference patterns), indicating proper placement.
2. Loading the Sample:
   • Mix the Sample: Vigorously mix the cell suspension to achieve a homogeneous distribution.
   • Filling the Chamber:
     • Use a pipette to draw the sample.
     • Touch the pipette tip to the edge of the cover glass and allow the chamber to fill by capillary action.
     • Avoid overfilling or introducing air bubbles.
3. Microscopy:
   • Placement: Place the loaded chamber on the microscope stage.
   • Focus: Use low power to locate the grid and then switch to the desired magnification (usually 10× or 40× objective).
   • Adjust Lighting: Optimize illumination for clear visibility of cells and grid lines.
4. Counting Cells:
   • Selection of Squares: Choose the appropriate squares to count based on cell density.
     • For sparse samples, you may need to count more squares for accuracy.
   • Counting Rules:
     • Count cells touching the top and left boundary lines of the square.
     • Do not count cells touching the bottom and right boundary lines to avoid double-counting.
   • Repeat Counts: Perform counts on multiple areas to increase statistical reliability.
5. Formula to Calculate Cell Concentration:Cell Concentration (cells/mL)=Total Cells Counted×Dilution FactorNumber of Squares Counted×Volume of One Square (mL)\text{Cell Concentration (cells/mL)} = \frac{\text{Total Cells Counted} \times \text{Dilution Factor}}{\text{Number of Squares Counted} \times \text{Volume of One Square (mL)}}Cell Concentration (cells/mL)=Number of Squares Counted×Volume of One Square (mL)Total Cells Counted×Dilution Factor​
   • Volume of One Small Square:Volume=Area×Depth=(0.05 mm×0.05 mm)×0.1 mm=0.00025 mm3=2.5×10−7 mL\text{Volume} = \text{Area} \times \text{Depth} = (0.05 \text{ mm} \times 0.05 \text{ mm}) \times 0.1 \text{ mm} = 0.00025 \text{ mm}^3 = 2.5 \times 10^{-7} \text{ mL}Volume=Area×Depth=(0.05 mm×0.05 mm)×0.1 mm=0.00025 mm3=2.5×10−7 mL
   • Example Calculation:
     If you counted 200 cells in 80 small squares:Cell Concentration=20080×2.5×10−7=2002×10−5=10,000,000 cells/mL\text{Cell Concentration} = \frac{200}{80 \times 2.5 \times 10^{-7}} = \frac{200}{2 \times 10^{-5}} = 10,000,000 \text{ cells/mL}Cell Concentration=80×2.5×10−7200​=2×10−5200​=10,000,000 cells/mL

Applications

• Hematology:
  • Counting red blood cells, white blood cells, and platelets.
• Microbiology:
  • Determining bacterial or yeast cell concentrations.
• Cell Culture:
  • Assessing cell viability and density before subculturing or experiments.
• Semen Analysis:
  • Quantifying sperm concentration.
• Parasitology:
  • Counting parasites in blood or other body fluids.
• Environmental Biology:
  • Analyzing cell counts in water samples.

Advantages

• High Accuracy and Precision:
  • Standardized dimensions allow for reproducible results.
• Cost-Effective:
  • Reusable with proper care, offering long-term utility.
• Versatility:
  • Suitable for a wide range of cell types and sizes.
• Ease of Use:
  • Straightforward methodology accessible to trained laboratory personnel.

Care and Maintenance

• Cleaning:
  • Gently clean with a mild detergent solution and rinse with distilled water.
  • Do not use abrasive materials that could scratch the glass surfaces.
• Handling:
  • Handle with care to avoid chips or cracks.
  • Store in a protective case when not in use.

Tips for Accurate Counting

1. Uniform Suspension:
   • Ensure the sample is well-mixed immediately before loading to prevent cell settling.
2. Avoiding Errors:
   • Check for air bubbles or overfilling, which can distort counts.
   • Use the correct cover glass thickness to maintain chamber depth.
3. Consistent Counting Technique:
   • Apply the same counting rules consistently across samples.
4. Replicates:
   • Perform multiple counts to calculate an average for increased accuracy.
5. Dilution Factor:
   • Account for any dilution of the sample in your calculations.

Comparison with Other Chambers

• Fuchs-Rosenthal Chamber:
  • Larger counting area, often used for cerebrospinal fluid analysis.
• Bürker Chamber:
  • Similar to Neubauer but with different grid patterns.
• Improved Neubauer vs. Original Neubauer:
  • The "Improved" version has a modified grid for easier counting and improved accuracy.

Conclusion

The Improved Neubauer Counting Chamber is an essential tool in laboratories for quantifying cells in a fluid sample accurately. Its precise construction and standardized grid facilitate reliable measurements, making it invaluable in clinical diagnostics, research, and various biological fields.