Common Cell Counting Issues

When dealing with cell counts, we encounter some common issues. Although automated cell counters are providing us a much time efficient method and accuracy, we still encounter some issues while using them. This post introduces the common issues and the quick solutions when using cell counters.

1. Overcounting of cells

• Problem: Hard to differentiate cells due to poor focus or debris, etc.

• Possible causes:

  • Incorrect focus settings.

  • Cells sample is contaminated or contains debris.

  • Dirty optic lens.

  • Any fingerprint or marks on the slide.

  • Air bubbles present with the sample.

• Solutions:

  • Adjust the focus when using manual focusing.

  • Use clean cell samples or wash the samples before use.

  • Clean the lens.

  • Prevent touching the slide where cells are loaded.

2. Presence of debris affecting the counting results

• Problem: Debris is being counted as cells, leading to overcounting.

• Possible causes:

  • Poor sample preparation or contamination.

• Solutions:

  • Wash the sample using proper solutions.

  • Use fluorescent cell counters to exclude debris from counting.

3. Undercounting of cells

• Problem: Clumped or aggregated cells are counted as one single large cell.

• Possible causes:

  • Insufficient mixing or cell handling errors.

• Solutions:

  • Gently pipette up and down to disperse cells.

  • Treat samples with reagents to prevent clumping, if necessary.

4. Over or undergaining of cell concentration

• Problem: The sample is either too concentrated or inadequate for accurate counting.

• Possible Causes:

  • Miscalculation of dilution or pipetting errors.

• Solutions:

  • Optimize the cell concentration to fall within the optimal range for the method (e.g., 10⁴–10⁶ cells/mL).

  • Recheck pipetting steps and dilutions.

  • Minimize sample loss.

    
    

5. Ambiguous detection of live and dead cells

• Problem: Live and dead cells are not clearly distinguishable.

• Possible Causes:

  • Incorrect staining method or

• Solutions:

  • Double-check the protocol for staining viability dyes like Trypan Blue or Propidium Iodide.

  • Ensure proper incubation time and temperature.

6. Low cell viability

• Problem: Low viability leading to inaccurate results in experiments

• Possible Causes:

  • Poor condition of cells, rough handling, unsuitable condition for cells to live, etc.

• Solutions:

  • Careful handling of cells under suitable conditions

  • Periodically check the condition of cells

    
    

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NanoEntek offers the perfect cell counter to meet your needs. If you're using a trypan blue-based cell counter and facing any of the challenges mentioned in this post, see our fluorescence cell counters below!