LimsPulse
Cell counter · Calibration procedure

Counting accuracy with reference beads

Counts certified polystyrene beads of known concentration and size several times, and checks the mean count, precision and size against the certificate.

Photo of a Cell counter
Photo: TimVickers, Public domain (cropped), via Wikimedia Commons
PURPOSE

Why this is checked

Beads are stable and uniform, so they test the counter itself (counting volume, focus, detection) without the biological variation of cells. Accuracy and precision with beads support confidence in cell counts, although they do not test live/dead classification.

Frequency

Typically monthly or quarterly, after service and before validation work, as defined in your SOP.

Learn how the Cell counter works
YOU WILL NEED

Standards & tools

  • Certified concentration and size bead standard (for example about 1 × 10⁶ beads/mL, nominal size in the cell range)
  • Bead certificate stating mean concentration with its uncertainty and mean size
BEFORE YOU START

Prerequisites

  • Counter clean and self-test passed
  • Beads in date, at room temperature and fully resuspended (vortex as instructed)
  • Bead protocol selected with dilution factor 1 (no stain) unless the supplier says otherwise
PROCEDURE

Step by step

  1. 1Vortex the bead vial for the time stated on the certificate.
  2. 2Load a slide with beads and count. Repeat to give at least 5 independent counts from fresh loads.
  3. 3Record concentration and mean size for each count.
  4. 4Calculate mean, standard deviation, % CV and recovery against the certified value.
  5. 5Compare the mean size with the certified size.
RECORD

Example observation table

ReplicateCount (beads/mL)
11.02 × 10⁶
20.98 × 10⁶
31.05 × 10⁶
41.01 × 10⁶
50.99 × 10⁶
Mean / SD / CV1.010 × 10⁶ / 0.027 × 10⁶ / 2.7%
CALCULATION

How to calculate the result

Formula
Recovery (%) = mean count ÷ certified count × 100; CV (%) = SD ÷ mean × 100
Worked example
  1. 1
    Mean = (1.02 + 0.98 + 1.05 + 1.01 + 0.99) ÷ 5 = 1.010 × 10⁶ beads/mL
  2. 2
    SD = 0.027 × 10⁶; CV = 0.027 ÷ 1.010 × 100 = 2.7%
  3. 3
    Certified value 1.00 × 10⁶ beads/mL → recovery = 1.010 ÷ 1.00 × 100 = 101.0%
ACCEPTANCE CRITERIA

Pass if

  • Mean count within the certificate range, typically about ±10% of the certified value
  • CV typically ≤ 5–10%, as set in your SOP
  • Mean size within the tolerance stated on the certificate
IF IT FAILS

What to do

  • Check bead resuspension and slide loading; repeat with a new vial if needed.
  • If still failing, label the counter out of use for reportable counts and call service.
  • Assess counts made since the last passing check.
In LimsPulse · Calibration Management

LimsPulse schedules this calibration, records the result against limits, and blocks the instrument everywhere if it fails or goes overdue.

See how