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SOP-026: Mycoplasma Testing of Cell Cultures

Purpose​

To routinely screen mammalian cell cultures for mycoplasma contamination and ensure cultures remain suitable for research and experimental applications.

Scope​

This SOP applies to routine mycoplasma screening of mammalian cell cultures using laboratory-approved detection methods.

Responsibilities​

The operator is responsible for:

  • Performing routine mycoplasma testing according to laboratory schedules.
  • Collecting representative samples.
  • Preventing cross-contamination during sample handling.
  • Recording and reporting test results.

Required Materials​

Reagents​

  • Mycoplasma detection kit or laboratory-approved testing reagents
  • Sterile PBS (if required)

Equipment​

  • Class II Biosafety Cabinet
  • Micropipettes and sterile tips
  • Sterile microcentrifuge tubes
  • Laboratory equipment appropriate for the selected detection method
  • Personal protective equipment (PPE)

Procedure​

1. Select Cultures for Testing​

Identify cultures requiring routine screening according to laboratory policy.

Confirm sample identity before collection.

2. Collect the Sample​

Collect the required sample using aseptic technique.

Avoid contaminating the culture during sampling.

Transfer the sample into a properly labeled sterile container.

3. Perform the Test​

Carry out the mycoplasma detection assay according to the manufacturer's instructions or validated laboratory protocol.

Include appropriate positive and negative controls when required.

4. Interpret Results​

Review the assay results according to the criteria established for the selected detection method.

If contamination is detected, remove the affected culture from routine use until appropriate corrective action has been taken.

5. Record the Results​

Document all findings immediately after completing the assay.

Acceptance Criteria​

ParameterAcceptance Criterion
Sample identificationVerified
ControlsPerform as expected
Test resultNegative for mycoplasma
DocumentationComplete

Critical Control Points​

  • Use aseptic technique during sample collection.
  • Test cultures at regular intervals.
  • Include appropriate assay controls.
  • Test newly received cell lines before routine use.
  • Retest cultures if contamination is suspected.

Documentation​

Record the following:

  • Date
  • Cell line
  • Passage number
  • Detection method
  • Test result
  • Operator initials
  • Corrective actions (if applicable)

Quality Control​

Verify:

  • Samples are correctly labeled.
  • Controls perform within expected limits.
  • Results are recorded accurately.
  • Laboratory testing schedule is maintained.

Troubleshooting​

ObservationPossible CauseCorrective Action
Positive resultMycoplasma contaminationIsolate the culture and follow laboratory contamination procedures
Invalid controlAssay failureRepeat the test with fresh reagents
Inconsistent resultsSample handling errorCollect a new sample and repeat the assay
Unexpected positive resultCross-contamination during samplingReview aseptic technique and repeat testing

Safety Considerations​

  • Handle all cultures as potentially biohazardous.
  • Wear appropriate personal protective equipment (PPE).
  • Perform sample collection in a certified Class II Biosafety Cabinet.
  • Dispose of assay materials according to institutional biosafety procedures.
  • Report confirmed contamination immediately.