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SOP-019: Operation of an Inverted Microscope

Purpose​

To ensure the safe and standardized operation of an inverted microscope for routine examination of mammalian cell cultures.

Scope​

This SOP applies to the routine use of inverted bright-field or phase-contrast microscopes for monitoring mammalian cell cultures.

Responsibilities​

The operator is responsible for:

  • Verifying microscope functionality before use.
  • Handling objectives and optical components correctly.
  • Preventing contamination of the microscope.
  • Cleaning the instrument after use.

Required Materials​

Reagents​

  • Lens cleaning solution (if required)
  • Lens tissue

Equipment​

  • Inverted microscope
  • Cell culture vessels
  • Laboratory wipes
  • Personal protective equipment (PPE)

Procedure​

1. Pre-Use Inspection​

Before use, verify that:

  • The microscope is clean.
  • Objectives and eyepieces are free of dust or residue.
  • Illumination is functioning correctly.
  • The stage moves smoothly.

2. Position the Sample​

Place the culture vessel securely on the microscope stage.

Ensure that the vessel is stable before focusing.

3. Focus the Sample​

Begin observation using the lowest magnification objective.

Bring the cells into focus using the coarse focus control, followed by the fine focus control.

Increase magnification only after the specimen has been properly focused.

4. Examine the Culture​

Assess the culture for:

  • Cell morphology
  • Cell density
  • Cell attachment (adherent cultures)
  • Cell aggregation (suspension cultures)
  • Evidence of contamination

Record observations when required.

5. Complete the Procedure​

Remove the culture vessel.

Return the lowest magnification objective to the viewing position.

Switch off the illumination.

Clean the stage if contamination is present.

Cover the microscope after use.

Acceptance Criteria​

ParameterAcceptance Criterion
Image qualityClear and well focused
Optical componentsClean and undamaged
Stage movementSmooth and unrestricted
IlluminationUniform and stable

Critical Control Points​

  • Begin focusing with the lowest magnification objective.
  • Avoid contact between the objective lens and the culture vessel.
  • Handle focusing controls gently.
  • Keep optical components clean.
  • Return the microscope to its storage position after use.

Documentation​

Record the following when applicable:

  • Date
  • Cell line
  • Observations
  • Magnification used (if required)
  • Operator initials

Quality Control​

Verify:

  • Image quality is satisfactory.
  • Objectives are clean.
  • Illumination functions normally.
  • Stage movement is unrestricted.
  • Microscope is cleaned after use.

Troubleshooting​

ObservationPossible CauseCorrective Action
Blurred imageDirty objective or incorrect focusClean the objective and refocus
Poor illuminationLamp or illumination settings incorrectVerify illumination settings and light source
Difficulty focusingIncorrect objective selection or stage positionReturn to the lowest magnification and refocus
Stage movement restrictedMechanical obstructionRemove obstructions and inspect the stage

Safety Considerations​

  • Wear appropriate personal protective equipment (PPE).
  • Handle culture vessels carefully to avoid spills.
  • Clean any biological contamination immediately.
  • Use only approved lens tissue for cleaning optical components.
  • Disconnect the microscope from the power supply before maintenance.