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SOP-005: Preparation of Complete Culture Medium

Purpose​

To prepare sterile complete culture medium by supplementing basal medium with the appropriate components according to the requirements of the cell line.

Scope​

This SOP applies to the preparation of complete media for routine mammalian cell culture.

Responsibilities​

The operator is responsible for:

  • Using sterile technique throughout media preparation.
  • Selecting the correct basal medium and supplements.
  • Preparing media according to the required formulation.
  • Properly labeling and storing prepared media.
  • Recording preparation details.

Required Materials​

Reagents​

  • Basal culture medium
  • Required supplements (e.g., FBS, GlutaMAX, antibiotics, growth factors)
  • Sterile PBS (if required)

Equipment​

  • Class II Biosafety Cabinet
  • Sterile serological pipettes
  • Pipette aid
  • Micropipettes and sterile tips
  • Sterile filter units (if applicable)
  • Labels and laboratory marker

Procedure​

1. Preparation​

Bring all components to the recommended working temperature if required by the protocol.

Inspect all reagents for:

  • Correct labeling
  • Expiry date
  • Cloudiness
  • Precipitation
  • Evidence of contamination

Do not use compromised reagents.

2. Prepare the Biosafety Cabinet​

Disinfect the work surface.

Arrange all materials to minimize unnecessary movement during preparation.

Maintain aseptic technique throughout the procedure.

3. Add Supplements​

Add supplements to the basal medium according to the formulation specified for the cell line.

Mix gently after each addition.

Avoid vigorous shaking to minimize foaming.

4. Mix the Medium​

Invert the bottle gently several times until the supplements are evenly distributed.

Do not vortex.

5. Label the Medium​

Label the container with:

  • Medium name
  • Supplement composition (optional)
  • Preparation date
  • Expiry date (according to laboratory policy)
  • Operator initials

6. Storage​

Store prepared complete medium under the recommended conditions for the formulation.

Protect light-sensitive media or supplements from prolonged light exposure.

Acceptance Criteria​

ParameterAcceptance Criterion
Medium appearanceClear and free of particles
SterilityNo visible contamination
LabelingComplete and legible
Supplement additionCorrect formulation

Critical Control Points​

  • Maintain aseptic technique throughout preparation.
  • Verify all reagent identities before use.
  • Use only sterile pipettes and tips.
  • Add supplements in the correct amounts.
  • Avoid repeated warming and cooling of media whenever possible.

Documentation​

Record the following:

  • Date of preparation
  • Medium formulation
  • Lot numbers of critical supplements (if required)
  • Operator initials
  • Expiry date

Quality Control​

Before use, verify:

  • Medium is clear.
  • No precipitation is present.
  • No evidence of microbial contamination.
  • Correct labeling.
  • Proper storage conditions have been maintained.

Troubleshooting​

ObservationPossible CauseCorrective Action
Medium appears cloudyPossible contaminationDiscard and prepare fresh medium
Visible precipitateImproper storage or incompatible supplementsVerify reagent compatibility and storage conditions
Incorrect formulationSupplement omitted or added incorrectlyPrepare a fresh batch using the correct formulation
Poor cell growthIncorrect medium compositionConfirm medium formulation for the specific cell line

Safety Considerations​

  • Perform all procedures in a certified Class II Biosafety Cabinet.
  • Wear appropriate personal protective equipment (PPE).
  • Handle all biological reagents according to institutional biosafety guidelines.
  • Dispose of waste using approved biohazard procedures.