Best Practice Guidance: Microbiological Media preparation, storage, quality check and incubation

Media Preparation –

• Culture media are essential for microbiological testing and may be prepared in-house or obtained commercially.
• The term “prepared media” includes both in-house and commercially sourced media.
• Ensuring the quality of prepared media is critical for accurate microbiological results.
• Proper media preparation, storage, and quality control are essential for maintaining media quality.
• Select the correct media or components using accepted sources or references for formulas.
• Follow the manufacturer’s instructions for preparation, including specific requirements like heating, additives, and pH adjustment.
• Ready-made media are typically accompanied by:
  • A certificate of analysis
  • Expiration date and recommended storage conditions
  • Information on quality control organisms
  • Performance testing results and acceptance criteria
• Similar quality control criteria should be established for in-house prepared media.
• Use Purified Water (preferred) for media preparation; deionized or distilled water may be used if appropriate.
• Avoid using lower-quality water for media preparation.
• Record the volume of water used in each batch.
• Use a calibrated balance suitable for the weight range of the ingredients.
• Always use clean weighing containers and tools (e.g., spatulas) to prevent contamination.
• Record the weight of each component used.
• Thoroughly dissolve dehydrated media in water before dispensing and sterilization.
• If heating is required to dissolve media:
  • Avoid overheating, as media are generally heat-sensitive.
  • Watch for signs of overheating, like darkening due to Maillard reaction.
• Use equipment that allows for controlled heating and consistent mixing.
• Mix thoroughly when adding supplements to the media.
• Ensure all glassware is thoroughly cleaned and rinsed to remove detergents and residues.
• Perform sterilization using:
  • Manufacturer-provided parameters
  • User-validated conditions
• Preferred sterilization method is moist heat sterilization (autoclaving), unless media contain heat-labile components.
• Filtration may be appropriate for sterilizing heat-sensitive media.
• Validate the sterilization method via sterility and growth promotion testing.
• Ensure autoclave cycle validation for uniform heat distribution based on load and volume.
• Common autoclaving condition: 121°C for 15 minutes, adjusted based on container size and load.
• Avoid prolonged holding or slow heating/cooling in autoclaves, as it may degrade media.
• Improper sterilization may result in:
  • Color change
  • Loss of clarity
  • Altered gel strength
  • pH drift
• Unless pre-sterilization pH testing is specified, confirm pH after cooling to room temperature (20°–25°C).
• Use:
  • Flat pH probe for agar surfaces
  • Immersion probe for liquid media
• Acceptable pH range: ±0.2 of the manufacturer’s stated value, unless otherwise validated.
• Inspect prepared media for:
  • Cracked containers or lids
  • Unequal filling
  • Cracked/dimpled surfaces on solid media
  • Hemolysis
  • Excessive darkening or discoloration
  • Crystal formation (freezing-related)
  • Excessive bubbles
  • Redox indicator status (if applicable)
  • Lot number and expiration date
  • Sterility
  • General cleanliness (e.g., lid should not stick to plate)

Media Storage –

• Understand the manufacturer’s or supplier’s transport and storage methods to ensure media quality upon delivery.
• Media manufacturers should use transport and storage conditions that:
  • Minimize moisture loss
  • Control temperature
  • Prevent microbial contamination
  • Provide mechanical protection
• Properly label media with:
  • Batch or lot numbers
  • Preparation and expiration dates
  • Media identification
• Store media according to manufacturer’s instructions; in-house prepared media should be stored under validated conditions.
• Do not store agar plates at or below 0°C to avoid freezing damage to the gel structure.
• Protect stored media from:
  • Exposure to light
  • Excessive temperature
  • Rapid temperature fluctuations that may cause condensation
• Consider storing agar plates in sealed packages or containers to prevent moisture loss.
• Remelting of solid media containers (without heat-labile components) should be performed only once to avoid quality degradation.
• Remelting is best done using a heated water bath or other suitable methods that preserve media quality.
• Avoid using microwave ovens or heating plates for remelting unless proper precautions are taken to prevent overheating and injury risks.
• After remelting, hold molten agar medium in a monitored water bath at 45°–50°C.
• Do not hold molten media for more than 8 hours, or as determined appropriate for the medium composition.
• Prevent water from the water bath from mixing with the sterile media when pouring; wipe the container exterior dry before pouring.
• Dispose of used or expired cultured media following local biological hazard safety procedures.

Quality Control Testing –

• Media may be prepared in-house or purchased commercially; lot-to-lot variability from raw materials must be considered.
• Performance of media depends on proper preparation and storage; improper handling can lead to poor microbial growth or unreliable results.
• Quality control tests should be performed on all prepared media, including swabs or media strips.
• Routine tests for in-house prepared media include:
  • pH measurement
  • Growth promotion testing
  • Inhibition testing
  • Indicative properties testing (as appropriate)
• Periodic stability checks are recommended to confirm expiration dating until validated.
• For media not terminally sterilized in final packaging (e.g., irradiated plates), inspect representative samples for microbial contamination before use.
• Sterility inspection involves detecting turbidity in liquid media or colonies on plated media.
• Incubate media samples during sterility testing at the same temperature and for the maximum incubation time used in testing (e.g., 5 days if test incubation is 3–5 days).
• Every batch of in-house sterilized media should undergo growth promotion testing.
• Select test organisms based on compendial references, manufacturer recommendations, or representative environmental isolates (not as formal requirements).
• Growth promotion is considered satisfactory if visible growth is comparable to a previously approved batch of the same medium formula and volume.
• Quantitative growth comparisons require adherence to appropriate guidelines considering biological variability.
• Media expiration dates must be supported by growth promotion testing showing acceptable performance up to expiration.
• Investigate and implement corrective actions for any media batch that fails growth promotion testing; such batches are unsuitable for use.
• Diagnostic reagents (e.g., Gram stain, oxidase, coagulase) should undergo quality control testing using appropriate standard microorganisms before diagnostic use.
• Media used in sterility tests and environmental monitoring require special care:
  • Preferably double-wrapped and terminally sterilized.
  • If not terminally sterilized, perform 100% pre-incubation and inspection before use in critical areas.
  • Growth promotion testing should confirm that pre-incubation does not affect microbial recovery.
• Verify raised agar levels in surface contact plates used for environmental monitoring.

Incubation Times –

• Incubation times less than 3 days should be expressed in hours (e.g., incubate at 30°–35°C for 18–72 hours).
• Incubation times longer than 72 hours should be expressed in days (e.g., incubate at 30°–35°C for 3–5 days).
• For incubation times expressed in hours, incubate for the minimum specified time and use microbiological judgment if extending beyond that time.
• For incubation times expressed in days, if incubation starts in the morning or afternoon, conclude the incubation at the same time of day (morning or afternoon).