Best Practices for Preparation of Culture Media

Culture media are fundamental in diagnostic microbiology. Nearly all media are commercially available as ready-to-use agar plates or tubes of broth. Laboratories in high-income countries procure ready-to-use culture media from specialized manufacturers. 

Culture media can be prepared in-house by dissolving a specified amount of dehydrated media powder in distilled or deionized water, following the manufacturer’s recommendations. Laboratories in low and middle-income countries prefer preparing needed supplies of agars and broths using media powder instead of purchasing ready-to-use media. 

In-house culture media preparation has inherent challenges, so this blog post tries to tackle this issue by compiling best practices for media preparation. 

Manual preparation of culture media is labor intensive, so many companies have developed automated culture media preparation systems. If your lab can afford to purchase them, automated culture media preparation systems can save you manual labor and time.

Read more: Automated Culture Media Preparation and Dispenser System.

Selection of Media Types and Manufacturer 

When selecting culture media, look for products from reputable manufacturers supported by your national reference laboratory. Carefully check the product’s name, compositions, instructions for use, and shelf life. Look for humidity-proof packaging (screw cap, peel-off seal).

All bacteriology laboratories may need a minimal supply of basic nutrient media (such as nutrient agar or tryptic soy agar), enriched media (such as blood agar and chocolate agar), and selective media for Gram-positive (Columbia CNA agar), and Gram-negative bacteria (MacConkey agar). 

There is a plethora of media with similar applications. Depending on the setting, you may need to keep stock of a few media for epidemic preparedness (e.g., TCBS  in cholera risk settings). Get help from a clinical microbiologist to select the appropriate culture media needed for your laboratory. 

Procurement, Storage, and Stock Management 

Ensure your suppliers (both primary and backup) are reliable and provide after-sales support. Up on reception of goods, check and record;

  1. product name and lot number
  2. quantity received 
  3. expiry date and remaining shelf life 
  4. package integrity
  5. order processing time
  6. correctness of order delivery

At all times, follow the manufacturer’s instructions about storage conditions. For example, growth supplements such as egg yolk or antibiotic solutions should be stored at 2-8°C, whereas the base medium may be stored at room temperature, not exceeding 30°C.

Dehydrated media are stored in a cool, dry place, protected from light and dust. Monitor the storage area’s temperature and humidity. Do not store dehydrated media in the same room used for steam sterilizing, boiling materials, cleaning glassware, etc.

Organize the stock according to a first-in, first-out system, rank media in a logical order and separate opened from closed containers. 

Dehydrated culture media
Dehydrated culture media

The shelf life of dehydrated powder is usually several years. However, dehydrated media are hygroscopic (i.e., it absorbs water) and quickly deteriorate when exposed to moisture. When exposed to moisture, a hard mass is formed, which alters the chemical and microbiological properties of the medium. This can be a serious problem for tropical countries with humid climates.

Therefore, record the opening date on the container and use the product for a maximum of 6 months after opening unless otherwise specified by the manufacturer. After this period, visual and performance checks are required.  When not in use, keep containers tightly closed and seal the caps with adhesive tape. 

Discard products when expired, when the powder is clumped, discolored, or not free flowing, or in case of quality issues; record product details and reasons for discarding.

Preparation of Culture Media

Materials/Equipment Requirement

  1. Pyrex flask (conical flask) at least double the size of the batch
  2. Magnetic Stir Bar
  3. Aluminum Foil
  4. Weighing machine
  5. Dehydrated culture media
  6. Spatula
  7. Graduated cylinder
  8. Distilled or deionized water
  9. Hot plate stirrer
  10. Autoclave
  11. Sterile Petri plates, and
  12. Infrared no-touch thermometer
Preparation of Culture Media
Preparation of culture media (Image source)

Calculation and Weighing

The dehydrated media should be calculated and weighed in a damp-free environment (low humidity), preferably under a laboratory fume hood. Ensure table, balance, and materials are clean, dust-free, and powder-free. 

Balance

Top loading (precision) balance is suitable for weighing media. Position the balance on a level, stable, vibration-free table. Calibrate the balance at regular intervals (e.g., annually) and verify control weights before use.

Weighing of Culture Media

Materials and PPE

To prevent the risk of inhaling fine particles of dehydrated media, wear a dust mask while handling dehydrated media. Wear gloves to avoid skin contact. Wear safety goggles (e.g., for sodium deoxycholate) while using media with components that can cause skin and eye irritation (read product instructions). Wash hands before and after working with dehydrated culture media. 

Calculate

Follow the manufacturer’s instructions. Prepare a table with pre-defined weights and volumes for a fixed number of plates. To prepare a solid medium with a depth of 4 mm in a Petri plate of 9 cm diameter, a volume of 20-25 mL is needed. Record product name, lot number, preparation date, weight and volume of water, and operator identification in a logbook. 

Weighing

To facilitate weighing, use materials with pre-defined volume. Open the container and take the required amount of powder. Close the container immediately to protect it from humidity. Do not put the excess powder back into the bottle.

Mixing with Water and Heating

Boiling is often required to dissolve media powder, but specific manufacturers’ instructions printed in media package inserts should be followed exactly.  Use fresh water prepared by distillation, deionization, or reverse osmosis. The presence of copper ions, high conductivity, and high pH may significantly alter the quality of in-house prepared media. Do not use tap water as it affects selectivity and pH. 

Conical flasks containing dissolved culture media (image source)

The quality of glassware used for pouring media is also an important factor. Use Erlenmeyers, bottles, and graduated cylinders made of borosilicate glass to measure water and mix with media powder. If using reusable glassware, only borosilicate glassware should be used because soda glass can leach alkali into the media and change the pH of the medium, which may affect growth.

Procedure for dissolving the powder in water

  1. Pour half of the required water into the flask, and add the pre-weighed powder. Pre-heating the water to 50-60°C  may facilitate dissolution. 
  2. Stir or rotate for a few minutes (do not shake!)
  3. Pour the rest of the water down the sides to dissolve any excess powder sticking to the flask walls (dry powder may not be sterilized in the autoclave)
  4. Heating is required for dissolving agar-containing media. Do not close the flasks tightly.
  5. Heat up to boiling, with frequent stirring, until the solution becomes clear. 
  6. Avoid boiling, overheating and foaming, scorching and burning, clumping, and inconsistent mixing. 

Sterilization

Most media require sterilization, so only bacteria from patient specimens will grow and not contaminants from water or powdered media. Some media cannot be autoclaved (e.g., SS agar, Cary Blair agar

Liquid media are distributed to individual tubes or bottles before sterilization. Autoclave a medium only when the ingredients are completely dissolved. Do not tighten the lids or caps completely. Agar media are sterilized in large flasks or bottles capped with either plastic screw caps or plugs before being placed in an autoclave.

Autoclave and spacing
Items are positioned in the autoclave with enough space for the steam to pass through. Autoclave indicator tape and chemical indicators are used.

The timing of autoclave sterilization should start from the moment the temperature reaches 121°C and usually requires a minimum of 15 minutes.

The volume of the media in one sterilization batch should be kept small; ideally, only two liters should be autoclaved at a time. Sterilization indicators such as the Bowie Dick test and biological indicators such as spores of Bacillus stearothermophilus should be used to monitor the proper working of an autoclave.

Once the sterilization cycle is completed, molten agar is allowed to cool to approximately 50°C before being distributed to individual Petri plates (approximately 20 to 25 mL of molten agar per plate). 

Avoid

  • Over-sterilization causes precipitation, pH change, component destruction 
  • Under-sterilization results in contaminated medium

Ensure

  • The wrapping allows for steam penetration
  • There is enough space between the items to allow steam circulation
  • To identify sterilized items use indicator tape to label the flask (medium name/code, preparation date, initials). 
  • Use chemical indicators with each cycle (e.g., time-steam-temperature strips) and biological indicators on an interval basis to verify autoclave cycles. 

Note

  • Use a ‘cool down program’ or wait until the pressure drops sufficiently before opening (70-80°C) to avoid fast pressure drop (liquid can boil over, and caps can be blown off). 
  • Do not wait too long before unloading the autoclave; overheating may destroy media ingredients. 

Fine-tuning (supplements and pH)

If other ingredients are to be added (e.g., supplements such as sheep blood or specific vitamins, nutrients, growth promoters, or antibiotics), they should be incorporated when the molten agar has cooled, just before distribution to plates.

The quality of the blood plays an important role in the performance of the blood-containing media, e.g., hemolytic reactions are well distinguished in sheep blood-containing media. The blood’s concentration, homogeneity, viscosity, and color should be checked before it is used for media preparation. The certificate of analysis and sterility conditions should be considered for other additives.

Adding supplements:

  • Let the media cool down to 45-50°C before adding heat-labile supplements 
  • Filter-sterilize heat-labile supplements (not in the case of blood)
  • Let supplements come to room temperature before adding
  • Add sterile supplements aseptically 
  • Ensure adequate mixing 

Delicate media components that cannot withstand steam sterilization by autoclaving (e.g., serum, certain carbohydrate solutions, certain antibiotics, and other heat-labile substances) should be sterilized separately by membrane filtration. Passage of solutions through membrane filters with pores ranging in size from 0.2 to 0.45 um in diameter will not remove viruses but does effectively remove most bacterial and fungal contaminants. 

Verification of pH after sterilization (not before):

  • Verify pH (usually at 25°C) with a pH meter before dispensing 
  • Take 20 mL out of the flask, let it cool down, and measure pH. Keep the flask in a water bath
  • Compare to the pH range specified in the manufacturer’s instructions. If pH is ok, continue with dispensing. 
  • The pH of ready-made dehydrated media should not require adjustment. In case of deviation, verify water, dehydrated medium, glassware, and procedure and start again.

Dispensing of Prepared Culture Media 

Cool down the culture media in a water bath (45-50°C) or hot plate stirrer before dispensing to minimize condensation. You can check the temperature with an infrared no-touch thermometer. Dispensing at too high temperature leads to excessive evaporation. If media stay too long in a water bath, it may cause precipitation (reheat, but do not overheat). Do not use cold water to cool down agar media, as it may lead to flakes or cloud formation. 

Dispensing culture media

If using reusable glass Petri plates, sterilize them at 160°C for 2 hours in a hot air oven. Allow the oven to cool to 50°C before opening (to avoid cracking glassware). Dispense the media aseptically in a draught-free room (with closed windows), and avoid fans or climate control.  Work close to the flame or in a biological safety cabinet

Procedure for dispensing agar media in plates

  1. Flame sterilize the neck of the flask before and between pouring
  2. Mix the culture media gently by rotating the flask before dispensing
  3. Dispense on a level surface
  4. For antimicrobial susceptibility testing, agar depth should be 4 ± 0.5 mm: Circular Petri plate of 90 mm: about 25 mL
  5. Use sterile, graduated pipettes or media distribution syringe/pump
  6. Avoid forming air bubbles (flame surface or use a heated loop to remove them). 
Dispensing and drying of Culture Media
Dispensing and Drying of Culture Media

Dispensing of agar or liquid media in tubes

  1. Use tubes with lids that allow ventilation (e.g., screw caps), do not tighten completely. 
  2. Put tubes in an autoclavable rack.
  3. Mix gently by rotating the flask before dispensing
  4. Dispense the correct volume per tube. Use sterile, graduated pipettes or a media distribution syringe/pump
  5. Close screw caps tightly after autoclaving

Drying of plates/tubes

  1. Dry for several hours at room temperature (up to 24 hours) to remove condensation. 
  2. Selective media: ± 30 minutes with lid ajar. If contamination risk: keep lids closed. 
  3. Dry before packing to prevent condensation on the lids. Avoid over-drying (cracks). 
  4. Drying depends on the type of media:
    1. For agar slants, let dry in a sloped position to give a butt of 2.5-3 cm deep and a slope of 2-2.5 cm long. Use a standardized and validated rack. 
    2. For agar, semi-solid tubes, and liquid media: let dry in a rack (vertical position).
Preparation of Culture Media

Packing and Storage

Label individual plates with name (abbreviated/code), preparation date, and lot number. Wrap the plates in sealed, labeled plastic bags, a maximum of ten plates per bag, to avoid moisture. Store them upside down, at 2-8°C, in the dark following manufacturer’s instructions.

Shelf life

Agar media in Petri plates. 

  • Blood agar: 7 days; with unstable additives: 2-5 days
  • Most selective media: 5-7 days
  • Nutrient agar without blood: 2-4 weeks

In tubes 

  • Simple, non-selective broths and agars: 6 months
  • Selective media: 3 weeks (2-8 weeks)
  • Selenite broth: 2-3 months

Quality Control

Finally, all media, whether purchased or prepared, must be subjected to stringent quality control before being used in the diagnostic setting. Quality control (QC) should be based on a pragmatic, risk-based approach. Newly prepared and dispensed media should be quarantined until they pass QC. Read more about quality control of culture media. 

Using Culture media

Bring the culture media to room temperature before use. Ensure there are no visible drops of water on the agar surface or inside the lid. If seen, do not shake off condensation water from the lid. 

Instead, dry plates for 20-30 minutes at 35-37°C with agar plates upside down and agar base resting at an angle on the lid (if necessary, dry plates at 20-25°C overnight). 

Do not over-dry plates (cracks in surface, surface wrinkled). 

Visual sterility check before use is a mandatory step. Check the plates for contamination or growth of colonies. 

Disposal of used culture media

For disposal of used culture media, follow in-country or WHO guidelines, which recommend inactivation and subsequent incineration before disposal. Inactivation (so-called ‘destruction’ or Level III inactivation) should be done in the laboratory, preferably by autoclaving. 

Most Common Errors and Possible Causes 

Clumped and free-flowing media
Clumped and free-flowing media

Clumping of dehydrated culture media

  • Humidity too high during storage
  • The container was left open for too long or was not closed tightly after opening 
  • Dehydrated culture medium beyond the shelf life

Wrong pH

  • pH meter not calibrated 
  • pH verification done on too hot medium (generally to be done at 25°C) 
  • Use of poor-quality water or container 
  • Use of chemically contaminated containers Incomplete dissolution/mixture of medium
  • Dehydrated medium stored incorrectly (e.g., not tightly closed) or beyond the shelf life

Incomplete solubility

  • Use of inadequate water
  • Inadequate heating/inadequate timing for dissolution 
  • Insufficient soaking or incomplete mixing 
  • Flask too small to allow adequate mixing and/or convection

Darkening, caramelization

  • Overheating: excessive sterilization, heterogeneous mixing medium kept at 50°C for too long, repeated re-melting or too high temperature. 
  • Incomplete dissolution of medium

Incomplete gelling or soft agar

  • Incorrect proportions of product to water: error in weighing or over-dilution
  • Agar not properly dissolved: poor mixing prolonged storage at 50°C
  • Overheating of culture medium, possibly at low pH
  • Repeated re-melting causes overheating

Turbidity, precipitation

  • Poor quality of dehydrated media
  • Use of poor-quality of water or container
  • Overheating: excessive sterilization, heterogeneous mixing medium kept at 50°C for too long, repeated re-melting or too high temperature. 
  • Wrong pH 
  • Incomplete dissolution/mixture of medium 
  • Loss of water of the prepared culture medium due to evaporation 

Poor growth or loss of differential properties 

  • Incorrect or improperly maintained QC organisms used 
  • Overheating: excessive sterilization, heterogeneous mixing medium kept at 50°C for too long, repeated
  • re-melting or at too high temperature
  • Incomplete dissolution/mixture of medium
  • Inhibitory substances in water, container or inoculum Wrong PH

References and further readings

  1. Bailey & Scott’s Diagnostic Microbiology, Forbes, 11th edition
  2. Orekan J, Barbé B, Oeng S, Ronat JB, Letchford J, Jacobs J, Affolabi D, Hardy L. Culture media for clinical bacteriology in low- and middle-income countries: challenges, best practices for preparation and recommendations for improved access. Clin Microbiol Infect. 2021 Oct;27(10):1400-1408. doi: 10.1016/j.cmi.2021.05.016. Epub 2021 May 18. PMID: 34015533.

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

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