Culture media are pivotal in any microbiology laboratory for isolating, identifying, and sensitivity testing different pathogens. Most laboratories usually prepare culture media or purchase ready-to-use media from commercial suppliers for routine diagnostics and research purposes.
Ensure that the media is of good quality and capable of giving satisfactory results; different parameters such as growth-supporting characteristics, physical characteristics, and sterility testing are needed before using them.
Media performance is checked by inoculating standard strains and incubating them at the desired temperature. Whenever a new batch of media is prepared, 10% of samples should be projected to sterility testing.
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Preparation of Media
Ready-to-use media: If ready-to-use media plates/tubes are directly purchased from vendors, there is little opportunity to control media preparation beyond having faith in the supplier. Even if the manufacturer provides a well-prepared media, the transport, and storage conditions until use may degrade the quality.
In-house prepared media: Several factors should be considered while preparing media in-house.
Testing of Physical and Chemical Parameters
Physical parameters such as the color of the prepared medium, thickness, formation of excessive bubbles, uneven surface, shrinking of the media, cracks or crystallization in the media, and chemical parameters such as the pH of the medium should also be checked.
Perform visual checks to rule out the following anomalies;
- Agar detached from the plates
- Cracked or damaged plates
- Frozen or melted agar
- Changes in the expected color of the media
- Obvious contamination
- Unequal filling of the plates Insufficient amount of agar (<3 mm)
(Note: For Mueller-Hinton agar, the agar depth should be 4± 0.5 mm)
- Hemolysis of blood-containing media
- Excessive bubbles or rough surfaces
- Excessive moisture or dehydration
- Presence of precipitates
- The integrity of the packaging
- Presence of broken or cracked Petri plates or tubes
- Presence of leakage from the Petri plates or tubes
- Accuracy of the labeling
Testing the pH of Medium
The pH of most culture media is near neutral (an exception is alkaline peptone water). The simplest way of testing the pH of a culture medium is to use narrow-range pH papers or a pH meter. pH testing can be done during media preparation either before or after autoclaving using a well-calibrated pH meter.
Measuring pH of Broth
- Dip a narrow-range pH paper into a sample of the test medium at room temperature.
- Compare the color of the paper against the pH color chart.
Measuring pH of Agar
- To test the pH of an agar medium, pour a sample of the molten medium into a Petri plate and let it solidify.
- Lay a narrow range pH paper on its surface.
- Compare the color of the paper against the pH color chart.
Adjustment of pH
In most cases, adjustment of the pH won’t be needed. Minor pH adjustments can be made by using
- 0.1 mol/L sodium hydroxide when the medium is too acidic, and
- 0.1 mol/L hydrochloric acid when the medium is too alkaline.
- Use 1 mol/L (1N) sodium hydroxide to adjust the pH of alkaline peptone water.
Every media prepared, following stringent quality control parameters, is sterile. Incubate in-house prepared culture media for 48 hours at 35-37°C (varying from 24 h to 5 days, depending on the reference). For the batch with <100 units, a 2% sample must be subjected to sterility testing, but for the batch with >100 units, ten random units are sufficient to ascertain no growth or contamination.
If growth is seen after incubation (i.e., contamination), reject or discard the whole batch and prepare a new one. Do not re-use the plates used for sterility check. Note that an additional visual check should always be done before use.
After sterility testing, test the media for supporting growth and giving desired reactions. Use a standardized suspension of QC strains to inoculate the culture media.
Check and record growth (colony size and morphology), selectivity, and differentiation:
- Support of target organism: Use at least one organism, and record growth and biochemical reaction. e.g., If a strain of Escherichia coli ATCC 25922 is inoculated into a sterile MacConkey agar plate, after proper incubation, it should show lactose fermenting pink colonies.
- Selective media: Use at least one organism that is expected to grow and at least one organism that is not expected to grow
- Differential media: Use organisms that will display the intended growth and reactions (e.g., MacConkey agar: Shigella flexneri, Escherichia coli, and Staphylococcus aureus)
- Biochemical media (e.g., urease-test): Use at least one organism that will produce a negative reaction and one that will produce a positive reaction.
The results should be examined both qualitatively and quantitatively, and while testing new lots, both previous and new batches should be included. Perform disc diffusion to check Mueller-Hinton agar plates.
Use of QC Strains
Choose appropriate QC organisms based on standards, guidelines, or manufacturer’s instructions. Type culture collection organisms (e.g., ATCC) are recommended, but previously characterized clinical organisms or EQA strains shown to be phenotypically stable (documentation required) are also accepted. QC organisms should be correctly maintained and stored (refer to CLSI M22-A3).
A list of quality control (QC) strains to be tested against routinely used culture media is as follows. However, one strain can be inoculated on various other media. Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, and Pseudomonas aeruginosa ATCC 27853 are used as cumulative control for testing all media.
|S.no||Quality control strain||Media||Expected output|
|1.||Escherichia coli (ATCC® 25922™)||MacConkey agar||Lactose fermenting pink colonies|
|2.||Acinetobacter baumanii (ATCC® 19606™)||MacConkey agar||Non- Lactose fermenting pale colonies|
|3.||Staphylococcus aureus (ATCC® 25923™)||Mannitol Salt agar||Golden Yellow colonies|
|4 .||Streptococcus pyogenes (ATCC® 19615™)||Blood agar||Beta hemolytic, grey colonies|
|5.||Streptococcus pneumoniae||Blood agar||Alpha hemolytic, small greenish colonies|
|6.||Shigella sonnei (ATCC® 25931™)||XLD agar||Red pink colonies|
|7.||Salmonella enterica subsp. enterica (ATCC® 14028™)||XLD agar||Red pink colonies with black center|
|8.||Pseudomonas aeruginosa (ATCC® 27853™)||Mueller hinton Agar||Flat serrated greenish colonies|
|9.||Neisseria gonorrhoeae (ATCC® 49226™)||Thayer martin agar||Water droplet-like colonies|
|10.||Enterococcus faecalis (ATCC® 29212™)||Bile esculin agar||Small transparent colonies with brown-black halos.|
|11.||Escherichia coli (ATCC® 25922™)||EMB agar||Blue-black bull’s eye-like colonies with a green metallic sheen|
References and further readings
- Basu S, Pal A, Desai PK (2005) Quality control of culture media in a microbiology laboratory, Indian journal of medical microbiology,23:3;159-163
- ATCC: Quality control strains used in microbiology culture medium
- Quality Control of Microbiological Culture Media by Scott Sutton, Ph.D.
- 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.