Culture media play a pivotal role in any microbiology laboratory for isolation, identification and sensitivity testing of different pathogens. Most of the laboratories usually prepare their own media or purchase prepared media for routine diagnostics as well as research purposes.
However, to 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 mandatory.
Media performance is checked by inoculating standard strains and incubating it at the desired temperature. Whenever a new batch of media is prepared, 10% of samples should be projected to sterility testing.
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 the preparation of media, beyond having faith in the supplier. Even if the manufacturer provides a well-prepared media, the transport conditions, storage conditions until use may degrade the quality.
In house prepared media: Several factors should be considered while preparing media in-house (either from dehydrated powder or the components) such as
- Proper storage conditions (as instructed by the manufacturer) of media as well as its components. 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.
- De-ionized or distilled water should be used for preparation. Presence of copper ions, high conductivity and high pH may significantly alter the quality of in-house prepared media and needs to be pre-checked.
- Proper sterilization procedure should be carried out using a validated autoclave cycle (shown to hold the media at 121oC for 15 minutes). Temperature and pressure should also be constantly monitored. Heat labile substances such as growth additives, sugar solutions, glycerol should be sterilized separately using appropriate techniques such as filtration.
The volume of the media in one sterilization batch should be kept small; ideally, only two litres should be autoclaved at a time. Sterilization indicators such and Bowie Dick test and biological indicators such as spores of Bacillus stearothermophilus should be used to monitor the proper working/efficacy of the autoclave.
- The quality of glassware used for pouring media is also an important factor. Disposable Petri dishes or tubes should be ethylene oxide (EtO) sterilized or gamma-irradiated. If using re-usable 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.
- There are various additives used in the preparation of media such as blood, glycerol and growth promoters. The quality of the blood plays an important role in the performance of the blood containing media e.g. haemolytic reactions are well distinguished in sheep blood-containing media. The concentration, homogeneity, viscosity and colour of the blood should be checked before it is used for media preparation. For other additives, the certificate of analysis and sterility conditions should be considered.
Testing of Physical and Chemical Parameters: Physical parameters such as 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 pH of the medium should also be checked. pH testing can be done during media preparation either before or after autoclaving using a well-calibrated pH meter.
Growth and Sterility testing
Every media prepared, following stringent quality control parameters is sterile. At least 5-10% (5% if a small batch is prepared like 50 plates and 10% if a large batch is prepared 100-200 plates or tubes) of a new batch prepared should be incubated at 37°C for 18-24 hours to ascertain that there is no growth or contamination. If any colonies develop after incubation, the whole batch needs to be discarded.
After sterility testing, media are tested for supporting growth and giving desired reactions. For this purpose standard control strains of organisms are inoculated into the test medium and observed for typical reactions 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.
The results should be examined both qualitatively and quantitatively and while testing new lots, both previous batch and the new batch should be included.
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. Mostly, 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 haemolytic, grey colonies|
|5.||Streptococcus pneumoniae||Blood agar||Alpha haemolytic, 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 centre|
|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 green metallic sheen|
- 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.