Part of the fundamentals and standardization pillar of the TAAG Learning Center. It develops a point we mention in ISO/IEC 17025 accreditation and builds on traditional microbiological culture.
Table of contents
The culture medium is also tested
When a laboratory reports "Salmonella: not detected" or an Enterobacteriaceae count, it takes for granted that the medium it plated on worked. If a batch of selective agar also inhibits the target microorganism, or lets through the background flora it was supposed to suppress, the result is wrong even if the analyst did everything right. That is why food microbiology has a standard devoted exclusively to media: ISO 11133.
Its introduction sums up the goal in three ideas: to demonstrate that each batch of medium is acceptable, that the medium is fit for its intended use and that it produces consistent results. According to the standard itself, they are an essential part of the laboratory's internal quality control.
What ISO 11133 is and who it applies to
The current edition, ISO 11133:2014, replaced the technical specifications ISO/TS 11133-1:2009 and ISO/TS 11133-2:2003, and added media for water testing. It defines the terms of media quality assurance, sets requirements for preparing them and describes methods and criteria to evaluate their performance. It covers the media used to test food, animal feed, production environment samples and water for consumption or for use in food preparation.
It applies to those who manufacture or distribute dehydrated or ready-to-use media, to non-commercial bodies that supply them to third parties and to laboratories that prepare their own media. Amendment 1 of 2018 expressly added end users of ready-to-use media: buying prepared plates does not exempt the laboratory from checking them.
Two amendments and a new edition on the way
- Amendment 1 (2018): corrections and clarifications, including the inoculum levels for performance tests.
- Amendment 2 (2020): added Annex K, with the control strains for confirmation and characterization media and reagents.
- Second edition: the ISO/FDIS 11133 project, which will be renamed "culture media and reagents", is in the final voting stage at ISO and will replace the 2014 edition when it is published.
The ISO/TC 34/SC 9 committee also clarifies that microorganism-specific standards published after 2014 that include their own performance testing clause prevail over what ISO 11133:2014 indicates for those media.
The three properties that are evaluated
Productivity
This is the recovery level of the target microorganism on the medium under defined conditions. It answers the most basic question: if the microorganism is there, does the medium let it grow?
Selectivity
This is the degree of inhibition of a non-target microorganism on a selective medium. An agar for Salmonella that lets Escherichia coli grow freely produces saturated plates where the typical colonies are lost.
Specificity (electivity)
This is the demonstration that non-target microorganisms, if they do grow, do not show the same visual characteristics as the target: the color, halo or morphology the analyst uses to recognize a suspect colony. In chromogenic media it is the property that prevents one colony from being mistaken for another.
How they are tested
Quantitative test and productivity ratio
For solid enumeration media, the standard compares growth on the test medium with that on a reference medium, normally non-selective, such as tryptone soy agar (TSA). The result is the productivity ratio, PR = NS/N0: the count on the test medium divided by the count on the reference medium. Amendment 1 sets the inoculum at about 100 CFU to achieve sufficient precision, with a practical range of 80 to 120 CFU per plate and a minimum of 50.
The usual criterion is a PR of at least 0.5 for selective media and at least 0.7 for non-selective media. A concrete example from the standard: TBX agar for β-glucuronidase-positive E. coli is tested with E. coli strains, compared with TSA and must give PR ≥ 0.5 with blue colonies.
Qualitative tests
In liquid media and in some solid ones, the result is expressed on a simple scale: 0 (no growth or no turbidity), 1 (weak growth) and 2 (good growth). For selectivity, Amendment 1 requires inoculating at least 104 CFU of the non-target microorganism; for specificity on plates, at least 103 CFU.
Selective enrichment broths
Broths such as RVS for Salmonella are tested by inoculating the target, the non-target or a mixture of both, and then streaking onto an isolation medium (for example, XLD) and onto a non-selective medium. The selectivity of the broth is considered satisfactory if there is no growth of the non-target on the non-selective plate, or fewer than 10 or fewer than 100 CFU, as the relevant annex or the specific standard indicates. It is the same principle that decides the success of microbiological enrichment in a real sample.
Multipurpose and confirmation media
For a medium with several uses, such as buffered peptone water (BPW), Amendment 1 asks for at least one pre-enrichment test with a pathogen relevant to the laboratory, for example Salmonella. For confirmation media and reagents (Annex K), the test is qualitative because it starts from an isolated colony: the positive control is incubated for the minimum time allowed by the standard and the negative for the maximum. This verifies that the positive appears on time and that the negative does not react because of over-incubation. The confirmation of suspect colonies is explained in the article on presumptive and confirmed results.
Control strains: why the WDCM number matters
A performance test is only comparable between laboratories if everyone uses equivalent strains. ISO 11133 defines a precise chain:
- Reference strain: obtained directly from a collection that is a member of the World Federation for Culture Collections (WFCC) or the European organization ECCO, identified at least to genus and species level.
- Reference stock: identical cultures obtained by a single subculture of the reference strain.
- Stock culture: primary subculture of the reference stock.
- Working culture: the one used in the test, obtained from the reference stock, the stock culture or a reference material.
Each collection numbers its strains in its own way, so the same strain has different codes in each catalog. The World Data Centre for Microorganisms (WDCM) solved that problem with a unique identifier: the WDCM catalog of reference strains. For example, WDCM 00031 corresponds to Salmonella Typhimurium NCTC 12023 in the British collection.
An example from the official list
The ISO/TC 34/SC 9 committee publishes a list of control strains for each medium and reagent in its standards; the fourth edition dates from May 2025. For XLD agar in ISO 6579-1, the list indicates Salmonella Enteritidis WDCM 00030 or Typhimurium WDCM 00031, E. coli WDCM 00012 or 00013 and Enterococcus faecalis WDCM 00009 or 00087. A note warns that some national regulations may require another Salmonella serovar, and another marks with the letter "b" the strains that must be used as a minimum.
Beyond the microbiological test
Control of a medium starts before plating. ISO 11133 requires the manufacturer to document, among other data, the name and components of the medium, the technical data sheet, the batch number, the target pH of the complete medium, the storage conditions and the expiry date. A laboratory that prepares its own media must also take care of the water, weighing, sterilization, final pH and storage; ISO 7218:2024 completes these general requirements for the microbiology laboratory.
All of this fits within ISO/IEC 17025, which requires ensuring the suitability of externally provided products and monitoring the validity of results. A batch that fails the control is nonconforming work: it is necessary to assess which analyses may have been affected, as discussed in the article on ISO/IEC 17025 accreditation. In molecular methods the logic is the same, with their own controls, which are explained in the article on PCR controls.
Common mistakes
- Accepting the supplier's certificate as the only control, without verifying the batch in the laboratory.
- Using subcultured collection strains without records, unable to trace their origin back to the reference strain.
- Testing productivity with very high inocula, which hide a partial inhibition of the medium.
- Controlling productivity and forgetting selectivity or specificity in selective and chromogenic media.
- Not taking into account that the microorganism-specific standard may set its own strains and criteria that prevail over ISO 11133.
Conclusion
A culture medium is a reagent with measurable performance, not a neutral supply. ISO 11133 sets out how to demonstrate it: productivity against a reference medium, selectivity with high inocula of non-target organisms, specificity in differential media and control strains traceable through WDCM numbers. Checking each batch, recording the chain of subcultures and reviewing what the standard for each microorganism requires prevents a result from depending on a defective batch without anyone knowing.
About TAAG
Find out how TAAG complements the work of the culture laboratory with molecular detection kits that incorporate their own reaction controls.
Frequently asked questions
What is ISO 11133?
It is the international standard that defines how to prepare, produce, store and control the performance of the culture media used in the microbiology of food, animal feed, production environment and water. The current edition dates from 2014, with amendments from 2018 and 2020.
What is the difference between productivity, selectivity and specificity of a medium?
Productivity measures how much of the target microorganism the medium recovers. Selectivity measures how much it inhibits non-target ones. Specificity shows that the non-target organisms that do grow cannot be visually confused with the target.
What is a WDCM number?
It is a unique identifier assigned by the World Data Centre for Microorganisms to the strains recommended for quality control. It makes it possible to know that strains with different codes in different collections are, in fact, the same one.
Does a laboratory that buys ready-to-use plates have to check them?
Yes. Amendment 1 of 2018 expressly included end users of ready-to-use media in the scope of ISO 11133, and ISO/IEC 17025 requires verifying the suitability of externally provided supplies.