Elaine Nichols, QA manager at Oxoid Ltd looks at sourcing, validating and maintaining the quality of culture media as part of the quality system for microbiological testing
Supplier selection should logically be regarded as the first stage of a multi-layered total quality system encompassing every aspect of microbial testing, and when faced with a choice of suppliers in respect of microbiological culture media, pharmaceuticals manufacturers need to weigh up their relative merits in terms of product quality, service and support.
The first thing to establish is whether or not a supplier is genuinely "pharma-focused". Not all culture media suppliers manufacture products specifically for the pharmaceutical industry and may, therefore, lack a fundamental appreciation of relevant working practices and regulatory requirements. When considering candidate suppliers, it is essential to assess their knowledge of the standards and guidelines that govern the production of pharmaceuticals. They should also be demonstratively proactive in terms of monitoring the introduction of relevant new regulations as and when they arise.
Next, manufacturers need to be sure that products purchased will perform consistently to an agreed specification - and this is where assessment of the manufacturer's own quality systems come into the equation. ISO 9001:2000 registration is widely considered to provide watertight evidence of the efficacy of an organisation's quality management, but it really only tells a part of the story as far as the manufacture of culture media is concerned. The ISO 9001:2000 standard can be applied to quality management across a broad spectrum of industry sectors. There is, however, a more precisely targeted quality standard relating to the production of culture media - ISO 13485:2003.
ISO 13485:2003 is based directly on ISO 9001:2000, but it is oriented specifically towards the design, development, production and installation of medical devices and related services. For example, Oxoid's scope includes "the research, development and manufacture of laboratory diagnostics for the detection, isolation and identification of micro-organisms", and its requirements are directly relevant to the exacting standards of the pharmaceutical industry. ISO 13485:2003 is a "process" rather than a "product" standard that not only encompasses the operation of a quality management system, but also focuses on compliance with relevant regulatory requirements. Crucially, it extends the "document, implement and maintain" imperatives in ISO 9001:2000 to encompass the meeting of specific customer or industry requirements, placing strong emphasis on the use of procedures to control and regulate the way in which specific processes and activities should be performed.
Having shortlisted an appropriately registered supplier, some form of audit should still be carried out in order to ascertain essential practical details about a supplier's processes and products and whether they will meet individual company requirements. For example, how are the raw materials for their culture media sourced? What documentation and certification is provided in this respect? What information is provided about the performance and shelf life of the culture media? What specific quality control procedures are in place?
Key criteria for the selection of culture media may differ substantially depending on the products being tested. For this reason, the standard written questionnaires normally used for assessment of suppliers in general may not be sufficiently focused to provide information that is meaningful. A site visit to suppliers" premises is normally of greater value, yielding considerably more qualitative information than the usual "tick box" responses to standard audit questions.
Of course, supplier selection should not be focused exclusively on product quality - customer service considerations are very relevant, too. Continuing availability of technical support in respect of culture media products is also an important consideration. In a sense, this is a continuation of the quality system pertaining to the product manufacturing process, since it enables customers to maintain their own quality standards by ensuring that media are prepared, stored and utilised correctly.
Having selected a supplier, the next step is for the pharmaceutical manufacturer to validate the supplier's products for use in specific applications in the context of the manufacturer's own QC. While the methods for microbiological QC found with the Pharmacopoeias can be regarded as validated in respect of the applications specified, it remains the responsibility of individual pharmaceutical companies to prove that these official methods function in their environment, for their products.
Ideally, the quality certificates issued by the chosen supplier in respect of their products should provide all the data required for validation purposes, including product formulation and details of QC tests which have been performed using specified reference organisms, complete with quantitative results. If certificates can readily be downloaded from the supplier's website in respect of individual product batches, so much the better. "Pharma-focused" culture media suppliers will recognise that internal validation of microbiological test methods can be extremely costly for their customers in terms of time, money and staff resources, and strive to provide as much assistance as possible in this respect.
Nevertheless, users of culture media must still evaluate the suitability of the manufacturer's QC esting in relation to their own requirements and acceptance criteria. Consideration must also be given to the any effects that arise from the conditions in which the products are stored. Prepared media must be stored at the correct temperature and in the dark. The main challenge of validation is to fulfil regulatory requirements and demonstrate fitness for purpose within financial targets set by management. The following considerations may be helpful in defining suitable testing regimes.
The first task in designing a validation strategy is to define precisely what you are trying to achieve. This may be:
- To demonstrate compliance with regulatory requirements
- To show that a product is microbiologically safe
- To confirm the capability of a process
- To confirm that a method is robust and easy to use
- To show that a culture medium is capable of recovering all target organisms from a specified type of sample
- To show that a medium is capable of recovering all likely contaminants
- To show that a medium can be relied upon to demonstrate that specified micro-organisms are absent
- To demonstrate that the medium is suitable for use in enumeration of micro-organisms in or on a specified type of sample
Your defined objectives may relate to several different aspects of your organisation's internal QC requirements, depending on the nature of the tests for which the culture media are going to be used. In each category of testing, a range of different considerations will apply. If the media are intended for use in water testing, for example, your validation strategy will need to take account of things such as the location, volume and frequency of sampling; sample transportation and storage conditions; the filtration method to be used; incubation methods and how the micro-organisms present will be counted or identified.
When the testing is concerned with raw materials, consideration needs to be given to issues such as the development of a sampling plan, the way in which you will demonstrate the satisfactory performance of the media and how validation samples will be spiked. Similar considerations apply to the use of tests to demonstrate microbiological control of the overall manufacturing process, encompassing environmental monitoring of air, surfaces and operator hygiene, in addition to the validation of equipment cleaning.
In all cases, the exact nature of the validation performed will depend upon the degree of sensitivity required. QC validation of microbiological methods should not be focused solely on the performance of a single culture media product, but should also encompass all the supporting microbiological tests involved.
Unless working with culture media that have been supplied in a ready-prepared format, quality assurance of a laboratory's testing processes must begin at the preparation stage. Incorrect preparation of dehydrated culture media can drastically affect their performance in terms of reduced growth and recovery rates, atypical colonial morphology and physical characteristics, inhibition of target organisms or failure to inhibit competing flora. Incorrect preparation of a medium can also reduce its shelf life. It is vital to follow the manufacturer's instructions.
Media should always be ordered in container sizes and quantities that are appropriate for the requirements of normal usage. A medium in a large container that has been opened many times will deteriorate on storage. Instructions provided by the manufacturer with regard to storage should always be followed, whether the requirement is for dry conditions below 30°C away from any heat sources, or for refrigeration at 2-8°C. It is also vital to check the expiry date on the label, because the shelf life of some media are significantly shorter than others.
Complete instructions for the preparation of dehydrated culture media should always be provided by the manufacturer, and followed to the letter. As a general rule, it is wise only to prepare a week's requirement in advance. If storage of media is required then shelf life validation should be carried out. Water for reconstitution should be prepared by distillation, deionisation or reverse osmosis. Toxic metal ions such as copper must be absent. If the pH of the water is below 5.5, it should be heated to drive off CO2 and then re-checked. The conductivity of the water should ideally be below 15 micro siemens (µS).
Ideally, media should always be prepared in a vessel about twice the final volume of the prepared product to allow for adequate mixing. Glassware should always be rinsed before use. First of all, half the required volume of water should be poured into the vessel, followed by the weighed quantity of dehydrated medium. After agitating briskly for a few minutes, the rest of the water should then be poured down the sides of the vessel to wash any adherent medium back into solution. This is an important step, because dry culture media powder above the level of the water may not be sterilised in the autoclave and could potentially be a source of contamination.
Media containing agar should be heated to dissolve the agar before autoclaving. Most culture media will require final sterilisation in an autoclave at 121°C for 15 minutes.
When boiling or steaming culture media, care must always be taken not to over-heat or burn them. If media preparators or autoclaves are used for sterilisation purposes, it is important to ensure that heat-up and cool-down times are not excessive. To facilitate this, some autoclaves are now available with automatic "F0 modules" that allow the total heat to which a medium is exposed during sterilisation to be measured. The "F0" function is a useful statistical tool that can be used to compare the same sterilisation processes in different autoclaves.*
Difficulties frequently arise when a medium is overheated, leading to problems with chemo-oxidation. This can cause peroxides and superoxides to be formed and the medium may become toxic, resulting in reduced microbial growth and selectivity. Visible precipitation of the medium, atypical colouration, low pH and low gel strength may also occur.
When culture media are purchased pre-prepared, they should still be subjected to validation testing to ensure that their performance conforms to the required specification. In some laboratories, full QC testing is initially carried out on three batches of each prepared medium purchased. In the majority of cases, if the results are satisfactory, incoming QC is either removed or reduced. The frequency of full QC testing of incoming prepared media following validation is a matter for individual laboratories to determine, but the checks should encompass pH value, sterility, growth performance and stability testing.
Guidance on the selection of microbial cultures for use in validation tests is contained in the European and United States Pharmacopoeias, but there are several key considerations to bear in mind. Firstly, when spiking validation samples, it is important to ensure that the levels of contamination reflect those likely to be encountered in routine testing. The rigour of testing may be strengthened by inclusion of atypical strains, sub-lethally injured cells and competitor organisms that might give rise to false positives. If your laboratory already has its own culture collection of organisms that have been isolated from products or environmental samples in the past, these are worthy of inclusion, since the chances of them being encountered routinely are high.
The selection and validation of microbiological culture media is a crucial activity for laboratories, since conclusions drawn from the use of these products have a direct impact on quality standards in pharmaceuticals production - and, ultimately, on people's lives. Adoption of a total systems approach is the secret of success.