Preserving reference cultures for QC and compliance

Reference cultures play an integral part in the procedures that are involved in effective cleaning, disinfection and environmental monitoring of production plants. One of the most important elements of maintaining any collection of reference cultures is ensuring the genetic stability of the strains within it; working cultures should therefore be no more than five generations removed from the original reference culture, and need a system for preserving and maintaining reference cultures with their original characteristics

Reference cultures are freeze-dried in glass ampoules and sealed off under vacuum

Reference cultures and plant isolates have an important role in disinfectant testing and selection, validation protocols and environmental monitoring. NCIMB’s CEO Dr Carol Phillips looks at best practices for maintaining and preserving them.

Effective cleaning, disinfection and environmental monitoring of production plants are essential processes for manufacturers from a range of industry sectors and reference cultures play an integral part in the procedures that are involved in these processes. They are used for the preparation of quality control (QC) cultures used to test the efficacy of disinfectants and for QC of media used in environmental monitoring (EM) programmes, as well as providing a resource for tracing sources of contamination. Their vital role in ensuring the quality of products makes it important that the production, maintenance and preservation of reference strains are carried out to a high standard.

QC cultures

It is obviously key that culture media used in plant EM programmes are capable of consistently supporting growth of micro-organisms. For example, with respect to the EM of cleanrooms in vaccine manufacturing facilities, the World Health Organisation (WHO) states that the growth promotion properties of media used in EM programmes should be tested for a predefined list of organisms.1 This list should include a minimum of five unique microbial strains and the QC cultures used should be traceable to a recognised culture collection such as the UK’s National Collection of Industrial, Food and Marine Bacteria (NCIMB), the National Collection of Type Cultures, a culture collection of Public Health England (NCTC), or the American Type Culture Collection (ATCC).

Some labs may prepare QC cultures from reference strains for their own in-house use. One of the most important elements of maintaining any collection of reference cultures is ensuring the genetic stability of the strains within it, and continuous sub-culturing will generally give rise to a degree of genetic drift that will ultimately render the cultures unsuitable for use as QC strains. Consequently, it is good practice to ensure that working cultures are no more than five generations removed from the original reference culture or isolate, and to have a system for preserving and maintaining reference cultures with their original characteristics.

To this end, there should be documented procedures in place to ensure that each working culture can be traced back to the master culture from which it was derived. It is also important to ensure that working cultures are not used as replacements for depleted stock or master cultures.

Glycerol stock can be prepared for long-term low temperature storage and the batch used to prepare this stock should be checked for purity. Cultures for media testing can be prepared from these glycerol stocks. To reduce the opportunity for contamination, ideally the vial containing the glycerol should be discarded after use and the prepared inoculum should be tested for purity.

As the preparation of QC cultures is a labour intensive process, many companies prefer to buy ready prepared QC strains from licensed derivative manufacturers, and these are cost effective and easy to store. They also have advantages in terms of the wide range of easy-to-use formats that have been developed – screw top vials, water soluble discs and plastic snap vial formats, such as MicroSnap, which have a lower chamber containing freeze-dried culture and an upper chamber with resuscitation media and attached swab.

A source of reference cultures

The strains used for QC purposes generally represent a small proportion of the total number of strains held by culture collections. These collections play an important role in harnessing, describing and preserving microbial diversity for future generations of scientists. NCIMB is continuously expanding its collection through the addition of new accessions and the acquisition of private collections that might otherwise be lost – for example, if a key university researcher retires or a particular area of work within a company stops.

Historical culture collection records

When a new strain is deposited in the NCIMB collection, it undergoes a number of identification and purity checks, including sequencing of the 16s rDNA gene. 16s rDNA sequences are searched against the validated MicroSeq database and the public EMBL database for comparison. Detailed records are kept that include information on who the depositor was, where the strain was isolated from, general strain characteristics, the most suitable preservation methods and information on cultivation. Sequence information is also retained for future comparisons.

Umbrella organisations, such as the World Federation of Culture Collections (WFCC) and Common Access to Biological Resources and Information (CABRI), produce guide-lines for culture collections that include aspects such as handling and maintenance. The guidelines recommend that deposits in culture collections should be maintained by at least two different methods, at least one of which should be a metabolically inactive method, to minimise the risk of genetic change.

At NCIMB most of our deposits are preserved by freeze drying along with a variety of other methods as some species are better suited to individual preservation methods than others. While QC cultures are generally supplied with a limited shelf life, e.g. two years, reference cultures need to be preserved for much longer. Therefore, once freeze-dried, the reference cultures are preserved in a vacuum to maintain an inert environment and each ampoule is tested to ensure this is achieved.

Preserved cultures from the 1920s beside present day freeze dried reference cultures

NCIMB also has a programme to check the viability of deposits. This is an ongoing process that requires detailed record keeping, and the variation in the response of individual species to preservation methods means that some need to be checked more frequently than others.

Another key element of the guidelines for culture collections, which aims to minimise the risks to these important genetic resources, is to have an offsite backup, at least of the most important strains and their associated documentation, and NCIMB complies with this. This is something that any lab maintaining a collection of stock cultures should consider and culture collections, such as NCIMB, will often offer this kind of service.

No matter how well a cleanroom functions, there is potential for the introduction of contaminants. The EM programme is, therefore, an important element in cleanroom management. It provides information on the quality of the manufacturing environment; it enables identification of any potential sources of contamination; and it allows decisions to be made on how to deal with them.

Approaches used for EM have developed and evolved over the years, but the identification and documentation of the micro-organisms isolated remains an essential element of any sampling methodology. Manufacturers must be able to prove that their microbial media are suitable to recover environmental contaminants consistently and it is also important that disinfectants are shown to be effective against the facility microbial flora as well as QC cultures.

Therefore EM programmes should ideally include identification of micro-organisms found to species level and involve the establishment of a database of microbes present in the facility. Environmental isolates often correlate with contaminants, therefore this kind of detailed information about the overall environment can provide valuable information for any investigation. For example, it may highlight trends with respect to migration of micro-organisms from one part of the plant to another.

Identification to strain level can also be useful, especially when it comes to comparing contaminants and tracing where they might have come from. For strain identification genotypic analysis is often used – including the MLST technique, which uses sequence analysis of internal fragments of up to seven essential housekeeping genes that are examined against a public database.

Once a contaminant has been identified to the strain level, the detailed culture collection records – including information such as where the strain was isolated from – can be used to help work out the source of contamination. Having strain information can also help to determine whether repeat problems with the same species have come from the same source.

NCIMB also has an extensive collection of specific media recipes that can be used to optimise the use of reference strains.

Once an environmental isolate has been incorporated into a company’s QC procedure, the type strain of that species could also be used alongside it.

Maintaining in-house strains

Laboratories undertaking EM programmes should maintain an in-house collection of their own environmental isolates. Records should be kept for each culture, including the identity, source, date of isolation; media requirements, preservation, maintenance and storage conditions for the master, stock and working cultures; results of purity and identification tests and dates of preparation of stock and working cultures.

Checking for a vacuum in a freeze dried ampoule

To minimise the risk of losing strains in the event of equipment malfunction, for example, companies can follow similar guidelines to those set out for culture collections – using two methods of preservation and having an off-site back-up storage facility for strains. Freeze-drying can be outsourced if in-house facilities and expertise are not available and this is probably the case for most smaller labs.

The whole process of using environmental isolates for QC purposes can also be streamlined by having them prepared as bespoke QC cultures in one of the formats that have been developed to increase ease of use and minimise the risk of contamination during resuscitation.


1. Environmental Monitoring of Clean Rooms in Vaccine Manufacturing Facilities. Points to consider for manufacturers of human vaccines, Nov 2012 VQR, Quality, QSS, EMP Department, WHO, Geneva, Switzerland