Environmental Monitoring Microbiological Considerations And Controls

Tryptone Soy Agar (TSA) is the standard medium for microbial recovery in environmental monitoring programs. Incubation is at 30-35°C. Yeasts and moulds may also be specifically sought out. Sabouraud Agar (SA) incubated at 20-25°C is generally used for this purpose. Media used in antibiotic manufacturing facilities, particularly when they are solid dosage forms, should be reviewed for their capability to recover environmental microorganisms. Neutralizers may have to be included in the preparation of the media. ß-lactamase is included in media used in environmental monitoring programs for facilities manufacturing penicillins and cephalosporins.

The duration of incubation generally recommended is 48 to 72 hours. This is curious because it means that samples taken on Thursdays (as they surely must in at least some weeks) must be read on Saturday or Sunday, when most laboratories are not routinely staffed. Since the upper time limit on incubation is largely arbitrary, it makes more sense to specify incubation to be 48 to 96 hours.

The occurrence of spreading forms that can obscure other growths after lengthy incubation on agar is fairly unusual in pharmaceutical manufacturing environments.

There is a regulatory enthusiasm for manufacturers to include some consideration of anaerobic microorganisms in environmental monitoring programs, usually by incubating TSA in anaerobic conditions. Other media more suited to the recovery of anaerobes may be used.

Anaerobic environmental monitoring may be seen as fulfilment of a regulatory requirement. Obligate anaerobes are intrinsically unlikely to be present in most pharmaceutical manufacturing environments; oxygen is toxic to obligate anaerobes. Pharmaceutical clean rooms are continuously swept by filtered air, and surfaces are smooth and clean; consequently there should be few opportunities for anaerobes to survive, even fewer for them to be recovered by active or passive air sampling, or on contact plates.

In practical terms, pharmaceutical clean rooms should be "brainstormed" to determine any locations where anaerobic microorganisms may survive, e.g., where there is grease, or in oil sumps. These locations should preferably be engineered out of the clean room, but if this is impossible, they should be the focus of anaerobic environmental monitoring by swabbing.

Conversely, microaerophilic organisms (e.g., Propionibacterium spp.) are rarely isolated in routine environmental programs but are not unknown as sterility test contaminants on those rare occasions when sterility tests "fail." Swab enrichment in fluid thioglycollate medium enables an evaluation of the presence of these common human commensals in the manufacturing environment.

The media used for environmental monitoring should be demonstrably able to support the growth of microorganisms (pharmacopoeial types and local environmental isolates as indicated for media fills). Although it is not absolutely necessary that growth support checks should always be carried out on every autoclaved batch of media, every supplier's batch of dehydrated media should certainly be checked. Some FDA investigators insist that every autoclaved batch of environmental monitoring media is tested for growth support.

Environmental control media should be validated for their ability to support growth throughout their shelf lives. Agars are often prepared, sterilized and stored for days or weeks before melting and pouring as environmental monitoring plates.

Environmental media must be preincubated for sterility before they are taken into Grade A and Grade B aseptic areas. There are two reasons for this.

First, it is quite absurd, when faced with the very strict microbiological limits usually applied to these areas, to risk reacting to incidental contamination arising from media preparation.

Second, aseptic areas must not be compromised by taking contaminated agars into them. This means that considerable care should be taken to ensure that environmental plates are not cross-contaminated in microbiologically contaminated incubators. Plates should be poured and double-bagged in plastic in Grade A laminar air flow-protected areas, before preincubation. Where agar plates have to be dried, this should be done in the Grade A pouring areas prior to bagging.

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