ENVIRONMENT MONITORING BY SETTLE PLATES IN ASEPTIC FACILITY
Introduction:- Settle plate sampling is a direct method of assessing the likely number of microorganisms depositing onto the product or surface in a given time. It is based on the fact that, in the absence of any kind of influence, airborne microorganisms, typically attached to larger particles, will deposit onto open culture plates. Microorganisms are usually found in the air of occupied rooms rafted onto skin cells with very few present on their own. The average size of microbial particle will deposit, by gravity, onto surfaces at a rate of approximately 1 cm/s. In settle plate sampling Petri dishes containing agar medium are opened and exposed for a given period of time, thus allowing microbe-bearing particles to deposit onto them. Petri dishes which are 90 mm in diameter (approximate internal area 64 cm2) are most commonly used. The number of microbe bearing particles deposited onto the agar surface of the plate over the period of exposure is ascertained by incubation of the plate and counting the number of microbial colonies, more commonly known as colony forming units (cfu). The microbial deposition rate may be reported as the number depositing in a given area per unit time.
Sample equipment:- Solid growth media (e.g. settle and contact plates), prepared according to a recognised formulation. It may be preferable however, if materials are purchased from a commercial manufacturer since they will be manufactured to a suitable consistency and are available in a variety of formats (e.g. irradiated). Commercially available materials are batch prepared to an appropriate specification and quality controlled to test the growth capabilities of the media. Agar strips specific to certain test devices should be purchased from the manufacturer. Growth media should also be demonstrated as ‘growth supporting’ for the time and conditions under which exposure occurs. The quality of test materials should be checked thoroughly prior to using them. Spurious high counts may arise from the use of inadequately controlled microbiological test materials. Consideration should be given to purchasing irradiated materials for use in critical zones. Irradiated materials need not be pre-incubated and may be safely transferred to the critical zones before opening. If materials are obtained that are not irradiated from a commercial source then consideration should be given to pre-incubating to check media sterility prior to use. It is preferable to use a growth medium with low selectivity i.e. capable of supporting a broad spectrum of microorganisms including aerobes, anaerobes, fungi, yeast and moulds. The media types listed below have been found to be suitable:
Tryptone Soya Agar (TSA)
Columbia agar with horse/sheep blood
The recommended size of solid media is 90 mm in diameter (approximate internal area 64 cm²) for settle plates and 55 mm (surface area 25 cm²) for contact plates. The media may be modified and contain neutralising agents to inactivate residual surface disinfectant present on the surface to be tested. Other media may be used provided the growth supporting capability of the media has been tested.
Sample locations:- Areas where a microbiologically controlled environment is specified should be monitored. An assessment of the controlled environment should identify the processes taking place and all potential microbiological hazards, which may be expected to occur or to be introduced. The critical work zone of clean air devices should be monitored as these are the areas where previously sterilised materials are brought together and manipulated (processed) into finished dosage forms. Sample locations of plates in the critical work zone should be selected with reference to the actual work area and the position of filters. Monitoring critical areas should be carried out under “worst case” conditions for contamination with process equipment running and personnel performing normal operations. It is recommended that 2 settle plates are exposed for the whole working session. Sample locations for settle plates in clean rooms should include areas where there is little air movement (i.e. “dead spaces”) or where airflows converge or are excessively turbulent. Areas where these conditions are most likely to occur are:
- adjacent to doors
- in pass through hatches
- at low level return air grilles
- between HEPA’s in clean rooms
- in corners of rooms
Areas within the clean room where there is personnel activity or specific operations are carried out should also be subject to monitoring e.g. adjacent to bench areas where trays that have been passed into the clean room are held before being transferred into a clean air device.
Frequency of sampling:-
(a) At least sessionally – at all test sites within unidirectional airflow cabinet (UAFC)/isolator and transfer devices as practicable.
(b) Weekly – at all test sites in background environment and change facilities. Sampling in the ‘at rest’ condition may be continued at an agreed frequency to monitor baseline contamination levels.
Method of sampling:- The following specimen procedure for use and exposure of settle plates may be adopted:-
- Examine the plates for contamination prior to use.
- Assemble the plates required and ensure that the correct information is written on the base of the plate (the part containing the media) with ink or other marker. Do not mark the lid of the plate, as there is always a possibility of lids coming off and being replaced on the incorrect sample plate. The following details may be marked on each plate or recorded separately:
- operator who collected sample
- date and time of day sample taken
- area/location of sample
- position/sample number
- Transfer the plates into the area/room/cabinet where they are to be exposed as outlined in the appropriate transfer procedure.
- Enter the area to be tested by the appropriate procedure, if required.
- Place the plates in the appropriate positions with the lids still on.
- Raise lids to expose the surface of the medium, rest the lid on the very edge of the plate so that the entire agar surface is completely exposed. Take care not to put fingers on plates. Avoid passing anything over the top of plates being exposed, where possible.
- Leave plates exposed for the full work session. The exposure time should be recorded before sending the plates for incubation.
- After exposure:
- Replace lids of plates.
- Swab areas where plates have been exposed with a suitable disinfectant (e.g. sterile IMS 70% solution) to remove any trace of media or condensation from the lids, which may contaminate the clean room.
- Remove from area/room/cabinet
- Collect all plates exposed, and return to QA or microbiology for incubation. Ensure plates are secured in a suitable container.
- Complete and enclose the necessary documentation.
The SOP should also include:
- Outline process of how settle plates are obtained for your hospital or a brief statement on where they are obtained or collected from.
- Settle plates for later use should be stored in the recommended storage conditions with the medium uppermost. If refrigerated, the plates should be removed from the refrigerator half an hour before they are due to be exposed. Care should be taken to ensure that plates are used before the expiry date on the label. Plates should not be used after this date.
- Outline details of how settle plates are returned to the Microbiology department, where used. Details of transport and any other arrangements should be described. Also outline method of destruction of exposed plates with/without growth.
Incubation conditions:- Following testing the samples should be incubated as soon as possible (within 24 hours of sampling, same day is preferred) and should be held at room temperature with the medium uppermost until incubated or manipulated. If the medium is dropped or touched by an operator then this should be reported, the sample should be marked accordingly and treated as usual. Under no circumstances should samples that have been taken be refrigerated.
Incubation of samples, inverted, at 30 – 35°C for at least 2 days is suitable for the growth of bacteria. Incubation of samples, inverted, at 20 – 25°C for at least 5 days is suitable for the growth of mould and fungi. Other incubation conditions may be used if it can be shown that the conditions promote the growth of (all) microorganisms that may have been recovered during the sampling procedure. Incubation conditions should be monitored to ensure that the appropriate incubation temperature is maintained throughout the incubation phase.
Results and reading of samples:- After appropriate incubation microbiological contamination should grow into discrete macroscopic colonies that can be enumerated and the number of discrete colony forming units (cfu) can be counted on each sample. Record the number (per unit surface area) on the appropriate report. Separate colony counts may be tabulated for mould and bacteria. Colony types may be identified if this is considered appropriate. If cfu are not discrete (coincidence) entities or are Too Numerous To Count (TNTC – usually greater than 300 cfu per sample), record the result as TNTC. If one type of cfu tends to grow in a spreading manner, count this as “one spreading colony” and record it as such. All samples (contaminated or not) should be disposed of according to local procedures.
Note: For plates used in the single sieve to agar sampler, it is necessary to correct the cfu reading for the statistical possibility of multiple particles passing through the same hole. A correction table is provided in the operating manual for the sampler.
The following details should be recorded:
- Sample location.
- Date sample taken (length of time plate exposed, if appropriate, for settle plates).
- Number of colony forming units (cfu) per sample.
- Batch number and expiry of media.
- Operator responsible for exposure of samples.
- Operation being undertaken in cleanroom.
- Operator reading the result and date read.
- Person reviewing/approving/accepting results (Responsible Pharmacist/Quality Controller)
It is important that there is knowledge of the ‘normal’ background flora of a cleanroom facility and therefore a suitably qualified person should identify organisms. Any unusual organisms or deviation from ‘normal’ flora may require action.
(a) Limits for background environment for negative pressure isolators with type A, B, C1 transfer devices or background environment for positive pressure isolators with type A transfer devices.
(b) Limits for isolators to be sited in a grade D background or the background environment for negative pressure isolators with type C2, D, E, F transfer devices or the background environment for positive pressure isolators with type B, C1, D, E, F transfer devices.
Note 1: Values shown are average values. Individual settle plates may be exposed for less than 4 hours. If the settle plates are exposed for less than 4 hours, the action levels should be adjusted accordingly.
Action to be taken in the event of a result being on or over the action level:- Exceeding action levels on isolated occasions may not require more action than examination of control systems. However, the frequency of exceeding the limit should be examined and should be low. If the frequency is high or shows an upward trend then action should be taken which may include an increase in frequency of testing, observation of operator technique or investigation of cleaning procedures. Identification of the causative microorganism(s) may aid tracing the source of the contamination. Successive results greater than the action levels demand that appropriate action be taken to reduce contamination to within limits. Where a problem has been observed the contaminating microorganisms should be identified. Isolated instances require no more action than examination of control systems. If repeated contamination appears an investigation into the problem should take place and corrective action should be carried out which will rectify the problem. The corrective action should investigate the root cause of the problem and identify steps, with time scale, that will be taken to reduce the contamination levels to “normal”.
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