No need to ‘vent’ your frustration: Petri-dishes & 20 points for contamination control
Tim Sandle, Ph.D., CBiol, FIScT
Pharmaceutical Microbiologist & Contamination Control Consultant and Expert. Author, journalist, lecturer, editor, and scientist.
The humble Petri-dish is named after German bacteriologist Julius R. Petri (1852-1921) (1), whether or not the German bacteriologist was the inventor (this is a matter of historical dispute). Since advent of injection molding technology in the 1960s, Petri dishes tend be manufactured from clear polystyrene plastic. The weight of most Petri-dishes are within the 15 to 17 grams range, mostly cylindrical (or occasionally square). The Petri dish has remained largely unchanged over the past 50 years (2).
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The filled Petri-dish may not be the most exciting of microbiological topics and it might be considered, in 2023, that all important aspects are in control, this is not always the case. Plates can become contaminated; plates can crack or dry out; the agar can be substandard and so on.
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In some previous articles on LinkedIn I have looked at:
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This article looks at venting and includes the twenty things about Petri-dishes that really matter within GxP environments.
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Vented and unvented Petri-dishes
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Petri-dishes are supplied either vented or unvented and contain agar and chemicals to support (or inhibit) microbial growth (3). Vented petri dishes feature small plastic protrusions on the inside of the lid. This means the lid so that it does not sit completely flat to the base. The objective of venting is to allow gaseous exchange and evaporation; and objective of unvented plates is to encourage anaerobic growth (4). However, with venting there are variations within the number of vents and their design. Unpicking this choice is important since the relationship between temperature, humidity, and bacterial growth is complex (5).
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Vented Petri-dishes: How many vents?
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Increasing the number of vents can increase the rate of growth of many aerobic cultures. However, at the same time, the rate of evaporation will increase. This means that longer incubation times are not suitable.
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Single vented plates allow for a limited air flow which helps to minimize evaporation. This design is suited for longer incubation periods. Triple vent designs are suitable for short term work due to the increase in moisture evaporation. Some designs have more vents, including four- five- and six-vent designs. It is uncertain whether this number of vents confers any additional; advantage for pharmaceutical facility environmental monitoring.
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What does this mean in the pharmaceutical microbiology context?
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Where a method has been developed internally as the organisms are capable of rapid growth, as with some bioburden determinations, then tripe vent dishes have an advantage. Where incubation times are fixed, consideration needs to be given to the robustness of the agar across the intended incubation time in relation to the selected number of vents. For situations where prolonged incubation is required, a single vent design may be preferable.
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This is because of the risk of desiccation where cracks or shrinkage of agar can occur, creating a data integrity issue. This consideration is of importance when selecting filled Petri dishes for use as settle plates and their subsequent incubation (often within a hot and dry airflow).
Contamination risks with vented dishes
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As the lid does sit securely there is a risk of contamination. However, the basic design of the dish tends to maintain a good level of protection from contamination because microbial carrying particles would need to go up and over the dish’s wall to get inside. This scenario is less likely in normal airflow (6).
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Non-vented Petri-dishes
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Some Petri-dishes are non-vented, meaning that the lid sits flat on the base. Non-vented Petri-dishes have flat lid that prevents evaporation, encouraging long incubation period and these are suitable for anaerobic applications (7).
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When preparing these plates, the lack of air flow can result in excessive condensation on the lid. In addition, prepared media will take longer to set due to the restriction on air flow in the plate.
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Non-vented plates have applications beyond culturing anaerobes in the laboratory, such as with some forms of compressed gas sampling.
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Non-vented dishes: Contamination risks
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With non-vented dishes the lid fits quite flatly on the base. This is not a hermetic seal; however, the space between dish and lid is extremely small. This results in there being far less potential for external contamination compared with the vented dish.
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Non-vented plates will have longer shelf lives. For example a 60mm vented Petri Dish containing 10ml of agar medium typically dries out in 2-3 weeks; whereas a similar 60mm non-vented dish typically lasts 2-3 months (8).
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Contamination control of pre-filled Petri-dishes: 20 points for success
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There are other factors that add to the control of microbiological culture media for use in cleanrooms. These include the following 20 points:
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1.????? Containing sets of pre-filled Petri-dishes in triple packaging (any contamination control strategy, especially transferring items to the aseptic core, will require at least three layers as part of the transfer disinfection process).
2.????? Given the commonality of vapor and gas disinfection, the inner sleeve should be impenetrable to chlorine dioxide gas and hydrogen peroxide vapor.
3.????? The process of introducing culture media into the facility needs to be mapped out, especially the eventual transfer into barrier devices like isolators.
4.????? Equal care must be given to the removal of plates and their transfer back to incubation, such as the use of plate racks or specialized carrier bags.
5.????? Locking lids can help to minimize the risk of the post-sampled plate becoming contaminated; however, if the locking process closes the vents these will need to re-opened for incubation (assuming aerobic monitoring is the objective).
6.????? There should be a pouch containing a drying agent to minimize condensation.
7.????? Each plate should be barcoded to ensure traceability with a digital information system (such as LIMS).
8.????? For environmental monitoring, the Petri-dish should be transparent. Where colored tints are used, these should not interfere with the ability to count colonies in relation to the natural coloration of the agar and the pigment of any microbial colonies. Consideration needs to be made as to how this combination appears on a colony counter and the light-source (including automated colony counters in conjunction with rapid microbiological methods).
9.????? For use in Grade A and B (ISO classes 5 to 7) the media should be sterile (this is also useful for Grade C / ISO class 8 cleanrooms as well). The standard sterilization method for tryptone soya agar is irradiation.
10.? The agar should be stable, for a period of time, at room temperature.
11.? Where there is a concern about prion transmission risk, the culture media should be made from a non-animal sourced peptone (such a vegetable based peptone).
12.? The shelf-life should be appropriate for the user.
13.? The fill volume and gel-strength will need to be suitable, especially where the plates will be used as settle plates for placement under unidirectional airflow or used in active air-samplers. Here, fill volumes often need to be around 30 mL.
14.? Plates intended for use as finger plates or for surface monitoring will need to contain an appropriate neutralizer (suitable for all in-use surface disinfectants). Neutralizers such as lecithin, Polysorbate 80, histidine, and sodium thiosulfate are often the most suitable.
15.? Often plates containing a neutralizer are not suitable as settle plates due to their tendency to crack, therefore a process of differentiating plates with and without neutralizer should be in place (such as color tinted Petri-dishes).
16.? For the release of culture media, the test panel should include organisms appropriate to those found in the cleanroom (do not simply use the default panel recommended by the media manufacturer, look at your own cleanroom microbiota patterns).
17.? Growth promotion should be performed post-sterilization. Whether the user needs to perform growth promotion on each lot or delivery will depend on the how the supply chain has been assessed, together with an impact assessment of heat or cold shock occur during transportation.
18.? To facilitate stacking during incubation, slots on lid or base will ensure that stacks of plates are less prone to sliding off one another. Consideration needs to be given to how plates will be placed in the incubator to avoid condensations. For example, it is often that by placing the plates upside down, the evaporation rate of water is reduced, minimizing the risk of condensation. In addition, the inverted position helps to retain moisture within the dish, ensuring that the agar remains hydrated throughout the incubation period. It is also likely that contaminants present in the incubator from airborne microbial carrying particles will be less likely to fall directly onto the agar surface when the dish is inverted.
19.? Cross ribs on the base make the Petri-dish resilient and resistant to warpage (this is a design factor of more importance when the molten media is poured into the plate, as with bioburden determinations). The overall design should also ensure that the Petri-dish can be handled by a person using one hand(wearing a sanitized glove, of course to reduce the possibility of cross-contamination).
20.? Some Petri-dishes have grids or a digital scale on the base to facilitate colony counting and positioning on a colony counter; this can be useful for bioburden determination.
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Readers wishing to explore the issues of culture media and data integrity, looking at topics like counting, merged colonies, agar desiccation etc. can read Tidswell and Sandle ‘Microbiological Test Data-Assuring Data Integrity ’.
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References
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QA Team manager at Cambridge University Hospitals NHS Foundation Trust
8 个月While some aseptic units utilise plastic bags to incubate agar plates, the MHRA has said during recent inspections that this approach may impede the growth of aerobic bacteria. Some years ago, I recall talking to someone who was, I believe, a microbiologist, about the space between the lid and the agar being sufficient for the bacteria to have adequate oxygen and ventilation in order for it to grow. If plastic bags are used there is undoubtedly enough air? Some aseptic units chose to use plastic bags to avoid contamination from the incubator. I would welcome your thoughts on the MHRA’s recent position. Many thanks Prakash ?
Quality Control Biologist at Antibiotice SA Ia?i
1 年Helpful! A very good article! Interesting info (s) for microbiologists ??
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1 年Thank you
Sterility Assurance Specialist presso Latina Pharma Spa
1 年A very good article to help in choosing the most suitable plate for environmental monitoring