Incubating plates – challenges and lessons learnt

By Steve Brimble

Incubation of plates as part of environmental monitoring, product testing, batch control or for media or disinfection qualification is a vital part of the process which needs consideration. A failure relating to the incubation of plates is not only frustrating but time consuming and potentially puts the validity of results of the process at risk. Microbial growth and morphology is not only impacted by time of incubation but also the temperature they are exposed to, which can depend on the media used, which will be dictated by the incubation regime elected.

Equipment validation is critical

Incubators should have sufficient space and ability to maintain temperature within the unit, proving this is achieved though validation, undertaking sufficient temperature mapping to cover all shelves and corners of the incubators. The validation should identify any out of tolerance areas of the incubator and ensure these are not used, with fan assisted incubators the distribution of heat should be relatively universal however this can be compromised by the level of products placed in the incubator or the location of the fan, and the location of the fan and heat source must be identified to prevent risk to desiccation of plates. Maintenance and re-qualification schedules should be in place to ensure the equipment remains fit for purpose, poor maintenance or change of use of incubators can affect microbial growth if the change isn’t appropriately controlled. It is good practice to have separate incubators for different operational functions to remove any risk of cross contamination of plates. Plates from Environmental monitoring programmes should be placed in separate incubators to other growth promotion studies, especially if high numbers of bacteria are expected.

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Choosing the correct incubation regime

Dual incubation regimes must be validated, the variation in which time and temperature is used as the first phase and second phase might be dependent on the typical microflora within the area being monitored, dual incubation consists of an exposure at 30-35°C and an exposure at 20-25° for a defined period of time, but collectively would be 7 days. If dual incubation is used how this is managed either by dual incubators or removing plates from one incubator to another need’s consideration and the lag time from one temperature to another needs consideration. Based on the type of bacterial growth expected to be seen consideration should be given to what temperature is chosen first as some viable organisms, if stressed may not grow when exposed to higher temperature resulting a miss represented count. Over colonisation or spreading bacteria such as fungal should also be considered to ensure incubation temperature doesn’t allow a dominant organism to supress other background flora.

Does Incubation time affect results?

The incubation time and when to inspect plates can be critical. If plates are incubated for 5 days, these may need to be checked daily to enable accurate counts to be obtained. Leaving plates for the 5 days without inspection could impact on the accuracy of counts if plates become overgrown with dominant bacteria. Incubation time of plates can also impact on the morphology for example Staphylococcus aureus & Staphylococcus epidermidis will be different at 5 days compared to 24 hours, as well as variations in media formulation such as TSA against TSA with lecithin and tween as demonstrated here:

Staphylococcus epidermidis and Staphylococcus aureus incubated at 30-35°CStaphylococcus epidermidis and Staphylococcus aureus incubated at 30-35°CStaphylococcus epidermidis and Staphylococcus aureus incubated at 30-35°C

Staphylococcus epidermidis and Staphylococcus aureus incubated at 30-35°CStaphylococcus epidermidis and Staphylococcus aureus incubated at 30-35°C

Staphylococcus aureus incubated at 30-35°C

Staphylococcus aureus incubated at 30-35°CStaphylococcus aureus incubated at 30-35°CStaphylococcus aureus incubated at 30-35°C

Staphylococcus epidermidis incubated at 30-35°CStaphylococcus epidermidis incubated at 30-35°C

 

Staphylococcus epidermidis incubated at 30-35°CStaphylococcus epidermidis incubated at 30-35°C

Variation in process can also have an impact on incubation of plates, consideration on how plates are managed in relation to incubation during weekends when staff maybe absence should be known. This is also the case when public holidays occur where the incubation time maybe further increased.

Summary

The incubation of plates and the daily use of incubators may not always be a consideration when risk assessing process. Mitigation of risk is an easy process and achieved though planned maintenance and requalification however issues can occur through operator error. Understanding the morphology in relation to incubator time and temperature can help when reviewing root cause / corrective actions associated to out of trend data.

 

 

 

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