Selection of Incubation Conditions for Environment Monitoring Plates in Pharmaceutical Microbiology

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Environment monitoring plays an important role in observing and controlling the contamination in controlled areas. Selection of environment monitoring incubation condition is an important factor for the recovery of both bacteria and fungus.
Selection of proper incubation conditions for environment monitoring plates is very confusing and it confuses every microbiologist. A microbiologist must know the proper incubation conditions for recovery of microorganisms.
There was a time when two different media were used for the recovery of bacteria and fungus separately in environment monitoring. But now a days, single media plates are used for the recovery of both bacteria and fungi in environment monitoring. Selection of proper media for the recovery of bacteria and fungus is also another important factor. In pharmaceutical, Soyabean casein digest agar (SCDA) media is used for the environment monitoring. But for the recovery of bacteria and fungus two different incubation conditions are required.
For fungal growth low temperature of 20-25°C is required and for bacterial growth high temperature of 30-35°C is required. Total 5 days incubation period is commonly used for environment monitoring plates in which plates are incubated for 72 hours for fungal growth and 48 hours for bacterial growth. But what creates confusion?
Actually two different temperatures are required for the recovery of bacteria and fungus on single media plates and we don’t have to compromise with the growth of either one. But the problem is that at what temperature plates should be incubated first? Whether fungus should be recovered first or bacteria. Whether lower temperature is suitable first for the recovery of fungus or we should incubate first at higher temperature for the recovery of bacteria. What would be the impact of temperature on the recovery of bacterial and fungal growth if we incubate environment monitoring plates first at low temperature for fungus and then at higher temperature for bacteria and vice versa? These are the questions which comes in mind when selecting incubation conditions for environment monitoring. Currently two different incubation concepts are followed in pharmaceutical for environment monitoring plates.

EM plate

                                               SCDA media plate showing microbial growth
1.0 First incubate at 20-25°C temperature for 72 hours for fungal growth and then same plates transferred to 30-35°C for further 48 hours for bacterial growth.
2.0 First incubate at 30-35°C temperature for 48 hours for bacterial growth and then same plates transferred to 20-25°C for further 72 hours for fungal growth.
These two concepts are there but which incubation condition should be selected to recover both bacteria and fungus effectively?
If we see USP chapter number (1116) Microbiological control and monitoring of aseptic processing environment, it is mentioned that incubating at low temperature first may compromise the recovery of gram positive cocci that are important because they are often associated with humans. Here important thing is that in aseptic area, humans are the major source of product contamination and it is important to recover the microorganisms which are associated with humans.

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SCDA media plate showing microbial growth                      
So, if we incubate first at low temperature for fungal growth then we may compromise with the recovery of these human associated gram positive cocci. This is one of the reason which support the concept of incubating environment monitoring plates first at high temperature of 30-35°C for 48 hours for the recovery of bacteria and then at low temperature of 20-25°C for 72 hours for fungal growth.
Another important thing is that most of the fungus easily grow in a range of 20-35°C and incubating first at 30-35°C for 48 hours also results in the growth of fungus. When we incubate environment monitoring plates first at  30-35°C, fungal colonies grow in that condition and we can easily count the number of fungal cfu’s in initial stage of growth. But it would become difficult to count the fungal cfu’s when fungal colonies develop  properly and spread on the media plates. Spreading of fungal colonies results in merging of different fungal colonies and that causes difficulty to count the number of cfu’s properly.
This is the another factor which support the incubation of environment monitoring plates first at 30-35°C. So, its a good option to incubate environment monitoring plates first at 30-35°C for 48 hours and then 20-25°C for further 72 hours.

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