Effect of Incubation Strategy on Recovery of Bacteria and Molds in EM Testing

One of the most asked questions in microbial EM testing relates to incubation strategy and media choice.  

Whether to go single temperature or serial and if serial to go low to high or vice versa with use of one media, TSA, or two, TSA and SDA. Factors that can influence the choice are time to results (single temperature is usually faster than serial), cost (TSA alone is cheaper than TSA and SDA) and quality of microorganism recovery. There are few studies reporting recovery related to incubation choice however in those published a single 30-35°C incubation has been reported to give poorer mold recovery than serial. The data below summarizes a small study performed in our facility.

Three strategies were evaluated using standard TSA LP80 media, single temperature at 30-35°C, and 25-30°C for 5 days and a serial protocol with 72 hours at 20-25°C followed by 48 hours at 30-35°C. A smaller evaluation was performed to review the recovery of molds with SDA media at 20-25°C for 5 days as the control.

The samples were taken from close to the manufacturing floor entrances, from un-controlled areas to ensure high enough cell counts to get suitable data sizes. At each site 3 surface contact plates and 1 active air sample (1m³ SAS 180) were taken making 12 points for each day. At each point 3 replicate samples were taken, 1 cassette for each incubation temperature tested. For experiment 2 an SDA sample was also taken at the same time. Samples were taken in Dec-Jan and then 9 months later in Aug-Sept to cover seasonal variation. All samples were incubated on the Growth Direct™. Following incubation, the data series for each cassette was recovered to determine the cfu counts. For experiment 2, three analysts also counted the number of mold colonies present on each cassette and the mean value was calculated for each sample.

Figure 1. Total number of Bacterial colonies detected at each incubation strategy over the 3 days of study 1 in Dec-Jan

Figure 2. Total number of Bacterial colonies detected at each incubation strategy over the 3 days of study 2 in Aug-Sept

Overall the single temperature at 25-30°C appeared to have the least variation by day and may have recovered more colonies than the other 2 methods. There is a fairly high variation in this type of testing, so a larger panel would need to be studied to get a firmer data set.

Mold detection

The frequency of mold colonies occurring at each test point was recorded over the course of the study. Results are shown in Figure 3. As can be seen The SDA recovered the most colonies as expected. Serial incubation recovered the next highest number then single temperature at 25-30°C and finally 30-35°C. The recovery at 30-35°C was less than 1% of that on SDA.

Figure 3. Total number of mold colonies detected at each incubation strategy over the 3 days of study

Conclusion

If cost is the driving factor then single media, TSA, with a serial incubation (low to high) is the best option. If speed is the main requirement then the single temperature at 25-30°C is best. If overall recovery is the requirement then 2 media, TSA and SDA is superior.

To learn more about this study or how Rapid Micro Biosystems can help your company, please contact Sales. 

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