Method validation in preservation of sterile agar plates beyond the manufacturers’ recommendations for use in a compounding unit

5 October 2022

M. Caudron, N.D. Alloh, A. Nowak, A. Fillatre, M. Royer, L. Mustapha, F. Marçon
CHU Amiens, France

The use of agar plates (AP) was considered for microbiological monitoring of our class A isolator, in accordance with current good compounding practices. Per production campaign, we only use 2 out of the 10 agar plates packed in a triple sterile packaging. Our supplier does not guarantee their fertility beyond 2 days in these conditions; the use of a gastight box could extend their conservation over 5 days.

Study the fertility of the AP beyond the 2 days recommended by the supplier thanks to a hermetic device initially intended for the microbiological culture in anaerobic conditions.

Material and methods
The AP were placed inside our isolator at room temperature (22°C ± 2°C) in a gastight box (BD® Gazpak EZ) (n=15) and outside the isolator (n=15) for 5 days after opening the primary packaging, then exposed for 4 hours in ambient air before inoculation.
Fertility test was performed with 5 micro-organisms according to the European Pharmacopoeia and manufacturer’s quality test: 3 bacteria (Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus) and 2 fungi (Aspergillus brasiliensis, Candida albicans). Low inocula (<100 CFU) were plated in triplicate. Control agar plates were used to determine the agar coverage rate (ratio of the number of test AP CFU / number of control AP CFU). Change in AP weight was measured as a secondary endpoint to assess water loss in the agar plates. The observed variations were compared using a Student’s t test ( ≤ 0.05)

The mean recovery rate of each germ is higher than 70% with P.aeruginosa = 158%, B.subtilis = 94%, S.aureus = 100%, A.brasiliensis = 83%, and C.albicans = 79%. Outside the isolator, a significant loss of the average weight of the AP was observed 5 days after opening the primary packaging (p = 0.02), contrary to those kept in the hermetic box (p = 0.43). This weight loss is also significant outside the isolator after exposure for 4 hours in ambient air (p < 0.001), in contrast to the AP exposed inside the isolator (p = 0.43).

Conclusion and relevance
The results of the fertility test are congruent with the expected results suggesting that the use of an airtight plate allows preserving the fertility of agar plates 5 days after opening the primary packaging in our study conditions.

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