Comparison of Incubation Conditions for Microbial Contaminant Isolation in Microbiological Environmental Monitoring

Abstract

INTRODUCTION. Microbiological environmental monitoring programmes for clean rooms for pharmaceutical production vary depending on the regulatory document. This is particularly evident in the experimental conditions, including the culture media used for sampling, as well as the temperature and time of incubation. To harmonise quality control procedures, it is necessary to develop a unified strategy for processing microbiological samples.AIM. This study aimed to investigate the optimal conditions for sample incubation during microbiological monitoring of clean rooms.MATERIALS AND METHODS. The study compared several culture conditions for indicator microorganisms, including Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 9027, Candida albicans ATCC 10231, Aspergillus brasiliensis ATCC 16404, Aspergillus fumigatus F-62, Aspergillus terreus F-1269, and Penicillium chrysogenum F-3 (the latter three strains were obtained from the Russian National Collection of Industrial Microorganisms), as well as for environmental isolates, including Staphylococcus epidermidis, Kocuria rosea, Micrococcus luteus, Bacillus spp., and Sphingomonas paucimobilis. The culture media used were trypticase soy agar (TSA), Sabouraud's dextrose chloramphenicol agar (SDCA), and Reasoner’s 2A agar (R2A). The incubation regimes used were as follows: 2 days at 30–35 ºC and then 3 days at 20–25 ºC; 3 days at 20–25 ºC and then 2 days at 30–35 ºC; 48–72 hours at 30–35 ºC (for aerobic bacteria); 5–7 days at 20–25 ºC (for yeasts and moulds).RESULTS. The comparison showed no statistically significant differences between the results obtained with TSA and R2A under different temperatureconditions. The germination rates of environmental isolates grown on TSA were significantly lower (by 19–37%) in the two-tiered incubation scheme that started at a lower temperature. The study identified groups of microorganisms requiring special culture conditions for microbiological environmental monitoring (bacteria with suppressed physiological functions and moulds).CONCLUSIONS. The study confirmed the need to standardise microbiological testing used in environmental monitoring and to provide for its proper regulation by drafting a general monograph on the matter. The authors demonstrated the applicability of both TSA and R2A as culture media for microbiological environmental monitoring. Currently, the use of a two-tiered incubation scheme with one non-selective culture medium requires validation on a case-by-case basis. Although the sequence of temperature levels did not affect the germination rates of microorganisms significantly, the incubation regime starting at a higher temperature (30–35 ºС) was determined as preferable for bacterial environmental isolates.

Keywords

• microbiological monitoring
• incubation
• two-tiered incubation scheme
• temperature regime
• clean rooms
• culture media
• trypticase soy agar
• reasoner’s 2a agar
• moulds