Microbiological quality control of pharmaceutical water systems is of importance in ensuring that trends in contamination are detected and responded to. This is not least because water is a niche environment for many types of microorganisms and a vector for their transfer. Trending relates to actual microbial counts recorded, incidents and the types of species recovered. To facilitate species identification, microorganisms need to be subcultured from the isolation medium (Reasoner’s 2A (R2A) agar in Europe). Transfer onto the wrong media can result in the microorganism not growing. This paper describes research into three different media for subculturing: low nutrient (R2A); highly nutritious (tryptone soya agar) and medium nutrient (R3A) and concludes that a higher recovery is obtained where R3A agar is used.
Microbiologists have been using agar growth medium for over 120 years. It revolutionized microbiol-ogy in the 1890s when microbiologists were seeking effective methods to isolate microorganisms, which led to the successful cultivation of microorganisms as single clones. But there has been a dis-parity between total cell counts and cultivable cell counts on plates, often referred to as the “great plate count anomaly,” that has long been a phenomenon that still remains unsolved. Here, we report that a common practice microbiologists have employed to prepare agar medium has a hidden pitfall: when phosphate was autoclaved together with agar to prepare solid growth media (PT medium), total colony counts were remarkably lower than those grown on agar plates in which phosphate and agar were separately autoclaved and mixed right before solidification (PS medium). We used a pure culture of Gemmatimonas aurantiaca T-27T and three representative sources of environmental sam-ples, soil, sediment, and water, as inocula and compared colony counts between PT and PS agar plates. There were higher numbers of CFU on PS medium than on PT medium using G. aurantiaca or any of the environmental samples. Chemical analysis of PT agar plates suggested that hydrogen per-oxide was contributing to growth inhibition. Comparison of 454 pyrosequences of the environmental samples to the isolates revealed that taxa grown on PS medium were more reflective of the original community structure than those grown on PT medium. Moreover, more hitherto-uncultivated mi-crobes grew on PS than on PT medium.
In relation to a growth in reported incidents of fungal contamination of pharmaceutical products, there has been a developing interest by U.S. and U.K. regulators concerning the risk of fungi. This paper describes a study undertaken to examine the suitability of different commercially available mycological agars for the environmental monitoring of pharmaceutical-grade cleanrooms. Five agars were evaluated in relation to the detection of both numbers and different species of fungi (yeasts and moulds). The objective was to determine if one mycological medium is more suitable than an-other. Data was collected using different sampling techniques (settle plates, active air samples, and contact plates) from different locations within representative cleanrooms. Samples were taken over a 3 month time period. The study results indicated that fungi are not distributed evenly across clean-rooms and that that the prevalence of fungi partly relates to the room design and operation. In rela-tion to the different agar types, the study indicated that Sabouraud dextrose agar was the most ef-fective at detecting the widest number of different types of isolates, and that Sabouraud dextrose agar and malt extract agar were the most efficient in terms of the numbers of recovered isolates. Other media, notably potato dextrose agar, was relatively less effective.
Current guidelines for air sampling for bacteria and fungi in compounding pharmacies require the use of a medium for each type of organism. U.S. Pharmacopeia (USP) chapter <797> (http://www.pbm.va.gov/linksotherresources/docs/USP797PharmaceuticalCompoundingSterileCompounding.pdf) calls for tryptic soy agar with polysorbate and lecithin (TSApl) for bacteria and malt extract agar (MEA) for fungi. In contrast, the Controlled Environment Testing Association (CETA), the professional organization for individuals who certify hoods and clean rooms, states in its 2012 certifi-cation application guide (http://www.cetainternational.org/reference/CAG-009v3.pdf?sid=1267) that a single-plate method is acceptable, implying that it is not always necessary to use an additional me-dium specifically for fungi. In this study, we reviewed 5.5 years of data from our laboratory to deter-mine the utility of TSApl versus yeast malt extract agar (YMEA) for the isolation of fungi. Our findings, from 2,073 air samples obtained from compounding pharmacies, demonstrated that the YMEA yield-ed >2.5 times more fungal isolates than TSApl.
Colony counting by spreading bacterial suspensions on plating media by various techniques is of gen-eral concern. Comparative studies between hand plating (Drigalski-spatula technique) for different time intervals and spiral plating resulted in significant differences in colony counts. Lower counts of Gram-negative bacteria were obtained by using hand plating for more than 10s, compared with short time hand plating (5s) or spiral plating. Colony counting of Gram-positive bacteria showed no differ-ences between both techniques. Further characterisation of Escherichia coli cells spread with the Drigalski-spatula technique by electron microscopy revealed a large number of damaged cells com-pared to control samples. The data clearly shows that the mechanical forces during hand plating are sufficient to damage E. coli cells.
Enterobacteriaceae Enrichment Broth (Eeb) should not be subjected to temperatures above 100°C, because of decomposition of brilliant green, and the subsequent loss of selectivity. Visible light spec-trometry seems to be a useful tool in the routine quality control of the medium.