Environmental monitoring, a critical process within the pharmaceutical and biotechnology industries, is used to determine the microbial and particulate content of cleanroom air and surfaces. Results are used for quality assurance, to identify conditions contributing to excessive microbial and particulate levels, and to alert personnel to conditions exceeding classification.
Surface monitoring of isolators and cleanrooms is typically conducted using special petri dishes containing growth medium. Filled with agar that forms a convex surface, contact plates are pressed against the flat surface to be monitored including floors, walls, equipment, garments, and gowned personnel. Viable microorganisms present on the surface will adhere to the agar surface that protrudes above the rim of the plate. Microorganisms that are present on the surface being monitored will then grow with the proper incubation conditions, allowing identification and enumeration. Contact plates can be employed to establish and monitor the microbial load of dry, sanitized surfaces. For this purpose, the medium contains neutralizing agents, which inactivate residual disinfectants on the surface to be tested.
The growth medium typically contained within these contact plates is tryptic soy agar (TSA). TSA contains peptones of animal origins and, as such, has the potential risk to transfer animal spongiform encephalopathy (TSE/BSE) to surfaces during monitoring.
For the manufacturing of medicinal products, the European Medicines Agency (EMA), European Pharmacopoeia (EP), and the U.S. Pharmacopeia (USP) recommend use of materials from “non TSE-relevant animal species,” or materials derived from “non-animal origin” to minimize the risk of transmitting TSE/BSE. Interspecies transmission has been reported justifying cautious handling with biological materials used for manufacturing drugs.
The results below compare a vegetable-based contact agar (EMD Millipore) with conventional TSA agar for cleanroom surface monitoring. The vegetable-based agar is comprised of high quality peptones of non-animal origin, which minimizes the risk of TSE/BSE transmission during surface monitoring. Vegetable contact agar is a complex medium for the cultivation and isolation of fastidious bacteria, yeast, and molds.
Table 1. Comparison of the composition of vegetable contact agar and tryptic soy agar.
In this study, vegetable contact agar and tryptic soy agar were prepared according to the recommendations of the EP and EP/USP, respectively (EP 5.2.8; EP 2.6.12; USP<61>). Growth-promoting properties and the neutralization of select disinfectants with the vegetable contact agar are completely comparable to TSA.
Determination of recovery rates
Overnight cultures of the test strains in brain/heart infusion broth were diluted in NaCl peptone buffer to adjust the inoculum to 10 to 100 colony forming units (CFU). The suspensions were spread on reference plates (three plates) and the test plates (two plates each) with a Drigalsky spatula. For Aspergillus brasiliensis (ATCC 16404) and Penicillium commune, spore suspensions were used for inoculation. For Bacillus subtilis (ATCC 6633), both spore suspensions and vegetative cultures were used. Bacterial test strains were incubated for 22 ± 2 hours at 30 to 35 C. Candida albicans was incubated for 46 ± 2 hours at 20 to 25 C, and A. brasiliensis was incubated for 72 ± 2 hours at 20 to 25 C.
Recovery rates were calculated as follows:
Average CFU x 100
Recovery (%) = —————————
Average CFU (reference plates)
The growth promoting properties of the test plates are defined as sufficient if the recovery on test plates is greater than or equal to 50% compared to the reference plates.
Recovery in the presence of disinfectant
All disinfectants were diluted according to the manufacturer’s instructions. A total of 0.025 to 0.1 mL of ready-to-use preparations were plated onto each test plate. All plates were dried for 20 minutes prior to inoculating 10 to 100 CFU of the test strains.
All bacterial test strains were incubated for one day at 34 ± 1 C. C. albicans was incubated for two days at 22.5 ± 2.5 C, and A. brasiliensis for three days at 22.5 ± 2.5 C.
Neutralization efficiency was calculated as follows:
Average CFU (test plates with disinfectant) x 100
Recovery (%) = ——————————————-
Average CFU (test plates without disinfectant)
Figure 1 shows the recovery rates for 16 test strains and select environmental isolates on vegetable contact agar (heipha Ref. 840) and TSA (heipha Ref. 820). One lot each of Ref. 840 and Ref. 820 was tested. Results show that the recovery rates for freshly produced vegetable contact agar are greater than or equal to 50% and for most test strains greater than or equal to 70%. In total, three lots of the vegetable contact agar were tested validating the results shown in Figure 1 (data not shown). Importantly, the vegetable contact agar maintains good growth promoting properties after nine months of storage (Figure 2).
Figure 1. Growth promotion test. Recovery of test strains and select isolates on vegetable contact agar (fresh plates; Ref. 840) and TSA (plates two weeks old; Ref. 820). (All figures and tables: EMD Millipore)
Table 2 lists the disinfectants used for testing of the neutralization efficiency of the vegetable contact agar. Sample results shown in Figure 2 indicate vegetable contact agar is suitable for surface monitoring in the presence of select disinfectants.
Figure 2. Recovery of test strains and select isolates from vegetable contact agar after nine months of storage.
In summary, a vegetable-based contact agar demonstrates comparable results to TSA with respect to growth promoting properties and growth promotion of microbial organisms in the presence of select disinfectants.
Yvonne Berghöfer-Hochheimer, PhD, has more than 20 years’ experience in molecular biology and microbiology in both academia and industry. Her research in gene regulation covered aerobic and anaerobic bacteria, archaea, HIV, and mammalian cells.
Reiner Hedderich is responsible for the quality control of raw materials. He holds a doctorate in microbiology and has a strong background in microbial physiology. Hedderich served as a research associate at the Max Planck Institute for Terrestrial Microbiology. Contact: firstname.lastname@example.org
This article appeared in the October 2012 issue of Controlled Environments.