FRESH PERSPECTIVES
Figure 1 - Millifl ex® Quantum Protocol Overview
and incubated at 32.5 +/- 2.5 °C in aerobic conditions for diff erent incubation times.
After incubation, the membrane was disconnected from the media cassette using the Removal Rack and placed onto a Millifl ex® liquid cassette pre-saturated with 2mL of the Quantum staining solution. The stained membrane was incubated at 32.5 +/- 2.5 °C for 30 min then read using the Millifl ex® Quantum reader. The stained membrane was re-incubated on a new R2A agar media cassette to determine microbial recovery after staining. Refer to Figure 1 for an overview of the Millifl ex® Quantum protocol.
The compendial method was performed in parallel and incubated for up to 7 days for fi nal visual counting of colony forming units (CFU).
For each incubation time point using the Millifl ex® Quantum system, the average fl uorescence count and average Colony Forming Unit (CFU) count obtained after re-incubation of stained membranes were compared to the average count of the Millifl ex® compendial method. The fl uorescence recovery and re-incubation recovery parameters were calculated as follows:
to-fi nal result in comparison with the compendial method. Water sample 1 required 30hr incubation and water sample 2 required 24hr incubation in order to achieve > 70% recovery as compared to the compendial method. After re-incubation of the stained membranes, >70% recovery was achieved for both types of purifi ed water samples as compared to the compendial method. Refer to Table 2 for results and Table 3 for a comparison of colonies after staining and re-incubation [1].
Time-to-Final Result of Diff erent Water Isolates
Table 4 contains a list of micro-organisms recovered from diff erent water sources and the respective time-to-fi nal result using the Millifl ex® Quantum system as compared to the traditional method. Organisms were recovered anywhere from 24 to 72 hours depending upon the strain.
Interpretation
The results of this study showed that: 1) using the Millifl ex® Quantum System, micro-colonies could be detected after 24 hours, whereas
Acceptance criteria for each of these parameters are set to 70% or greater, which is the lower limit of acceptance to replace the conventional method by an alternative one (General Information Chapters United States Pharmacopeia <1223> and European Pharmacopeia 5.1.6) [3-5].
Results
Lab Water Lab water samples were incubated for 24 and 30 hours to determine whether or not contamination could be detected quicker using the rapid method as compared to the traditional method. After 24hr incubation, micro-colonies were detected using the Millifl ex® Quantum system whereas no visible colonies were detected using the traditional method. The traditional method required a minimum of 30hr incubation in order to visually detect macro-colonies, but they were extremely small and only a third of the count as compared to the rapid method. Refer to Table 1 for results.
Purifi ed Water
Purifi ed water samples, each from a diff erent process of a pharmaceutical plant, were incubated for 24 and 30 hours to evaluate the reduction of time-
52 American Pharmaceutical Review | Fresh Perspectives 2013
Table 1 - Detection of contamination in lab water after diff erent incubation times; results are an average of three membrane replicates
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