Rapid Microbial Testing - Pharmaceutical Technology

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PharmTech Europe

Rapid Microbial Testing
In this technical forum, experts describe different methods of rapid microbial testing and their applications.


Pharmaceutical Technology
Volume 36, Issue 5, pp. s14-s19

Results. Both methods were used to detect bacteria in Chinese hamster ovary (CHO) cell cultures. Epifluorescence microscopy was limited by filterability, media interference, and nonrobustness issues, whereas microcolonies fluorescent staining method enabled consistent detection of Bacillus cereus, Staphylococcus epidermidis, and Propionibacterium acnes after eight, nine, and 48 hours of incubation.


FIgure 2
With epifluorescence microscopy, nonspiked treated CHO cells yielded a great deal of fluorescent debris and a very high background (see Figure 2A), which interferes with the detection of B. cereus (see Figure 2B). Because observation was difficult in the presence of cells, B. cereus was spiked in CHO medium. The fluorescent background was removed, but it was still difficult to observe bacteria on the polycarbonate filter. The detection of the microorganisms was only possible when increasing the number of contaminants retained on the filter by tenfold (see Figure 2C). Microorganisms spiked in the mammalian cell culture were easily detected with the MFSM without any background or media interference (see Figure 2, D–F). Background noise was minimized as a result of lysing the CHO cells before the filtration using the mammalian cell lysis solution; this buffer eliminates CHO cells while minimizing the impact on microorganisms. Similar results were obtained for both methods with S. epidermidis and P. acnes (data not shown).

As a result of the nondestructive feature of the MFSM, stained membranes could be reincubated on culture media to yield visible colonies that can be collected for further identification using existing identification methodologies (i.e., biochemical, morphological, nucleic-acid analytics, etc.). The ability to identify the microorganism can accelerate the root cause analysis and implementation of the corrective/preventative action (CAPA) plan.

References

1. A. Onadipe and K. Ulvedal, PDA J. Pharm. Sci. Technol. 55, 337–345 (2001).

2. R.T. Noble and S.B. Weisberg, J. Water Health 3, 381–392 (2005).

3. S. Flint et al., J. Appl. Microbiol. 102, 909–915 (2007).

4. R. Chollet et al., J. Rapid Methods Autom. Microbiol. 16, 256–272 (2008).

5. U. M. Rodrigues and R.G. Kroll, J. Appl. Bacteriol. 59, 493–499 (1985).

6. J. Moldenhauer, (2008) "Overview of rapid microbiological methods" in Principles of Bacterial Detection: Biosensors, Recognition Receptors and Microsystems M. Zourob, S. Elwary, and A. Turner, Eds. (Springer New York, 2008) pp. 49–79.

7. Y. Motoyama et al., Transfusion 48, 2364–2369 (2008).

8. Y. Motoyama et al., J. Health Sci. 55, 726–731 (2009).

9. J.T. Lisle et al., Appl. Environ. Microbiol. 70, 5343–5348 (2004).

10. K. Mignon-Godefroy, J.G. Guillet, and C. Butor, Cytometry 27, 336–344 (1997).

11. S. Asano et al., J. Biosci. Bioeng. 108, 124–129 (2009).


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