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
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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.
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