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FSC, Forward Scatter; SSC, Side Scatter; FL1, Green Fluorescence; FL2, orange-red fluorescence

FSC, Forward Scatter; SSC, Side Scatter; FL1, Green Fluorescence; FL2, orange-red fluorescence

4. Notes

1. PI is light-sensitive and toxic to cells. Protect cells from light after applying the PI viability stain. Incubate and perform FCM analysis within 30 min of application of PI, as the stain will kill cells with extended exposure.

2. The major advantage of the use of fluorophores and FCM over culture-based procedures for assessing viability is the rapid analysis and estimation of the efficacy of various antimicrobials on microorganisms. The disadvantages of FCM procedures include underestimation of lethality owing to the inability of viability fluorophores to discriminate between viable and less damaged cells (i.e., viable but nonrecoverable) after exposure to some antimicrobials (3). In our FCM procedures, PI is capable of discriminating between viable intact cells and dead cells after treatment with most of the chosen antimicrobials; however, with certain antimicrobial exposures, lysis of cells resulted in an overestimated viability. Lysed particles are not stained with PI and therefore are estimated as viable; however, after careful examination of FSC (i.e., measurement of size) histogram plots we were able to discriminate between the lysed particles from intact cells and determine the discrepancy in the staining procedure.

3. It is generally recognized that the physiological state of the microorganism will determine its relative susceptibility to antimicrobials. The choice of media, particularly for the production of cysts of Acanthamoeba, will affect susceptibilities. Cysts induced from axenic cultures, particularly via increased concentration of MgSO4, may show increased susceptibility to antimicrobials. Inclusion of a standard disinfection system as a control is advised.

Acknowledgment

Supported in part by NIH GM579450.

References

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