Heat and chemical treatments affect the viability, morphology and physiology of Staphylococcus aureus and its subsequent antibody-labelling for flow cytometric analysis

The effects of heat and chemical treatments on Staphylococcus aureus viability and physiology and their subsequent effects on antibody binding ability and cell morphology were measured. Treatments included lethal and sub-lethal heat, exposure to organic acids, salt, sodium hydroxide and freeze-thawing. Strain-related differences in viability were noted depending on treatment and were reflected in changes in physiology as monitored by flow cytometry (FCM) using three different staining protocols: SYTO 9/Propidium Iodide; DiOC 2 (3); or calcein acetoxymethyl ester (calcein-AM)/PI. Treatments which resulted in significant losses in viability as measured by plate counting were reflected better by the first two staining combinations, as intracellular calcein-AM uptake may have been impaired by certain treatments. FCM analysis using labelling by commercial anti- S. aureus antibodies indicated that differences in cell physiology as a result of treatments influenced immunofluorescence detection. The ratio of the mean fluorescence intensity of stained cells to that of unstained cells (MFI/MFI [us] ) varied with treatment, five of which, including freeze-thaw, citric acid, oxalic acid, NaCl and NaOH, resulted in significantly lower fluorescence values compared to controls. IMPORTANCE FCM data indicated that cells conventionally considered to be dead and which would not give rise to CFUs in a plate count assay, e.g. cells heated to 80°C, were labelled by antibody staining. This finding suggests that without the inclusion of a live/dead discriminating dye these cells would be erroneously detected as viable within an FCM assay. Reductions in antibody staining due to physico-chemical treatment was strain-related, reflecting the complexity of the phenomenon under study and illustrating that substantial validation of any new antibody detection-based method, including physiological staining and cell sorting should be undertaken. Researchers should be aware of physico-chemical treatments causing false negatives: in this study freeze-thawing severely reduced antibody binding without affecting the viability of a substantial percentage of cells. Scanning electron microscopy (SEM) carried out on treated cells revealed a range of morphological changes resulting from physico-chemical treatments which may have hindered antibody binding.