Heat and Chemical Treatments Affect the Viability, Morphology, and Physiology of Staphylococcus aureus and Its Subsequent Antibody Labeling for Flow Cytometric Analysis

Abstract

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 sublethal heat; exposure to organic acids, salt, and 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 (PI), DiOC₂(3), or calcein acetoxymethyl ester (calcein-AM)/PI. Treatments that 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 labeling 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 intensities of stained cells to those of unstained cells [MFI/MFI_(us)] varied with treatment, five of these treatments, including freeze-thaw, citric acid, oxalic acid, NaCl, and NaOH treatments, 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 CFU in a plate count assay, e.g., cells heated to 80°C, were labeled 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 physicochemical treatment were 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 physicochemical treatments causing false-negative results: in this study, freeze-thawing severely reduced antibody binding without affecting the viability of a substantial percentage of cells. Scanning electron microscopy carried out on treated cells revealed a range of morphological changes resulting from physicochemical treatments which may have hindered antibody binding.

Keywords: Staphylococcus aureus; antibody-based detection; cell viability; flow cytometry; food-borne pathogens; morphology; physiology.

FIG 1

Mean log₁₀ reduction expressed as CFU/ml for S. aureus strain 1 (light gray) or strain 2 (dark gray) after the following treatments (all for 10 min): 50°C, heated to 50°C; 60°C, heated to 60°C; 70°C, heated to 70°C; 80°C, heated to 80°C; citric acid, exposure to 1% (wt/vol) citric acid solution; oxalic acid, exposure to 1% (wt/vol) oxalic acid solution; NaCl, exposure to 1% (wt/vol) NaCl solution; and NaOH, exposure to 2.5% (wt/vol) NaOH solution. Freezing, cells frozen overnight and thawed at room temperature for 1 h before plating.

FIG 2

Cytographs of S. aureus strain 1 exponential-phase live control cells stained with SYTO 9 and PI (a) and DiOC₂(3) (b) and of heat-treated dead control cells (95°C for 30 min) stained with SYTO 9 and PI (c) and CCCP-treated cells (depolarized) stained with DiOC₂(3) (d).

FIG 3

Cytographs of S. aureus strain 1 exponential-phase live control cells (a) and heat-killed control cells (95°C for 30 min) (b) stained with calcein-AM and PI. (c) Overlay of green fluorescence distributions of calcein-AM- and PI-stained cells subjected to various treatments. For an explanation of the labels, see Fig. 1.

FIG 4

Overlays of the PE fluorescence distributions of S. aureus exposed to various physical or chemical treatments (for an explanation of the labels, see Fig. 1) and stained with one of a pair of commercial anti-S. aureus antibodies, followed by staining with R-PE-conjugated secondary antibody. The left panel shows the average MFI/MFI(_us) PE fluorescence values for the various treatments for both pairs of strains and antibodies.

FIG 5

Comparison of the MFI/MFI(_us) of the PE fluorescence of S. aureus strains subjected to various physicochemical treatments (for an explanation of the labels, see Fig. 1, and for an explanation of the bars' color coding, see Fig. 4) and stained with primary antibody A or B, followed by staining with R-PE-conjugated secondary antibody, with log₁₀ plate counts obtained after each treatment. The red line is an illustration of the log₁₀ counts for strain 1, while the orange line represents strain 2.

FIG 6

Scanning electron micrographs of S. aureus cells. (A) Exponential-phase live control cells; (B) stationary-phase cells; (C) cells after freeze-thaw treatment; (D) cells after heating at 80°C for 10 min; (E) cells exposed to 2.5% (wt/vol) sodium hydroxide for 10 min; (F) cells exposed to 1% (wt/vol) citric acid for 10 min; (G) cells exposed to 1% (wt/vol) oxalic acid for 10 min; (H) cells exposed to 1% (wt/vol) sodium chloride for 10 min.