Depending on the size of the pores one wishes to produce in plasma membranes, the choice will probably fall on one of the three toxins discussed above. S. aureus alpha-toxin should be tried first when pores of 1-1.5 nm diameter are required. This is generally the case when Ca2+ and nucleotide dependence of a given process is being studied. If alpha-toxin does not work, this is probably due to the fact that the toxin either does not produce pores, or that the pores are too small. In this case, high concentrations of alpha-toxin should be tried. If this still does not work, we recommend the use of HlyA. When very large pores are to be created, e.g. for introduction of antibodies into the cells, SLO or another member of this toxin family are the agents of choice. SLO preparations need to be checked for presence of protease contaminants. Tetanolysin currently offers advantages since it is protease-free, and the size of the pores can probably be controlled by varying the toxin dose. Methods for assessing the size of pores created by such agents have been published in the recent literature, and the appropriate papers can be consulted whenever the need arises.

Incubation of human keratinocytes with nanomolar concentrations of Staphylococcus aureus alpha-toxin leads to irreversible depletion of cellular ATP. The toxin forms hexamers in the target cell membranes, and rapid transmembrane flux of K+, Na+, and 86Rb is observed. Unexpectedly, pores formed in keratinocytes through application of low but lethal doses of alpha-toxin appeared to be considerably smaller than those formed in erythrocyte membranes. They permitted neither rapid influx of Ca 2 or propidium iodide, nor efflux of carboxyfluorescein. Larger pores allowing flux of all three markers did form when the toxin was applied at high concentrations. Flux of monovalent ions and reduction in cellular ATP levels evoked by low toxin doses correlated temporally with a fall in oxygen consumption, which was interpreted to reflect breakdown of mitochondrial respiration. The lethal event could not be thwarted by manipulating the extracellular K+ or Ca21 concentrations. Realization that alpha-toxin may form very small pores in nucleated cells is important for future research on cellular toxin effects and membrane repair processes.

We describe a simple microtiter method for determining the susceptibility of Candida albicans and hyphal forms of Aspergillus fumigatus against antifungal agents. The assay measures mitochondrial respiration by determining reduction of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to formazan, a process that is enhanced in the presence of menadione. C. albicans or conidial suspensions of A. fumigatus are seeded into microtiter plates. Hyphal outgrowth of Aspergillus spp. was achieved by a 12 to 14-h culture at 30؇C. Antifungal agents (amphotericin B, fluconazole, itraconazole) were added to the cultures for 24 h. Thereafter, incubations were continued for 3 h in the presence of MTT plus 0.1 mM menadione. Formazan formation was quantified photometrically after extraction of the formazan with acid isopropanol. Well-defined dose-response curves reflecting impairment of mitochondrial function by the antifungal agents were obtained. With C. albicans, the results correlated excellently with the MIC determinations performed according to the standard macrodilution procedure. In confirmation of a recent report, it was found that fluconazole was unable to exert its fungistatic action on a sensitive C. albicans strain in the presence of serum. The presented method can easily be integrated in the standard repertoire of a diagnostic microbiology laboratory and should prove useful as a means to assess the antifungal action of various agents on yeasts and filamentous fungi in the presence and absence of serum proteins or body fluids.

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We describe a simple, rapid, automated procedure for measuring opsonophagocytosis and killing of Candida albicans by human peripheral blood leukocytes. Yeast cells are labelled by allowing uptake and cleavage of membrane-permeable bis-carboxyethyl-carboxyfluorescein pentaacetoxymethylester to its membrane-impermeable fluorescent derivative bis-carboxyethyl-carboxyfluorescein. The yeast cells are added to cell-rich plasma obtained after dextran sedimentation of erythrocytes. Opsonophagocytosis and killing are quantified by using automated fluorescent cell analysis, and the following parameters can be obtained: (i) relative percentage of phagocytes that participate in opsonophagocytosis, (ii) relative percentage of yeast cells that become associated with phagocytes, and (iii) percentage of killing of C. albicans. The first two parameters are obtained through the additional use of a phycoerythrin-conjugated monoclonal antibody that selectively labels monocytes and polymorphonuclear granulocytes in peripheral blood. Killing is assessed by solubilizing blood cells with deoxycholate to liberate yeast cells from the phagocytes. Viable yeast cells retain carboxyfluorescein, but nonviable cells lose the fluorescent marker; thus, the reduction in number of fluorescent particles directly reflects phagocytic killing. Results obtained by the present method correlated excellently with parallel enumerations by colony counting. Test results with seven healthy individuals revealed a marked dissociation between the process of opsonophagocytosis, which was essentially complete after 20 min at 37°C, and killing rates, which were 48% 11% and 63% 9% (standard deviation) after 1 and 2 h, respectively, when yeast cell-to-phagocyte ratios were in the range of 0.5:1 to 2:1. The described assay is unrivaled in simplicity, rapidity, and reproducibility and generates results for a large number of samples within hours.