Assay of Staphylococcal  -Haemolysin with Fish Erythrocytes

Abstract

Delta-haemolysin has a much lower specific activity than the other haemolysins, and in low dilutions lyses a wide range of erythrocytes; it is usually assayed with human or horse erythrocytes and these have indicated as many as 300 haemolytic units per mg (HU per mg) in purified preparations (Heatley, 1971 ; Kreger et al., 1971). Heatley (1971) found that ,8-haemolysin was capable of interfering with the assay of 8-haemolysin when human erythrocytes were used. Lysis of bacterial protoplasts (Bernheimer et al., 1972) provides a more sensitive assay (610 units per mg) but is less convenient when a large number of samples is screened. The release of radioactively labelled nucleotides from tissue-culture cells (Thelestam, Mollby and Wadstrom, 1973) also provides a sensitive assay (approximately 600 units per mg purified protein) in which interference by other haemolysins does not occur. The simple haemolytic assay is still routinely used for 8-haemolysin, but a more specific and sensitive assay would be desirable. The work described here resulted from a preliminary survey of the sensitivity of marine fish erythrocytes to staphylococcal haemolysins and we describe the use of fish erythrocytes for the assay of staphylococcal 8-haemoly sin. MATERIALS AND METHODSStaphylococcal strains. Staphylococcus aureus strains NCTC10345 and NCTC7121 (Wood 46) were obtained from Professor J. P. Arbuthnott. The remainder, obtained from Dr S. McKay, comprised S. uureus strain BB, a ,fl-toxinogenic bovine-mastitis isolate, and clinical isolates of S. aureus (strains SM9, SM10, SM14, JK21) and S. epidermidis (strain SM6). Cultures were maintained on nutrient agar slopes.

Binding of 125I-Alpha Toxin of Staphylococcus Aureus to Erythrocytes

“…Moreover, the results of attempts to elute membrane-bound toxin by ionic manipulations by Fiissle et al (1 98 1) led these authors to conclude that manipulations of the toxin which lead to loss of haemolytic activity may alter membrane-binding activity. In the present studies, binding to rabbit and horse erythrocytes was independent of temperature over the range 1-45"C, as was cell lysis induced by staphylococcal 6 lysin, a lytic agent of low specificity whose action is thought to be due to surface-active properties (Chao and Birkbeck, 1978). Cassidy and Harshman (1976a), however, found that binding to rabbit erythrocytes was temperature dependent with maximum binding at 24°C.…”

“…Alpha-toxin was assayed by haemolytic titration against rabbit erythrocytes according to the method of Bernheimer and Schwartz (1963). The level of contamination of the a-toxin fraction with staphylococcal 6 lysin was estimated by titration against cod erythrocytes (Birkbeck, Chao and Arbuthnott, 1974;Chao and Birkbeck, 1978) and with staphylococcal /? lysin by titration against sheep erythrocytes (Low et al, 1974).…”

Binding of radiolabeled Staphylococcus aureus delta-toxin to human erythrocytes

“…One liter of medium contained 4.5 g of KCI, 0.55 g of NaH2PO4, 2.5 g of glucose, t Present address: Microbiology Laboratories, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642. 2 mmol of L-isoleucine, and 20 mmol each of L-leucine, L-serine, L-phenylalanine, L-histidine, L-lysine, L-proline, L-threonine, L-aspartate, L-glutamate, L-methionine, L-asparagine, L-cysteine, L-tryptophan, L-analine, L-arginine, glycine, L-valine, and L-tyrosine. The pH was adjusted to 7.2 with 4 M NaOH, and the medium was filtered through Whatman no.…”

Effect of phenethyl alcohol on Staphylococcus aureus alpha-lysin production

“…Delta-lysin was assayed by its hemolytic activity against a 1% (vol/vol) suspension of washed cod erythrocytes (6).…”