Differentiation of Staphylococcal and Micrococcal Proteinases by Electrophoresis

Abstract

The electrophoretic separation of the proteinases produced by staphylococci and micrococci was studied in four buffers. The duration of electrophoresis was based on the migration of a marker dye for a predetermined distance. The migration distances of the enzymes and dye were measured, and enzyme-dye values were calculated. A comparison of enzyme-dye values showed that complete separation of eight serologically different proteinases did not occur in any one buffer; however, in most instances, their relative order of migration was the same in all buffers. Certain strains of Staphylococcus epidermidis produced two proteinases that were different serologically as well as electrophoretically. Staphylococcus aureus strains, on the other hand, produced up to four proteinases that were serologically the same. The proteinases of staphylococci and micrococci can be best characterized by both electrophoretic and serological methods. The serological differentiation of extracellular proteinases has been used for classifying organisms of the family Micrococcaceae (7, 13). Electrophoresis has been used for the separation and identification of various proteinases (9; 0. Sandvik, Ph.D. thesis, Veterinary College of Norway, Oslo, 1962). Because eight serologically different proteinases of staphylococci and micrococci have been identified, we wanted to determine whether the enzymes also differed in their electrophoretic mobilities. The possibility existed that a preliminary identification of a proteinase could be made by electrophoresis and then confirmed with a specific antiserum. This procedure would obviate the need to test each proteinase with all eight antisera before an identification was made and thus conserve the antisera. This report describes the separation of staphylococcal and micrococcal proteinases by electrophoresis with different buffers and support media. MATERIALS AND METHODS Cultures. Fifteen cultures of Staphylococcus aureus, 67 of Staphylococcus epidermidis, and 11 of Micrococcus sp. were used in this study. All cultures were classified by the method of Baird-Parker (3) and by serological differentiation of their proteolytic enzymes (12, 13). Proteinase production by the different organisms was as follows: group A by S. aureus; groups B, C, D, F, and H by S. epidermidis; and groups E and G by Micrococcus sp. Five cultures classified as proteinase groups A, B, C, D, and E were 76E obtained from Olav Sandvik, Veterinary College of Norway, Oslo, and the other cultures, including strains of groups F, G, and H, were isolated at the National Animal Disease Center, Ames, Iowa. All cultures were isolated from milk samples of infected bovine udders except six cultures of S. aureus; five of these six were of porcine origin obtained from Richard Shuman, and one was of avian origin obtained from Kenneth Heddleston of this Center. Enzyme production. Heart infusion agar (Difco

Differentiation of staphylococci on the basis of nuclease properties

“…Nuclease production. The enzymes used for antibody production and in enzymoserological examinations were produced by growing the microorganisms on a dialysis membrane covering heart infusion agar (19). For testing of nuclease activity and thermostability, the enzymes were produced by overnight growth in heart infusion broth followed by removal of the cells by centrifugation.…”