SUMMARYStrains of Staphylococcus epidermidis isolated from bovine udders were differentiated by comparing biochemical tests, serological typing of proteolytic enzymes, colonial morphology, and the spectrophotometric analysis of pigments extracted with methanol. The cultures were divided into proteinasepositive and proteinase-negative groups, based on their proteolytic activity on skim milk agar, staphylococcus medium no. 110 (Difco), and gelatin. Most of the proteinase-negative cultures produced acetoin, whereas the opposite was true for the proteinase-positive cultures. A further subdivision of the cultures in each group was made by using Baird-Parker's (1963) biochemical subgrouping scheme. The proteinase-positive cultures were also subdivided by serological typing of their proteolytic enzymes into five groups, B, F, G, H, and NR, a non-reacting group. Approximately three-quarters of the cultures in the first four proteinase groups could be placed in one of Baird-Parker's biochemical subgroups. The cultures in the NR group, however, were sufficiently distinctive in their biochemical reactions to be considered as a separate biochemical subgroup. The colonies formed by the cultures were classified into five types, each consisting of two to four forms. No absolute relationship was found between the spectral absorption curves of the pigments and proteinase groups, although there were differences in the types of absorption curves that predominated in each proteinase group. Nine cultures which produced coagulase were more closely related to S. epidermidis than to S. aureus in that they did not utilize mannitol anaerobically or produce a-or ,!?-type haemolysis, they were non-pigmented and were less active biochemically. Also, serological typing of the proteolytic enzymes of three of these cultures resulted in one being classified as group G and two as group H. Group H contained only these two cultures. In contrast 27 cultures of S. aureus were classified as group A. Except for coagulase production, the biochemical reactions and the serological grouping of the proteolytic enzymes suggested a classification of S. epidermidis for the nine cultures.

phenyltetrazolium chloride added, produced three main types of colonies: rough, intermediate, and mucoid. These colonies were yellow to amber in color and produced slight yellow zones in the medium. Rough colonies were flat, dry, and spreading, with a cut-glass appearance. Intermediate-type colonies varied considerably, but could be divided into two general subtypes. Intermediate-rough colonies had the cut-glass appearance characteristic of rough colonies, but were much more compact and raised, with irregular edges. Intermediate-smooth colonies had a slight cut-glass appearance, but were smooth and entire. Mucoid-type colonies also appeared in two subtypes. Mucoid A colonies were mucoid hemispheres. Mucoid B colonies, after incubation at 37 C for 24 hr, appeared as small, intermediate colonies. However, during a 24-hr holding period at room temperature, mucuslike material proliferated around the colonies. A fourth type of colony was red with blue surrounding medium. Only mucoid-type cultures could not be serologically 0-grouped.

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