Use of Phenethyl Alcohol in Media for Isolation of Anaerobic Bacteria.

negative strains according to the slide clumping reaction, were the test organisms. In the clumping factor-positive series, there were 13 coagoulase-positive (including the Smith strain) and three coagulase-negative organisms. The clumping factor-negative group was composed of eight coagulase-positive and six coagulase-negative staphylococci. Of the coagulase-positive, clumping factor-negative organisms, four were originally obtained through the courtesy of 1\I. W. Fisher and one (the mouse virulent, diffuse colony-forming variant of the Smith strain) was kindly supplied by G. A. Hunt. All other strains were isolated from patients and personnel at this Medical Center. Saline suspensions of cells harvested from 18-hr Trypticase Soy Agar cultures were airdried on slides until no visible moisture remained. A solution of 1% Armour fibrinogen in phosphate-buffered saline, or 0.15%N rehydrated Warner-Chilcott fibrinogen, was then applied. This method gave the same results as when smears were made from fibrinogen-cell suspensicns except that in the latter instance clumping of susceptible cells proved to be a disadvantage. After a 30-min incubation in a moist chamber, the slides were treated according to the usual indirect fluorescent-antibody procedure. Control preparations included smears in which fibrinogen solution was replaced by (i) phosphate-buffered saline, (ii) an undiluted anti-S. aureus (clumping factor-positive) immune rabbit serum, and (iii) an undiluted normal bovine serum with an S. aureus (Smith strain) agglutinating titer of 1:160. Smears were examined with a Leitz Ortholux ultraviolet microscope assembly. Of the 30 strains tested, none fluoresced when cells were treated with bovine fibrinogen (Armour or Warner-Chileott) and fluorescein isothiocyanate-labeled antibovine globulin. Conversely, cells of all strains fluoresced after treatment with the undiluted immune rabbit serum and labeled antirabbit globulin. When undiluted, Smith strain-agglutinating, normal bovine serum was used with antibovine globulin conjugate, fluorescence was observed with (i) 12 of 13 coagulase-positive, clumping factor-positive strains; (ii) six of eight coagulase-positive, clumping factor-negative strains; (iii) one of three coagulase-negative, clumping factorpositive strains; and (iv) four of six coagulasenegative, clumping factor-negative strains. Control preparations in which cells were exposed only to fluorescein-labeled antirabbit or antibovine globulin were negative. Autofluoreseence of cells was not a problem. Regardless of their free coagulase activity, all cells which gave a positive slide-clumping reaction, but none of the clumping factor-negative cells, produced compact colonies in soft agar medium containing 0.1 % bovine fibrinogen (Armour or WarnerChilcott). These results do not support the idea that staphylococcus-fibrinogen reactions are essentially antigen-antibody reactions. Neither do they negate the theory that clumping factor activity depends upon an accessory factor rather than upon fibrinogen itself.