The sera of several animals were examined for suitability in coagulase testing. The assay for coagulase-reacting factor (CRF) activities of the whole sera indicated the following relative concentrations of CRF: human > pig > rabbit > horse > bovine, chicken, and lamb. Human, pig, and rabbit sera had adequate amounts of CRF for coagulase testing. The plasmin activities of the different sera, arranged from the strongest to the weakest, were as follows: rabbit > human > lamb >horse > bovine, chicken, and pig. Fibrinolysis was observed when rabbit, human, lamb, or horse sera were incorporated into coagulase test agars. Pig serum was superior to the other sera for use in the plate test for coagulase since it had adequate amounts of CRF and the plasminogen-plasmin system was not activated by staphylokinase or staphylococcal Muller factor. Heparinized pig plasma was more suitable than citrated pig plasma since citrate interfered with the growth of Staphylococcus aureus, and the use of heparinized plasma prevented false-positive coagulase reactions due to citrate utilization.

Egg white trypsin inhibitor activated coagulase clotting when added to a final concentration between 2 and 60 mg/ml. The greatest increase in clotting rate was observed in reaction mixtures containing the lowest concentrations of serum and plasma. Maximal activation was reached with 40 mg of trypsin inhibitor per ml when either serum or plasma was used as the source of coagulase-reacting factor (CRF). The increased rate of clotting is partly due to inhibition of plasmin. Freezing and thawing reduced plasma clotting inhibition. Soybean trypsin inhibitor also activated the coagulase reaction. The increased rate of clotting was observed with a coagulase preparation from organisms which produced plasminogen activators and with the culture supernatant fraction from organisms which did not activate plasminogen to plasmin. The tube test for coagulase could be made more sensitive for some strains of staphylococci by increasing the concentration of CRF (added as plasma or serum) by adding trypsin inhibitor, or both.

Fibrin halos developed around coagulase-positive Staplylococcus aureus colonies, and deoxyribonuclease production was detected by using a developing solution containing mg of methyl green per ml.

A selective, differential plating medium was developed for the isolation and identification of coagulase-positive and mannitol-fermenting staphylococci. Coagulase produced by growing Staphylococcus aureus caused an opaque zone of fibrin to form around each colony. Several strains of S. aureus produced a visible coagulase reaction by 8 hr, and all strains gave a positive reaction before 12 hr. Mannitol fermentation was usually observed between 12 and 36 hr. Rabbit serum was filtered through Sephadex G-100 to obtain plasminand plasminogen-free coagulase-reacting factor (CRF). False-negative reactions, caused by staphylokinase and staphylococcal Miller factor action on plasminogen, were eliminated when this CRF was used. False-positive reactions by lipolytic, coagulase-negative staphylococci were reduced, since gel filtration removed the serum lipoprotein which served as a pnrmary source of opacity. The addition of 75 jAg of polymyxin B per ml selectively retarded the growth of S. epidermidis and minimiz false-positive reactions caused by citrateutilizing gram-negative rods. The preparation, characteristics, and use of the medium are presented.