Graduate Thesis Or Dissertation

 

Improved coagulase tests for Staphylococcus aureus Public Deposited

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  • The need for better procedures for detecting Staphylococcus aureus prompted an investigation to improve coagulase testing. The rates of coagulase clotting were studied by using varying concentrations of trypsin inhibitor and coagulase-reacting factor (CRF). Egg white trypsin inhibitor activated coagulase clotting when added to a final concentration between 2 and 60 rmg/ml. Maximal activation was reached with 40 mg/ml of trypsin inhibitor when either serum or plasma was used as the source of CRF. The increased rates of clotting were partly due to the inhibition of plasmin (E. C. 3.4.4. 14). 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 typsin inhibitor, or both. The plate test revealed the production of coagulase by the formation of opaque fibrin halos around growing colonies of S, aureus, Rabbit serum was filtered through Sephadex G-100 to obtain plasmin and plasminogen-free CRF. False-negative reactions, caused by staphylokinase and staphylococcal Milller factor action on plasminogen, were eliminated when this CRF was used in the plate test. False-positive reactions by lipolytic, coagulase-negative staphylococci were reduced since gel filtration removed the serum lipoprotein which served as a primary source of opacity. The addition of 75 μg/ml polymyxin B selectively retarded the growth of S. epidermidis and minimized false-positive reactions caused by citrate-utilizing gram-negative rods. Polymyxin-Coagulase-Mannitol-Agar and Polymyxin- Coagulase-DNase-Agar were prepared with plasminogen-free CRF. These media were superior to other selective media for rapidly recovering coagulase-positive staphylococci from naturally contaminated and seeded frozen foods. Since the preparation of plasminogen-free CRF may be too difficult for routine use in some laboratories, the sera of several animals were examined for their suitability in coagulase testing. The assay of 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 > pig, bovine and chicken. 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 coagulase testing. Preliminary experiments indicated that heparinized pig plasma was more suitable than citrated pig plasma since citrate interfered with growth of S. aureus, The use of heparinized plasma prevented false-positive coagulase reactions due to citrate utilization.
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