Appendix 



I. CHROMOGENIC CEPHALOSPORIN TEST FOR (3-LACTAMASE PRODUCTION 



Results of degradation studies and the chromogenic cephalosporin test have documented 

 that the Legionnaires' disease bacterium produces a /3-lactamase. The enzyme is more active on 

 cephalosporin than on penicillin, and is not at all active on cefoxitin or cefuroxime (newer 

 cephalosporins not yet approved in the United States but used in other countries). 



The procedure for performing the chromogenic cephalosporin test for j3-lactamase pro- 

 duction follows. 



Reagents 



1 . Phosphate Buffer, pH 7.0 



a. M/ 1 5 KH: PO4 9.07 g/liter distilled water 



b. M/ 1 5 Na. HPO4 .... 9.46 g/liter distilled water 



c. Mix 39.2 ml of the potassium phosphate solution with 60.8 ml of the sodium 

 phosphate solution. This buffer is stable for several weeks at room temperature. 



2. Cephalosporin Substrate 



a. Cephalosporin 87/312 is available only from: 

 Glaxo, Ltd. 



Greenford, Middlesex UB6 OHE 

 England 



b. Dissolve 10 mg of cephalosporin 87/312 in 1 ml of dimethylsulfoxide (DMSO). 

 Dilute with phosphate buffer, pH 7.0, to a concentration of 500 jUg/ml. The sub- 

 strate is yellow when viewed in a microdilution plate, but larger volumes may 

 appear more orange. However, the red reaction which constitutes a positive test 

 result is easily discerned. This substrate is stable at 4°-10°C for many weeks. 



B. Procedure 



1. In a Microdilution Plate or Small Tube: 



a. Place 0.05 ml of cephalosporin substrate in a well of a microdilution plate (or in 

 a small tube). 



b. Use a loop to remove several colonies of the test organism from the agar surface. 

 Make a turbid cell suspension in the cephalosporin substrate. 



c. Mix the substrate and cells for 1 min. Observe for color change immediately, 

 after 10 min, and after 1 h. 



d. If the test organism produces j3-lactamase, the color of the substrate will change 

 from yellow to red. |3-lactamase-producing Haemophilus influenzae or Neisseria 

 gonorrhoeae isolates usually turn the substrate red in less than 10 min. but 

 staphylococci may take an hour. 



e. Run the test with a known |3-lactamase-producing strain and a non j3-lactamase- 

 producing strain of N. gonorrhoeae. H. influenzae, or Staphylococcus aureus. 



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