Cultural and Biochemical Characterization of the Legionnaires' Disease Bacterium 



beyond the edge of the bacterial growtli. Tliis reaction is considered to be a positive test and 

 suggests that starch has been utilized. The LDB is positive by this starch utilization test. 



B. Catalase 



Inoculate an F-G or CYE agar slant heavily; incubate at 35°C for 48-72 h. Examine the 

 growth for catalase activity by adding 2 to 3 ml of 3% hydrogen peroxide to the agar slant. 

 Bubbles should appear vv'ithin 30 sec. The LDB catalase reaction is positive and can be speeded up 

 if the growth is "broken up" slightly with a sterile capillary pipette. The "sticky" consistency of 

 the growth apparently reduces contact between the peroxide and the LDB cells. 



C. Gekitinase 



This test can be performed with a photographic film or a gelatin cupule. Prepare a turbid 

 suspension in broth (Mueller-Hinton or heart infusion) by emulsifying some growth from an F-G 

 agar plate. 



1 . Fihii method 



Place a strip (0.5 x 1.5 cm) of photographic film (Kodak, plus-X) into 0.5 ml of the 

 turbid cell suspension in a screw-capped tube (13 x 100 mm). Incubate at 35°C in air plus 

 2.5% CO, . The emulsion on the film should be digested within 48-72 h if the isolate is the 

 LDB. 



2. Cupule method 



Inoculate an API (Analytab Products Inc.) gelatin cupule with the turbid cell suspen- 

 sion. Incubate at 35°C in air plus 2.5% CO, . Gelatin should be digested within 48-72 h if 

 the isolate is the LDB. 



D. (3-Lactamase 



Inoculate the test organism heavily on the medium developed by Martin and Lewis (5) to 

 detect penicillinase-producing A'fme/va ^o/7on7;ofat'. Incubate at 35°C in air plus 2.5% CO2 for 7 

 days. Although the LDB will not grow well on this medium, it will inactivate the penicillin 

 contained in the medium and allow growth of the Sarcina lutea that is also in the medium. 

 Growth of the S. lutea indicates lactamase activity by the test organism. All LDB identified to 

 date produce jS-lactamase. In addition, /3-lactamase is a consistent characteristic of all LDB (7) 

 examined with the chromogenic cephalosporin test (see Appendix). 



E. Nitrate 



Potassium nitrate (0.2%) is incoiporated into agar slants in which Fikles" enrichment is 

 substituted for hemoglobin. (Reduction of nitrate by Pseudoinoiuis aeruginosa and Yersinia 

 enterocolitica can be readily demonstrated with the medium). The slants are inoculated heavily 

 and are incubated for 7 days. All LDB isolated to date are nitrate negative. 



F. Oxidase 



Wet a piece of filter paper with a 0.5'? solution of oxidase reagent ('')) (tetramethyl- 

 p-phenylenediamine dihydrochloride) which has been freshly prepared or refrigerated tor less 

 than a week. Test the suspect isolate for oxidase activity by transferring some growth from an 

 F-G agar plate with a platinum loop; rub the growth into the previously prepared filter paper. A 

 dark blue color should appear in 9-10 sec, indicating a weakly positive test if the isolate is the 

 LDB. 



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