Primary Isolation Media and Methods 



II. Examination of Cultures 



Examine all ciiltiu-es macroscopically and microscopically each day. 



A. MH-IH Agai : 



Examine plates for macroscopic growth. Colonies which are visible before 3 days 

 of incubation probably are not LDB, because colonies of LDB do not usually appear 

 on this medium before 5 days of incubation. Examine colonies suspected of being LDB 

 with a dissecting microscope. They should have a cut-glass appearance, and there 

 should be a distinctive brownish darkening of the surroundmg agar. 



B. F-G Agar: 



Examine this medium macroscopically with a desk lamp, allowing the light to pass 

 through the agar. In areas of heavy inoculum, confluent growth will be visible in 2 to 3 

 days, and there will be a distinctive brownish darkening of the colonies and the sur- 

 rounding agar. In liglitly inoculated areas, colonies of LDB should appear in 4 to 5 days 

 as white pin-point dots. These colonies grow larger after longer incubation. Examine 

 heavy growth in the dark with a long-wave (366 nm) ultraviolet light; a butter-yellow 

 color should be detectable in the 4- to 5-day growth. Examine suspected LDB colonies 

 microscopically by placing plates on the stage of a dissecting microscope and illuminat- 

 ing from one side with a light source held at an angle slightly > 10° to the horizontal. 

 LDB colonics will have a cut-glass texture. 



C. CYE Agar: 



Examine CYE plates as described for F-G plates. LDB colonies will appear in 3 to 

 5 days. Young colonies have a slight cut-glass texture which is rapidly lost after addi- 

 tional incubation. The browning of the agar is not visible on this black mediiun. 



D. CYE Diphasic Blood Culture Medium: 



Examine bottles daily for growth on the portion of agar slant extending above the 

 broth. Reinoculate this part of the slant daily by tilting the bottle so that broth flows 

 over it. Growth should be observed in about 6 to 8 days. Subculture colonies to CYE 

 agar. Keep bottles for 14 days before discarding as negative. 



in. Presumptive Identification of LDB Colonies 



Subculture colonies with a characteristic cut-glass appearance to both LDB-growth- 

 supporting media (F-G and CYE agar) and non-LDB growth-supporting medium (a blood 

 agar plate that does not contain L-cysteine). Incubate media at 35°C in air plus 2.59? CO, 

 for 5 days. Examine plates daily for growth. Test all cultures that grow on F-G and/or CYE 

 agar but not on blood agar. Cultures which grow on plain blood agar are not the LDB. A 

 culture that has typical morphological and biochemical characteristics, a compatable cellular 

 fatty acid profile, and a higli DNA-relatedness value to LDB strain Philadelphia 1 is the 

 LDB. 



Figure 1: Growth of the Legionnaires' disease bacterium (LDB). Suspensions of guinea pig spleen tissue infected 



with LDB strain Philadelphia 1 were inoculated onto CYE, F-G. and MH-IH agars. MH-IH and F-G agars received 



a 1/5,000 dilution: CYE agars received a 1/500,000 dilution. Plates were incubated at 35°C in air + 2.5% COj- 



Pictures show growth at 4, 6 and 10 days on CYE (black plates), F-G (clear plates), and MH-IH (red plates) 



media. 



Figure 2: Heavy growth of the LDB on F-G agar showing brown pigment production. 



Figure 3: The LDB on F-G agar showing cut-glass appearance of colonies resulting from oblique lighting. 



Figure 4: CYE diphasic blood culture medium; Wheaton bottle with a charcoal agar slant and brotli inoculated 



with blood. 



82 



