Primary Isolation Media and Methods 



days. Hold blood bottles for 2 wk, and tilt once each day so that the agar slant is inoculated 

 with any growth in the broth. Look for turbidity of the broth and growth on the slant 

 protruding from the broth. 



3. Store media in the dark, in sealed plastic containers, in a refrigerator. (Media have been used 

 successfully for growing the LDB after being stored for as long as 2 mon under these conditions.) 



Maintenance of Stock Cultures 



Stock cultures of the LDB have remained viable for several months in our laboratory when 

 stored as follows: 



1. Prepare CYE agar and pour (5 ml/tube) into 16- X 125-mm screw-capped tubes. Allow to 

 cool in a slanted position. 



2. Inoculate the surface of the CYE agar slants with strains of the LDB. 



3. Incubate the slants at 35°C for 2 days. 



4. Remove, tigliten the screw caps, and store in the dark at room temperature. 



Clinical Specimens 



Clinical specimens can be either liquid or solid. The initial preparation, media inoculation, 

 and subculturing of colonies thouglit to be LDB must all be done in a biological safety cabinet by 

 personnel protected with masks and gloves. 



I. Initial Processing and Media Inoculation 



A. Liquid specimens (pleural fluid, transtracheal aspirate, sputum, blood, etc.) re- 

 quire no special preparation before being inoculated directly on media. Inoculate each 

 container of F-G and CYE agar in two spots. Leave one spot undisturbed, and streak 

 the other with a bacteriological loop for isolated colonies. Inoculate blood specimens 

 directly into botdes of CYE diphasic medium. Use a sterile needle and syringe to put 5 

 ml of fresh blood into each bottle; mix well. 



B. Solid specimens (lung, spleen, liver) should be processed (sliced or ground) as 

 follows: 



1. To prepare sliced specimens, cut the tissue with a sterile scalpel, and slide the 

 freshly cut surface of the tissue over the surfaces of media to be inoculated. This 

 procedure must be followed for tissues obtained from patients who received 

 extensive antibiotic therapy. 



2. Prepare 10% suspensions of ground tissue by emulsifying approximately 1.0 

 gm of tissue in 9.0 ml of sterile phosphate buffered saline (PBS), pH 7.2. Use a 

 sterile mortar and pestle with sterile 60-mesh alundum as an abrasive. Mix well, 

 and dispense into several sterile screw-capped test tubes. Use one aliquot for steps 

 3 and 4. Quick freeze the remainder for future use. 



3. Inoculate CYE and F-G agars with one aliquot of the unfrozen suspension as 

 described for liquid specimens. 



4. Also, dilute the above 10% tissue suspension 1:50. Inoculate CYE and F-G 

 agars with 0.1 ml of this 0.2% suspension. Spread the inoculum over the entire 

 surface of each medium with a sterile glass spreading rod. 



C. Incubate aU inoculated CYE media aerobically in a humidified atmosphere at 

 35°C. Incubate the inoculated F-G agar at 35°C in air plus 2.5% CO2 • 



