Primary Isolation Media and Methods 



Adjust the complete medium to exactly pH 6.9 at 50°C by adding 4.0 to 4.5 ml of 1.0 N 

 KOH. The pH of this medium is critical. 



Pour 20-ml quantities of medium into sterile petri dishes ( 1 5- X 100-mm). Swirl the medium 

 between pouring plates to keep charcoal particles suspended. 



IV. CYE Diphasic Blood Culture Medium 



Agar Phase: 



Activated charcoal (Norit SG) 2.0 gm 



Agar(DIFCO) 17.0 gm 



'Distilled water 500.0 ml 



Broth Phase: 



Yeast extract (DIFCO) 20.0 gm 



L-cysteine HCI • H2 O 0.40 gm 



Fe(N03)3 ■ 9H,0 0.10 gm 



Distilled water 500.0 ml 



Prepare agar first. Combine ingredients, boil, and dispense as 20-ml aliquots into each of a 

 series of 125-ml Wheaton serum bottles. Stopper each bottle loosely with both a rubber stopper 

 and a metal cap. Autoclave the bottles at HTC for 20 min, cool to 50°C. remix the warm 

 charcoal and agar thoroughly, and place the bottles at an angle so that an agar slant with a 

 vertical height of 6.0 cm is formed. This procedure should leave a portion of the agar protruding 

 above the 25 ml of liquid-broth plus specimen-that are added later. 



Prepare the broth by first autoclaving the yeast extract and water at 12rC for 15 min. 

 Allow to cool, and then add fresh sterile solutions of L-cysteine HCI and ferric nitrate in that 

 order. Adjust the pH to 6.9 by adding 6.0 ml of 1 N KOH. Dispense the broth in 20-ml aliquots 

 into the Wheaton bottles of charcoal agar slants. Seal the bottles by crimping the metal caps over 

 the rubber stoppers. Check for sterility by preincubation at 35°C for 2 days. 



Quality Control 



Each batch of media must be tested for pH and ability to support the growth of the LDB. 

 Ideally, all media should be tested with a standardized tissue inoculum. However, since this 

 material is not generally available, stock culture inoculum is recommended with the understand- 

 ing that it is a minimal quality control test. 



1. The pH of the media must be 6.9 ± 0.05. Measure with a pH meter. To check solid media, 

 use a surface electrode (if available), or emulsify the agar from one plate in distilled water, and 

 measure the pH of the emulsion. The pH is critical. If given lots of ingredients do not conform to 

 the acceptable pH range, adjust the pH of the complete liquid medium by adding 1 N HCI or 1 N 

 KOH. 



2. Check for support of growth in the following manner: 



a. Prepare a standardized inoculum by emulsifying some actively growing LUB in sterile 

 distilled water to a turbidity of a McFarland ^4 standard. 



b. Seed the media with 0.05 ml of the standard moculum. Streak the plates for isolated 

 colonies, and incubate at 35°C aerobically (except for F-G agar, which is incubated in air 

 plus 2.5?; CO: ). 



c. Examine the media daily. On agar plates, growth should be present in the heavily 

 inoculated area after 2 days. Isolated colonies should be macroscopically visible in 4 to 5 



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