Primary Isolation Media and Methods 



II. F-G Agar 



Casein (acid liydrolysis) 17.5 gm 



Beef extractives 3.0 gm 



Starcii 1.5 gm 



Agar 1 7.0 gm 



L-cysteine HCl • H. 0.4 gm 



Ferric pyrophosphate, soluble* 0.25 gm 



Distilled water 1.0 liter 



* Available on request from Dr. Morris Suggs, Director. Biological Products Division, Center tor Disease Control, Atlanta, Georgia 

 30333. 



Baltimore Biological Laboratories (BBL) Mueller-Hinton (M-H) agar, lot #105621 , contains 

 all the ingredients of F-G agar except for L-cysteine HCl and ferric pyrophosphate, soluble, and 

 provides a source of casein, beef extractives, starch, and agar. (NOTE: Other lots of M-H agar 

 may vary too widely in formulation and stability to serve as the base for satisfactory F-G agar.) 



Add the casein (acid hydrolysate), beef extractives, starch, and agar (or their equivalent-38 

 gm of M-H agar) to 980 ml of distilled water, autoclave at 121°C for 15 niin, cool to 50°C in a 

 water bath, and hold until L-cysteine HCl and soluble ferric pyrophosphate are added. 



Prepare separate fresh solutions of L-cysteine HCl (0.4 gm in 10 ml distilled water) and 

 soluble ferric pyrophosphate (0.25 gm in 10 ml distilled water). Note the precuation for ferric 

 pyrophosphate stated below. Filter sterilize each solution separately. Add the L-cysteine HCl to 

 the agar mixture first, and then add the ferric pyrophosphate. 



Before pouring the warm medium into petri plates, adjust its pH so that the final pH of the 

 cooled medium will be 6.9. Pour 20 ml into each of a series of plastic petri dishes (15- X 

 100-mm). Allow agar plates to cool to room temperature. Measure the pH of a plate of cool agar 

 (procedure in the Quality Control section of this chapter). The final pH must be 6.85 to 6.95 to 

 consistently support growth of the LDB. 



Precaution: Soluble ferric pyrophosphate must be kept dry and stored in the dark; it is no 

 longer usable if its color changes from green to yellow or brown. Prepare fresh solution of the 

 compound each time it is needed for media. Do not heat over 60°C to dissolve. The mixture can 

 be readily dissolved by placing in a 50° C water bath. 



III. CYEAgar 



Yeast extract (DIFCO) 10.0 gm 



Activated charcoal (Norit SG)* 2.0 gm 



L-cysteine HCl ■ Hj O 0.4 gm 



Ferric pyrophosphate, soluble 0.25 gm 



Agar (DIFCO) . ~. 1 7.0 gm 



Distilled water 1 .0 hter 



Add all other ingredients of CYE agar except L-cysteine HCl and soluble ferric pyrophos- 

 phate to 980 ml of distilled water; dissolve by boiling; autoclave at I21°C for 15 min, and cool to 

 50°C in a water bath. 



Prepare separate fresh solutions of L-cysteine HCl (0.40 gm in 10 ml distilled water) and 

 soluble ferric pyrophosphate (0.25 gm in 10 ml distilled water). Filter sterilize each solution 

 separately. Add the L-cysteine HCl to the basal medium first, and then add the ferric pyrophos- 

 phate. Note the above precaution for ferric pyrophosphate. 



* Available through Sigma Chemical Co., St. Louis, MO., Catalogue # C5510. Activated charcoal, washed witli phosphoric and 

 sulfuric acids. 



79 



