92 DIFCO MANUAL 



for this purpose by "Diagnostic Procedures and Reagents"^ of the American 

 Public Health Association. Bacto-Cystine Heart Agar without enrichment sup- 

 ports excellent growth of Gram-negative cocci and other pathogenic micro- 

 organisms. 



Since P. tularensis was first isolated by McCoy and Chapin,^ many media 

 have been described for its cultivation. A large number of the media first em- 

 ployed were difficult to prepare and contained egg or serum. Francis^ reported 

 Blood Dextrose Cystine Agar in his later investigation as being a satisfactory 

 medium for cultivating this fastidious organism. Shaw* added 0.05 per cent 

 cystine and 1 per cent dextrose to Bacto-Heart Infusion Agar for the cultivation 

 of P. tularensis, Shaw^ also showed that the amount of destruction of cystine in 

 the autoclave at 15 pounds pressure for 15 minutes would be small or negligible 

 as far as the bacteriological culture medium was concerned. 



Rhamy® found Francis' Blood Dextrose Cystine Agar to be excellent but often 

 it became contaminated due to the difficulties attendant to its preparation. In 

 his experience an autoclaved solution of Bacto-Hemoglobin added to Bacto- 

 Cystine Heart Agar proved to be entirely satisfactory for the cultivation of 

 P. tularensis. In three or four days the growth is sufficient for the preparation of 

 bacterial antigens. Because of its nutritional value, this medium may also be 

 used for cultivating many other organisms ordinarily difficult to grow. 



Bacto-Cystine Heart Agar was originally developed in collaboration with 

 Rhamy. As mentioned in the paper by Rhamy, referred to above, W. M. Simp- 

 son found this formula, with a reaction of pH 6.8, a most satisfactory medium 

 for the cultivation of this organism. Also cooperating in these preliminary trial 

 studies of the medium, Francis found a culture medium made with Bacto- 

 Cystine Heart Agar and Bacto-Hemoglobin entirely satisfactory for growing 

 P. tularensis. 



When used with Bacto-Hemoglobin, the medium is prepared for use as 

 follows: 



A. Suspend 10.2 grams Bacto-Cystine Heart Agar in 100 ml. cold distilled 

 water and heat to boiling to dissolve the medium completely. Sterilize in the 

 autoclave for 15 minutes at 15 pounds pressure (121°C.). 



B. Place 2 grams Bacto-Hemoglobin in a dry flask and add 100 ml. cold 

 distilled water, while the flask is being agitated vigorously. The hemoglobin 

 suspension is shaken intermittently for 10-15 minutes to break up all aggregates 

 and effect complete solution and sterilized in the autoclave for 15 minutes at 15 

 pounds pressure (121°C.). 



C. Both solutions are cooled to 50-60° C, mixed and poured into sterile petri 

 dishes or tubes. 



When a plain Cystine Dextrose Agar, without hemoglobin, is desired, the 

 medium is rehydrated by suspending 51 grams of Bacto-Cystine Heart Agar in 

 1000 ml. cold distilled water and heating to boiling to dissolve the medium com- 

 pletely. Distribute in tubes or flasks and sterilize in the autoclave for 15 minutes 

 at 15 pounds pressure (121°C.). The final medium will have a reaction of 

 pH 6.8. 



One pound of Bacto-Cystine Heart Agar will make 8.9 liters of the enriched, 

 or plain, medium. 



1 Diagnostic Procedures and Reagents, * Zentr. Bakt. I Abt. Orig., 118:216:1930. 

 3rd Edition: 259: 1950. ^ J. Lab. Clin. Med., 16:294:1930. 



2 J. Infectious Diseases, 10:61:1912. ^ Am. J. Clin. Path., 3:121:1933. 



3 J. Am. Med. Assoc, 91:1155:1928. 



