120 DIFCO MANUAL 



per cent Bacto-Agar. The carbohydrate media should be freshly prepared or 

 reheated and cooled without agitation before inoculation. The inoculum should 

 be fairly heavy and should be placed on the surface layer of the medium not to 

 exceed a depth of 0.5 cm. Bacto-Phenol Red Broth Base with 0.5 per cent added 

 carbohydrate and 0.15 per cent agar is suggested as a satisfactory medium for 

 fermentation determinations as given in Diagnostic Procedures and Reagents--. 

 Some laboratories prefer a more solid fermentation medium containing en- 

 richment substances. A satisfactory medium of such character can be prepared 

 by adding 0.8 per cent Bacto-Agar to Bacto-Phenol Red Broth Base, sterilizing 

 it in the autoclave for 15 minutes at 15 pounds pressure (121°G.), cooling below 

 60°G., and adding 0.5 per cent of the carbohydrate previously sterilized in 10 or 

 20 per cent solution, and 5 per cent sterile fresh rabbit serum. Sera from other 

 animals have not been found satisfactory. A similar medium for determination 

 of fermentation by N. gonorrhoeae has been described by Faber, Gonzales and 

 Pelczar.26 



Other Primary Isolation Media 



Some laboratories prefer to use a clear medium rather than a Ghocolate Agar 

 for culturing A^. gonorrhoeae. This may be accomplished by omitting the Bacto- 

 Hemoglobin and enriching the Proteose No. 3 Agar with other substances capa- 

 ble of supporting growth of the gonococcus. Peizer and Steffen-^ reported the 

 use of a horse plasma hemoglobin dextrose nile blue A enrichment which, when 

 added to Proteose No. 3 Agar, gave a clear medium and yielded excellent results 

 in the culturing of the gonococcus. Steinberg and Mollov-^ enriched Proteose 

 No. 3 Agar with starch and blood and produced a satisfactory, clear medium. 



Mueller and Hinton-^ described a casein hydrolysate infusion medium which 

 they reported to give good results in the primary culturing of the gonococcus. 

 This medium is available in the dehydrated form as Bacto-Mueller Hinton 

 Medium as discussed on page 93. 



Sulfonamide Resistance of N. Gonorrhoeae 



Goodale, Gould, Schwab and Winter^^ developed a culture technique for test- 

 ing the resistance of N. gonorrhoeae to sulfonamides. The method consisted of 

 inoculating plates of Mueller Hinton Medium containing 0.10, 0.25 and 0.50 

 mg. per cent of sulfathiazole, respectively. Susceptible strains fail to grow in the 

 presence of the sulfonamides, while resistant strains do grow. 



Nelson,^^ using the Bacto-Mueller Hinton Medium as Goodale, et al.^° had 

 described for checking sulfonamide resistant gonococci, obtained very close cor- 

 relation with the clinical picture on thousands of cases. Nelson also employed 

 the Proteose No. 3 Hemoglobin Agar in the same manner for sulfonamide re- 

 sistance tests with equally good results. Frisch, Edwards, and Edwards^^ found 

 that the Proteose No. 3 Hemoglobin Agar worked well as a basal medium for 

 testing the sulfonamide resistance of A^. gonorrhoeae. 



Mass Culture and Stock Strains 



Mass cultivation of newly isolated strains for vaccine production is readily 

 accomplished on Bacto-Dextrose Starch Agar as discussed on page 124. This 

 medium supports luxuriant growth of the organism, and colonies of the gono- 

 coccus frequently exceed 3-5 mm. in diameter. Bacto-Dextrose Starch Agar 

 cannot be recommended for cultural detection of the gonococcus due to over- 

 growth by extraneous organisms. Bacto-Dextrose Starch Agar, prepared in half 

 strength, is an ideal medium for maintaining stock cultures of the gonococcus. 

 In tubes of this medium the gonococci generally are viable after 6-8 weeks in- 

 cubation at 37°C. 



