DEHYDRATED CULTURE MEDIA 119 



sulfuric acid to the beaker and place cover on the can at once. One gram sodium 

 bicarbonate with 3 ml. of sulfuric acid diluted to 100 ml. with water will give 

 about a 10 per cent carbon dioxide atmosphere to a container of about 2.5 liters 

 capacity. 



Laboratory workers wishing more detailed information on optimum carbon 

 dioxide tensions should consult Ferguson^* and Morton.-^ 



Observation 



Remove plates from the containers after the incubation period, keeping them 

 in the inverted position until the time of examination to prevent flooding of the 

 plate with water of condensation. 



The following procedure as given by Carpenter^'-^^ {^ recommended: Make a 

 direct examination of the plates for colonies of the gonococcus. Typically such 

 colonies are convex, transparent, from 1 to 3 mm. in diameter, with undulate 

 margins. By their transparency and character of their margins they can usually 

 be differentiated from young colonies of streptococci and diphtheroids which 

 they simulate. Films are prepared from the selected colonies, stained and exam- 

 ined. Cultural confirmation of typical Gram-negative diplococci is made by 

 subculturing typical colonies on enriched Chocolate Agar for purification and 

 inoculation into the appropriate carbohydrate media. 



When no typical gonococcus colonies can be detected by direct inspection, the 

 culture is subjected to the oxidase test which is of especial value in detecting 

 colonies of A^. gonorrhoeae in mixed cultures. The test is based upon the pro- 

 duction of an enzyme, oxidase, by organisms belonging to the genus Neisseria. 

 From 1 to 2 ml. of a one per cent solution of para-aminodimethylaniline mono- 

 hydrochloride (dimethyl-p-phenylenediamine hydrochloride) or the oxalate 

 salt, are dropped on each primary plate culture and the plate tilted to spread 

 the reagent over the entire surface. The plate is observed for a period of 6-10 

 minutes for evidence of color change of the colonies. The series of color reac- 

 tions, i.e., pink, maroon, and black, identifies the colonies of Neisseria. Films are 

 made from the "oxidase-positive" colonies, stained and examined microscop- 

 ically. If subcultures are to be made for further identification, the colonies 

 should be picked as soon as they become pink, because the dye component is 

 toxic for the organisms. 



Confirmation 



In the routine diagnosis of gonococcal infections and in the release of patients 

 under treatment, the oxidase test followed by a confirming Gram stain, in the 

 hands of an experienced operator, is generally sufficient for the identification of 

 the gonococcus. The identity of the organism may be further confirmed by 

 studying the reactions of purified cultures on carbohydrate media. The follow- 

 ing table shows the characteristic reactions of A^. gonorrhoeae and other Neis- 

 seria which are occasionally encountered. 



Microorganism Dextrose Maltose Saccharose Lactose 



Neisseria catarrhalis — — — — 



Neisseria gonorrhoeae + — — — 



Neisseria meningitidis -f- + — — 



Neisseria sicca + + + — 



In the study of carbohydrate fermentation we recommend the use of Bacto- 

 Phenol Red Carbohydrate Broths, or of Bacto-Phenol Red Broth Base contain- 

 ing 0.5 per cent of the desired carbohydrate, and to which has been added 0.15 



