118 DIFGO MANUAL 



gonococcus and obtained 12 per cent more positive isolations on the Chocolate 

 Agar enriched with liver extract or yeast extract, than upon the unenriched 

 medium. Following this work, Lankford and Snell^^ identified the required 

 growth factor as glutamine. Lankford^^ called our attention to the fact that a 

 second growth factor, cocarboxylase, was required by a small percentage of 

 gonococcal strains and that cystine helped under certain conditions. With this 

 information, an extensive study to provide a satisfactory enrichment for supple- 

 menting the Chocolate Agar was undertaken, and resulted in the development 

 of Bacto-Supplement A and Bacto-Supplement B, as discussed on page 276. 



Nelson,i8 using the Bacto-Proteose No. 3 Agar and Bacto-Hemoglobin en- 

 riched with Bacto-Supplement A, increased his positive isolations 5 per cent 

 over the unenriched medium. In addition to increasing the number of positive 

 isolations, the efficiency of the medium was improved by reduction of extraneous 

 growth and an increase in size and number of gonococcal colonies. Rosenblatt, 

 Meyer, and Robbins^^ found the Chocolate Agar enriched with Bacto-Supple- 

 ment A superior to the unenriched medium. Morton and Leberman^'^ recom- 

 mended the use of Bacto-Supplement A in the Chocolate Agar as it restricted 

 growth of extraneous forms, gave rise to larger gonococcal colonies and in- 

 creased the positive isolations over that obtained on the unenriched medium. A 

 practical method for the isolation of the gonococcus using Chocolate Agar pre- 

 pared from Bacto-Proteose No. 3 Agar, Bacto-Hemoglobin and Bacto-Supple- 

 ment A, is given in detail by Morton^i. 



A recommended procedure for the cultural detection of the gonococcus is 

 described in detail below. 



Collection of Specimen and Inoculation 



Methods for preparing the patient, obtaining adequate specimens and cultur- 

 ing exudates suspected of harboring N. gonorrhoeae are described in detail by 

 Carpenter^'22 and Morton.^^ Specimens are usually collected on sterile cotton 

 swabs. These may be used to inoculate the Chocolate Agar directly or preferably 

 placed in 1-2 ml. of sterile broth. A broth consisting of 2 per cent Proteose 

 Peptone No. 3, 0.5 per cent sodium chloride and 1 per cent soluble starch and 

 adjusted to pH 7.2 is recommended as a suspending fluid. Specimens should be 

 inoculated onto Chocolate Agar as soon as possible after collection and in no 

 case should plating be delayed longer than 8 hours. Specimens not immediately 

 inoculated onto plates should be kept in the ice box. 



The exudate is suspended in the broth by rotating the swab and pressing 

 against the inside of the tube to remove as much material as possible, after 

 which the swab is discarded. About 0.05-0.1 ml. (1-2 drops) of the suspension 

 is then transferred onto sterile plates and smeared over the surface of the 

 medium with a bent glass rod. 



Incubation 



The plates after inoculation should be incubated in an inverted position at 

 35-37°C. for 36-48 hours. 



Best results are obtained in an atmosphere containing carbon dioxide. Cans 

 with a tight fitting cover or Novy jars are satisfactory containers for the plates 

 during incubation. Carbon dioxide can be supplied in one of the following pro- 

 cedures outlined by Christensen and Schoenlein.^s 



1. Place a lighted, smokeless candle near the top of the container, with the 

 plates and put the cover on the container. 



2. Replace about 10 per cent of the air in the container with carbon dioxide. 



3. Place sodium bicarbonate in a beaker in the container with the inverted 

 plates. Cover the sodium bicarbonate with cotton to reduce foaming. Add dilute 



