DEHYDRATED CULTURE MEDIA 141 



(b) Insert a loosely packed cotton plug, about 1 inch long^ into the tube, and 

 slowly force it down through the fecal mixture by means of a glass rod or pipette, 

 so that all the gross particles are carried to the bottom of the tube on the cotton 

 plug, and an opaque fluid rises through the cotton. A second cotton filtration may 

 be necessary, since it is essential that the supernatant fluid be free from gross par- 

 ticles. Such solid particles in the medium may support growth of the extraneous 

 organisms, giving pseudo-blackening which may be mistaken for typhoid colonies. 



Some workers may prefer to allow the gross solid particles of fecal suspension 

 to settle by gravity instead of removing them by filtration with cotton. In such 

 cases it is not advisable to allow the suspension to stand longer than 30 minutes 

 in order to obtain a supernatant fluid free from gross particles. Other methods of 

 preparing fecal suspensions that will give a liquid free from gross solid suspended 

 material without removing typhoid may also be employed. 



(c) Transfer about 5 ml. of the prepared fecal suspension to one petri dish 

 and 1 drop to a second dish. Add 20 ml. of Bismuth Sulfite Agar, cooled to 

 45 °C., to each dish, and mix thoroughly. It is necessary to use at least 20 ml. of 

 the medium to each 5 ml. of inoculum, for dilution of the medium beyond this 

 point will allow the development of extraneous fecal forms. 



(d) Incubate at 37°C. and observe after 24 hours for typical colonies as de- 

 scribed below. Frequently typical colonies develop within 24 hours incubation; 

 however, in all cases the plates should be incubated for at least 48 hours to allow 

 the development of all typhoid strains, before considering the specimen negative. 

 Specimens containing only a small number of typhoid should show isolated col- 

 onies from the 5 ml. inoculum, while those specimens containing increasingly 

 large numbers of typhoid organisms should show isolated colonies from the 1 

 drop inoculum in the poured plate or on the smear plate. 



In the examination of samples of urine, blood, sewage or other material, either 

 the poured plate or smear method with Bismuth Sulfite Agar may be used. It is 

 suggested that in examining blood specimens, the specimen first be inoculated 

 into a tube of broth and after preliminary incubation of 8-12 hours, smeared 

 onto the Bismuth Sulfite Agar plate. 



Description of Colonies 



Streak or Smear Plate 



The typical discrete surface typhoid colony is black and is surrounded by a 

 black or brownish-black zone which may be several times the size of the colony. 

 By reflected light, preferably daylight, this zone exhibits a distinctly characteristic 

 metallic sheen. Plates heavily seeded with typhoid may not show this reaction 

 except possibly near the margin of the mass inoculation. In these congested areas, 

 typhoid frequently appears as small light green colonies. This fact emphasizes the 

 importance of inoculating plates in such a manner as to have some sparsely popu- 

 lated areas with discrete typhoid colonies. 



Poured Plate 



Well isolated subsurface typhoid colonies are circular, jet black and well de- 

 fined. The size of the black colony may vary from 1 to 4 mm. in diameter de- 

 pending upon the particular strain, length of incubation and position of the 

 colony in the agar. Only those colonies growing very close to the surface or on 

 the surface will show a decided black metallic sheen. Plates containing typhoid 

 too numerous to permit the development of individual colonies give a black plate 

 or a plate dotted with black areas. Plates with about three hundred to a thousand 

 typhoid colonies will exhibit this appearance. When typhoid develops in a plate 



