498 APPLICATION- OF METHODS OF BACTERIOLOGY 



(a) Water . . 1000 c.c. 



Powdered agar-agar ." 15 grams 



Peptone (Witte) . 10 grains 



Meat Extract (Liebig) 3 grams 



Heat until the agar-agar is dissolved, keeping the mass to 

 100 c.c. volume by addition of water. This should require 

 about an hour over the flame, or less if the mass be dissolved 

 in the autoclave. Render just alkaline to litmus by the 

 addition of deci-normal sodium hydroxide solution. Filter 

 and decant to flasks containing 100 c.c. each. Sterilize. 



(6) Prepare a 10 per cent, solution of fuchsin in 96 per 

 cent, alcohol. 



(c) Prepare a 10 per cent, solution of sodium sulphite in 

 water. 



For the making of the plates mix 1 c.c. of (6) with 10 c.c. 

 of (c) and heat in the steam sterilizer (100° C.) for 20 minutes. 

 This decolorizes the fuchsin. To each 100 c.c. of the agar 

 prepared as (a), add 1 per cent, of chemically pure lactose 

 and heat in the steam sterilizer at 100° C. until the agar- 

 agar is completely liquefied and the lactose dissolved. To 

 each 100 c.c. of this lactose-agar add 1 c.c. of the decolorized, 

 fuchsin solution, mix thoroughly and while still fluid and 

 warm, pour into sterile Petri dishes; sufficient in each dish 

 to give a layer of from 3 to 5 mm. depth. Place these dishes, 

 with the covers removed, in the incubator until the agar- 

 agar has set; this will require about 30 minutes. They are 

 then ready for inoculation. The plates are now inoculated 

 by spreading evenly over the surface small quantities from 

 the primary "enriching" cultures. This is best done by, 

 the use of a bent glass rod that has been sterilized in the 

 flame and allowed to cool. ^ 



If typhoid bacilli be present they develop as tiny, trans- 

 parent, practically colorless colonies of from 1 to 2 mm. 



