132 DIFCO MANUAL 



investigation. Serial inoculation of several plates is recommended to insure suit- 

 able distribution of well isolated colonies. After inoculation, the plates should be 

 incubated at 35-37°C. for 16-18 hours and should be observed at the end of this 

 period because prolonged incubation may lead to confusion of results. 



The differential action of Bacto-MacConkey Agar is clear and distinct. Isolated 

 colonies of coliform bacteria are brick red in color and may be surrounded by a 

 zone of precipitated bile. This reaction is due to the action of the acids, produced 

 by fermentation of lactose, upon the bile salts and the subsequent absorption of 

 neutral red. Typhoid, paratyphoid and dysentery bacilli do not ferment lactose 

 and do not greatly alter the appearance of the medium. These colonies, in reality 

 giving an alkaline reaction, are uncolored and transparent. When growing in 

 proximity to coliform colonies, they have the appearance of clearing the areas of 

 precipitated bile. On plates which are not overcrowded the differentiation is 

 exceptionally distinct. A plate crowded with coli will appear red and opaque, 

 yet, if not too crowded, typhoid or other lactose non-fermenting organisms may 

 easily be detected by transmitted light. On such plates they will appear as small 

 transparent areas against the red background. A plate showing discrete colonies 

 is to be desired for isolation purposes. 



For the cultural detection of typhoid organisms in infected material, it is 

 recommended that Bacto-MacConkey Agar be used in conjunction with the more 

 selective media, Bacto-S S Agar and Bacto-Bismuth Sulfite Agar as discussed on 

 pages 134 and 139. 



A procedure designed to show the largest number of pathogens from a speci- 

 men would be: 



A. Streak or smear a large inoculum on one plate of S S Agar and one plate of 

 Bismuth Sulfite Agar. 



B. Streak or smear a light inoculum on one plate of MacConkey Agar. 



G. Prepare Bismuth Sulfite Agar poured plates with a 5 ml. and a one drop 



inoculum. 

 D. Enrich for 12-18 hours in Tetrathionate Broth or Selenite Broth, followed by 



streaking on one plate of Bismuth Sulfite Agar or Brilliant Green Agar and 



one plate of S S Agar. 



Saccharose in 1 per cent concentration may be added to isolation media, such 

 as Bacto-MacConkey Agar to permit the detection of certain members of the 

 coliform group which ferment saccharose more readily than lactose. This prin- 

 ciple was described by Holt-Harris and Teague^ and has been employed by many 

 other bacteriologists. In some laboratories pathogenic significance is assigned to 

 these organisms, and under such conditions, saccharose should not be added to 

 the medium. 



To rehydrate the medium, suspend 50 grams of Bacto-MacGonkey Agar in 

 1000 ml. of cold distilled water and heat to boiling to dissolve the medium com- 

 pletely. Distribute in tubes or flasks and sterilize by autoclaving for 15 minutes at 

 15 pounds pressure (121°G.). MacGonkey Agar inoculated the same day as re- 

 hydrated may be used without autoclave sterilization. Under these conditions 

 the medium need be heated only to boiling to dissolve it completely before pour- 

 ing into petri plates. The final reaction of the medium will be pH 7.1. 



One pound of Bacto-MacGonkey Agar will make 9 liters of medium. 



i Standard Methods for the Examination of ^ Diagnostic Procedures and Reagents, 3rd Edi- 



Water and Sewage, gth Edition: 228: 1946. tion:24, 195:1950. 



2 Standard Methods for the Examination of * J. Hyg., 5:333:1905. 



Dairy Products, gth Edition: 166: 1948. ^ Am. J. Pub. Health, 130:42:1940. 



*J. Infectious Diseases, 18:596:1916. 



