43 

 IV. THE DETECTION OF THE COLON GROUP IN WATER. 



Various methods have been suggested for the isolation of members 

 of the colon group. They are all based on the principle that all members 

 of this group of organisms are capable of decomposing lactose with acid 

 and gas production. Media are employed which contain lactose and some 

 indicator to show whether fermentation has taken place. 



Isolation may be accomplished (1) directly by plating on solid dif- 

 ferential media or (2) indirectly on solid differential media, after pre- 

 liminary enrichment in some fluid medium. 



The mediums most commonlly employed for direct isolation are: 



Litmus lactose agar. 



Phenol ated litmus lactose agar. 



Endo agar. 



Conradi Dragalski agar. 



MacConkey agar. 



Litmus lactose agar consists of nutriment agar, neutral to phenolph- 

 thalein, which contains sufficient litmus or azolitmin to give a distinct blue 

 color. Fermentation of lactose with the production of acid is indicated 

 by the formation of red colonies. 



The technique for direct isolation is merely to place definite quan- 

 tities of the test sample (1.0 c. c., 0.1 c. c., 0.01 c. c., etc) into petri dishes 

 to which are added about 10 c. c. of litmus lactose agar. (In some labor- 

 atories the litmus is added to the petri dish and then lactose agar poured 

 in.) The plates are incubated at 37 degrees to 40 degrees C. for 24 hours 

 after which members of the colon group will appear as red colonies on a 

 blue background. As there are other organisms, particularly the strep- 

 tococci, which are capable of fermenting lactose) with acid production, 

 the mere appearance of acid colonies is not absolute proof of the presence 

 of the colon group. In the process of sterilization, lactose is sometimes 

 broken down so that organisms other than the colon group may then 

 produce slightly acid reactions. They must therefore be examined fur- 

 ther. Suspicious colonies are fished to agar and a Gram stain is made 

 to insure that the organism is a Gram negative non-spore-forming rod. 

 From the agar slant various other tests (fermentation of carbohydrates, 

 gelatin liquefaction, milk coagulation, the V. P., methyl red, etc.) may be 

 carried out. 



Acid produced by the growth of fermenting forms diffuses through 

 the medium, often coloring considerable portions of the plate, making it 

 extremely difficult to detect the acid producing colony. The medium 

 exerts very little inhibitory action and overgrowths of non-colon forms 

 are not infrequent. These disadvantages may be overcome to some ex- 

 tent by (1) the use of porous tops to prevent spreaders, (2) by increasing 

 the concentration of agar to 3 percent thereby diminishing considerably 

 the diffusion of acid, and (3) by the addition of selective antiseptics to 

 inhibit the growth of forms other than the colon group. The use of the 

 higher temperature (37 degrees C.) and preferably 40 degrees C., exerts 



