ISOLATION OF THE VIBRIO 501 



in swarms all pointing the same way like fish in water ; but frequently micro- 

 scopical examination furnishes no certain evidence. Microscopical examina- 

 tion should always be supplemented by sowing cultures with traces of the 

 stools. 



Very pretty microscopical preparations can be made by double staining the film 

 by Gram's method using dilute carbol-fuchsin as a counterstain : the cholera vibrio 

 is red, and the gram-positive organisms violet. 



Dunbar's method. Dunbar applies the phenomenon of agglutination to the 

 detection of the vibrios in the stools. Prepare two hanging- drop cultures with a 

 trace of the suspected material diluted in a drop of peptone water, and add to one 

 a drop of an agglutinating serum. On microscopical examination, the vibrios are 

 motile in the preparation to which no serum was added and non-motile and agglu- 

 tinated in the other. Whether the method be reliable or no, the technique certainly 

 requires much care. 



Staining of sections. Sections of the intestine are best stained by Nicolle's 

 tannin method. 



B. Isolation. 



Stools. The method originally employed consisted in isolating the organism 

 from the stools by means of gelatin or agar plates ; but the plates are very 

 often overgrown with other organisms so that the vibrio cannot be isolated. 

 The following method is preferable. 



Metchnikoff 's method. 1. Prepare a number of tubes of gelatin-peptone-salt 

 solution (p. 33), and after sowing them with a trace of the suspected stool 

 incubate them at 37 C. 



2. After 3 or 4 hours the tubes will be cloudy and in about 7 hours a thin 

 pellicle will have formed on the surface of the medium. Examine a trace 

 of the film under the microscope : as a rule the vibrio will not be found in 

 pure culture but mixed with various other organisms. 



3. To obtain a pure culture sub-cultivate a trace of the pellicle in another 

 tube of the gelatin -peptone-salt solution, incubate for 6 or 7 hours and if 

 necessary (after microscopical examination of a film from the pellicle) sow 

 another tube in the same way ; it is however usually sufficient to plate on 

 agar after the first sub-cultivation. 



Liquefy a tube of agar and pour the medium into a Petri dish, allow it to 

 set and then sow surface stroke cultures with a trace of the material from the 

 pellicle. Incubate at 37 C. After a few hours, small delicate transparent 

 or opalescent but never opaque colonies are visible which can 'be picked off 

 after about 8 hours' incubation. The whole process of isolation thus takes 

 only about 15 hours. / 



Gelatin plates may also be prepared at the same time as the agar plates 

 and an examination of them subsequently will furnish a clue to identification. 



[Note. Instead of the peptone-gelatin medium, ordinary peptone water is 

 very commonly used for the isolation of the cholera vibrio and Ottolenghi has 

 recently suggested bile as an " enrichment " medium for the same purpose.] 



Water. (Metchnikoff's method.) Recommended. 1. Prepare a series of 

 conical flasks of 250 c.c. capacity or thereabouts. Measure 200 c.c. of water 

 into each and make a mark on the glass with a diamond. 



2. To each flask add the following solution : 



Water, - 



Peptone (Chapoteaut), 



Common salt, - 



Gelatin, - 



Solution of soda, 



50 c.c. 

 2 grams. 

 2 



4 

 Q.S. to make 



faintly alkaline. 

 Sterilize in the autoclave. 



