DIAGNOSIS OF CHOLERA 135 



3. At present the same methods are being used for cholera vaccines as for those of 

 typhoid. An emulsion of the vibrios in salt solution or a bouillon culture is subjected 

 to a temperature of S4C. for one hour. Three doses are injected seven to ten days 

 apart, going from 500,000,000 to 2,000,000,000. 



Among the Greek forces 0.45% of the inoculated and 1.9% of the noninoculated 

 were attacked. The inoculated, however, were sanitary troops and hence more 

 exposed to infection. 



For diagnosis: i. take a fleck of mucus, make a straight smear and 

 fix; stain. with a i :io carbol fuchsin. The comma-shaped organisms 

 appear as fish swimming in a stream. 



2. Inoculate a tube of peptone solution. The cholera spirilla grow 

 so rapidly, and being strong aerobes, they grow on the surface of the 

 fluid so that by taking a loopful from the surface, we may in three to 

 eight hours obtain a pure culture. Should there be a pellicle present, 

 this should be avoided in the transfer by tilting the tube slightly, so that 

 the material near the surface be obtained without touching the pellicle. 

 Inoculate a second tube from the surface of this first and, if necessary, 

 a third (enrichment method). 



3. Test for cholera red reaction. (Simply adding from 3 to 5 

 drops of concentrated chemically pure sulphuric acid to the first or 

 second peptone culture after eighteen to twenty-four hours' growth. 

 Some specimens of peptone do not give the reaction.) At times we 

 only get the cholera red when we have a pure culture of cholera. 



4. Smear a fleck of mucus or, better, the three-hour surface growth of 

 a peptone culture on a dry agar surface in a Petri dish. From colonies 

 developing, make agglutination and, if desired, cultural tests. It 

 is by immunity reactions that we identify cholera spirilla. The surface 

 moisture of plates is best dried by the filter-paper top. 



The cholera colony is easily distinguished from the ordinary faecal bacterial colonies 

 by its transparent, bluish-gray, delicate character. It emulsifies with the greatest 

 ease. A practical, quick method is to make smears from suspicious colonies, stain 

 for one minute with dilute carbol fuchsin and if vibrios are present to make 2 

 vaseline rings on a single slide allowing ample space at one end for handling the 

 preparation safely. Inside of one ring deposit with a platinum loop a drop of salt 

 solution and inside the ring nearest the end which is to be held by fingers or forceps, 

 deposit a loopful of i to 500 or i to 1000 dilution of cholera serum. The emulsion 

 in the salt solution remains uniformly turbid and under a low power of the micro- 

 scope (% inch) shows a scintillating motility. The emulsion made into the drop 

 of serum quickly shows a curdy agglutination and upon examination with the 

 %-inch objective shows clumping and absence of motility. ^ Cover-glasses placed 

 over the 2 vaseline rings assist in the study of the preparation. 



Cholera selective media are considered under "Culture Media." 



