Il6 STUDY AND IDENTIFICATION OF BACTERIA 



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 three to five 

 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 

 two 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 microscope 

 (2/3 in.) shows a scintillating motility. The emulsion made into the drop of serum 

 quickly shows a curdy agglutination and upon examination with the 2/3-in. objective 

 shows clumping and absence of motility. Cover-glasses placed over the two 

 vaseline rings assist in the study of the preparation. 



