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tube (3” by 3”) containing a few drops of sterile water, mix 
well, and place 6 sterile iron wire loops in the mixture—one 
wire is used to inoculate each of the 6 tubes containing the 
sugars. 
The tests that are recommended by MacConkey are sac- 
charose, dulcit, adonit, inulin. Two other tubes are also in- 
oculated, the one a glucose tube for the Vosges and Proskauer’s. 
reaction, and a broth tube for the indol reaction and for 
motility. 
The four sugar media are made up with sugar free peptone, 
bile salt, and neutral red, according to MacConkey’s formula. 
The glucose tube contains neither neutral red nor bile salt. 
These tubes are placed after inoculation into one rack and are 
incubated for 48 hours at 37°C. The broth tube, however, is. 
examined for motility after 18 hours’ growth of the organism. 
The reaction in the sugars is considered to have taken place if 
gas and acid are developed. Gas is not always very apparent. 
The Vosges and Proskauer reaction is completed by adding a 
few drops of a very concentrated solution of caustic potash 
after 48 hours’ (or more) incubation. The red colouration 
nearly always appears within an hour, but in some cases it is 
delayed. The tubes are, however, kept for 24 hours after the 
caustic potash has been added. The indol test is done with 
persulphate of potash and paradimethyl-amido-benzaldehyd. 
The method of recording the results of these changes in the 
sugars will be seen from the tables given of the various 
analyses, all tubes that give a definite reaction are marked 
as +, those that fail — ; it is very seldom that there is any doubt 
about the result, but when this does occur an extra 24 hours. 
in the incubator usually settles the matter definitely one 
way or the other. 
These may be taken as the working details of the method 
used in this book; the various media will be found in 
the table given in Appendix C. The method, it will be 
observed, does not differ materially from all others in daily 
use, the important improvement being that the organisms, 
that grow in the bile salt broth, are, by the use of MacConkey’s. 
system of passing each organism through certain fermentation 
tests, separated out into their individual species. 
