CHOLERA. . 155 



A small quantity of the material from the intestine is mixed with double 

 its quantity of faintly alkaline meat broth, which is then allowed to stand 

 at a temperature of 35 C. for about twelve hours ; at the end of that time 

 the comma bacilli have multiplied to such an extent, especially where they 

 are in contact with the air, that if a small portion of the pellicle from the 

 surface is stained and examined, as above, it is found to consist of an 

 almost pure cultivation of comma bacilli which are, however, som e what 

 shorter than those usually met with. If the cultivation be left undisturbed 

 for a longer period the bacilli generally grow larger, and eventually spirilla 

 may be formed, but in the course of a few days the other bacteria grow 

 so luxuriantly that the comma bacilli no longer predominate, and in many 

 cases they are almost entirely "overgrown." From the earlier " maass" 

 cultivation so prepared, in which the relative proportion of cholera bacilli 

 is enormously increased, "plate" cultivations may now readily be made. 

 The plate method as employed by Koch is the best, and the one least open to 

 fallacy for obtaining pure cultivations. It is carried out as follows : On a 

 sterilized platinum needle a drop of the above fluid, of the cholera discharge 

 or of the contents of the small intestine is taken. This is introduced into 

 a test tube one-third full of nutrient gelatine, liquefied at as low a tem- 

 perature as possible by immersing the lower portion of the tube in warm 

 water. The seeded gelatine is carefully shaken in order that the organisms 

 may be disposed widely and equally throughout its substance ; three 

 small drops of this are then taken on a looped platinum wire and added 

 to a second tube, which is similarly shaken, and from this second tube five 

 drops are transferred to a third tube. After the contents of each tube have 

 been thoroughly mixed and the seed material taken for the next tube, the 

 remainder is poured on to a glass plate previously sterilized ; each of the 

 three plates is carefully labelled and placed under a bell jar in which the 

 air has been thoroughly sterilized (see Appendix). The plate from tube 

 No. I is found to contain a large number of organisms, that from tube 

 No. 2 contains a much smaller number, and that from tube No. 3 a much 

 smaller number still, so that as the organisms are developed (one colony 

 from each organism or group of organisms) there is sure to be sufficient 

 space on one or other of the plates between the different groups to allow 

 of a careful study of the growths as they extend, and at the same time there 

 is a sufficient number of points to ensure the appearance of several growths 

 of each kind of organism that was present in the original seed material. 



If plates made according to Koch's method be kept in 

 a chamber in which the temperature is maintained at 

 a little over 20 C. small greyish or white points are first 

 seen ; examined with a low power lens, each of these has 

 a slight yellow tinge and a somewhat wavy margin ; as 

 the " colony " increases in size its colour becomes slightly 

 deeper, the margins become more and more crenated, 

 and the surface somewhat granular. Slow liquefaction 

 is now found to be taking place, and small funnel-shaped 

 depressions, any one of which seldom measures more 

 than ip in diameter, are seen in the gelatine ; at the 



