396 BACTERIA PATHOGENIC TO MAN 



whitish, viscid mass. Later, the funnel increases in depth and diame- 

 ter, and at the end of from four to six days may reach the edge of the 

 test-tube; in from eight to fourteen days the upper two-thirds of the 

 gelatin is completely liquefied. (See Fig. 124.) Freshly isolated cholera 

 vibrios liquefy gelatin more rapidly than old laboratory cultures; a 

 certain variation, under some circumstances, in the characteristic lique- 

 faction of the gelatin even in fresh cultures, should be borne in mind 

 in making a diagnosis. Such variations in cultural peculiarities occur 

 also with other bacteria. 



Upon the surface of agar the comma bacillus develops a moist, shin- 

 ing, grayish-yellow layer. In agar-plate cultures, for diagnostic pur- 

 poses, the growth of separated colonies is of great importance. The 

 nutrient agar after pouring in the plates and solidifying should be 

 slightly dried on the surface by putting the uncovered plate face down- 

 ward on the shelf of the incubator at 37 C. for thirty minutes, or at 

 60 C. for five minutes. The cholera colonies develop fairly charac- 

 teristically, being more transparent than those of most other bacteria 

 except the cholera-like vibrios. Blood serum is rapidly liquefied at 

 the temperature of the incubator. On potato at incubator temperature 

 a moist growth of a dirty-brown color occurs. Milk is not coagulated. 

 In bouillon the growth is rapid arid abundant; in the incubator at the 

 end of ten to sixteen hours the liquid is diffusely clouded, and on the 

 surface a wrinkled membranous layer is often formed. In general 

 the spirillum grows in any liquid containing a small quantity of organic 

 matter and having a slightly alkaline reaction. An acid reaction of 

 the culture medium prevents its development, as a rule; but it has the 

 power of gradually accommodating itself to the presence of vegetable 

 acids. Abundant development occurs in bouillon which has been 

 diluted with eight to ten parts of water and in simple peptone solution. 



The comma bacillus belongs to the class of aerobic organisms, inas- 

 much as it grows readily only in the presence of oxygen, and that it 

 develops active motility only when a certain amount of oxygen is 

 present. It does not grow in the total absence of oxygen, but a small 

 quantity of oxygen is all that is required for its development, as in the 

 intestines. This need of oxygen tends to send the spirilla to the surface 

 of fluid culture media. 



CHOLERA-RED REACTION. When a small quantity of chemically pure 

 sulphuric acid is added to a twenty-four-hour bouillon culture of the 

 cholera bacillus containing peptone a reddish-violet color is produced. 

 Brieger separated the pigment formed in this reaction the so-called 

 cholera-red and showed that it was indol, and that the reaction was 

 nothing more than the well-known indol reaction. Salkowski and Petri 

 then demonstrated that the cholera bacilli produced in thin bouillon 

 cultures, along with indol, nitrites by reducing the nitrates contained 

 in small quantities in the culture media. They showed that it is the 

 nitric acid, liberated by the addition of sulphuric acid to the culture, 

 which gives rise to the indol, the red body upon which the cholera 

 reaction depends. For a long time it was belie ved that this nitroso- 



