356 METHODS OF OBTAINING PURE CULTURES 



Blood, serum, and ascitic or hydrocele fluid are substances that are frequently 

 used to stimulate the growth of certain organisms ; glucose and other sugars, 

 extracts of vegetable and animal tissues, and certain salts such as potassium 

 nitrate, are likewise used for the same purpose. On the other hand certain aniUne 

 dyes, phenol, telluric acid, bile salts, and numerous other substances are used 

 for inhibiting the growth of various organisms. Gentian violet, in a concen- 

 tration of about 1-10,000, suppresses the growth of most Gram-positive organisms, 

 while allowing most Gram-negative organisms to develop. Used at 1-500,000 

 it is of value in separating streptococci from staphylococci, since the latter organ- 

 isms are inhibited by this concentration. Brilliant green is often used for pre- 

 venting the growth of lactose-fermenting organisms in cultures from the stools. 

 It is added to the faecal suspension in broth in a concentration of about 1-150,000 ; 

 the culture is incubated, and plated out on a suitable medium after 18 to 24 hours. 

 Many Gram-negative bacteria are inhibited by potassium tellurite in a concen- 

 tration of 1-80,000 or more, while penicillin exercises an inhibitory action mainly 

 on Gram-positive bacteria (Fleming 1932). 



L. Indicator Media. — These are media that contain an indicator which changes 

 colour when a certain organism or group of organisms develops. Thus, if it is 

 known that the organism which it is desired to cultivate produces H2S, lead acetate 

 may be added to the medium ; the colonies of the organism are coloured brown, 

 owing to the production of lead sulphide, and can be readily picked off for identi- 

 fication. The diphtheria bacillus reduces sodium tellurite, whereas many of the 

 organisms likely to be associated with it in a throat swab do not. When this 

 substance is added to the medium, the colonies of the diphtheria and of the diph- 

 theroid bacilli are coloured black, whereas those of the streptococci and numerous 

 other organisms are colourless. Litmus and neutral red are two dyes that are 

 frequently used to indicate the production of acid from some carbohydrate incor- 

 porated in the medium. Colonies of organisms that ferment the sugar are coloured 

 red owing to the production of acid, whereas those that do not do so take on the 

 alkaline colour of the dye — blue and yellow respectively. Blood is a very useful 

 indicator. Some organisms produce no alteration in it, others form from it a 

 green pigment, while others lyse it completely. It is usually added to agar in a 

 concentration of 5 per cent. The colonies of the first class leave the medium 

 unchanged ; those of the second class are surrounded by a greenish ring ; those 

 of the third class by a perfectly clear transparent ring. It is used particularly in 

 the differentiation of the streptococci. 



Selective and indicator media are frequently combined. Thus in MacConkey's 

 medium, bile salts are added to inhibit the growth of organisms other than those 

 capable of multiplying freely in the intestine, and lactose and neutral red are added 

 to distinguish the lactose-fermenting coliform organisms from the non-lactose- 

 fermenting group. 



M. Pathogenicity Methods. — The introduction of the pathogenicity method of 

 separating organisms from one another we owe to Koch (1880). By this means he 

 succeeded in separating streptococci from Erysipelothrix muriseptica. When the 

 mixed culture was injected into the ear of a house mouse, Ery. muriseptica proli- 

 ferated, invaded the blood stream, and could be obtained in pure culture from the 

 heart's blood after death ; the streptococcus proliferated locally but did not invade 

 the blood stream. As it was mixed in the local lesion with Ery. muriseptica it 

 could not be obtained in pure culture. But Koch found that if the mixed culture 



