MALACHITE GREEN MEDIA 409 



Roth's technique. Prepare broth in the ordinary way and add sufficient soda 

 solution to give a permanent pink colour with phenol- phthalein. Add 80-100 c.c. 

 of a 1 per cent, solution of caffeine to every 100 c.c. of broth. 



Sow the fluid with the material to be examined and incubate at 37 C. for 24 hours, 

 then plate traces of the culture on gelatin. 



Picker's technique. To 100 c.c. of a 3 per cent, peptone-meat-broth add 0'6 gram 

 of pure caffeine and 0*00007 gram of crystal- violet (0'7 c.c. of a O'Ol per cent, solution). 

 Sow the fluid with the suspected material, incubate at 37 C. for 12 or 13 hours and 

 sow Conradi-Drigalski plates with the culture obtained. 



Lubenau's technique. Lubenau sows in 100 c.c. of Ficker's broth containing 

 - 3 per cent, of caffeine, incubates for 13 hours, adds 100 c.c. of broth containing 0*6 

 per cent, of caffeine, incubates for a second period of 13 hours and adds 100 c.c. of 

 broth containing 0*9 per cent, of caffeine. Before and after the second addition of 

 broth he sows surface plate cultures on litmus-lactose-caffeine-agar for purposes 

 of isolation. 



Lubenau's caffeine-cigar. Prepare a litre of 6 per cent, peptone- beef -broth, 

 dissolve 4060 grams of agar in the broth, make neutral to litmus, heat to 120 C., 

 filter and sterilize. After sterilization and while the agar is still hot add 60 c.c. of 

 litmus solution, 5 grams of lactose and finally 110 c.c. of a 6 per cent, solution of 

 pure caffeine. Distribute in Petri dishes. 



Gathgens' technique. To a litre of Endo's medium add 33 c.c. of a 10 per cent, 

 solution of pure caffeine. Distribute in tubes hi quantities of 15 c.c. which can 

 afterwards be used to prepare plates, 



9. Malachite green media. 



Loeffler has stated that the addition of a certain quantity of malachite 

 green to culture media impedes the development of the colon bacillus while 

 having no effect on the growth of the typhoid and paratyphoid bacilli. 



Unfortunately malachite green media are not so selective as Loeffler believed, 

 for though the growth of a large number of organisms (streptococci, staphy- 

 lococci, cholera vibrios, etc.) is inhibited the colon bacillus will often grow 

 (Kiralyfi). 



These media are difficult to prepare ; if too much green be added the 

 typhoid bacillus is inhibited, if too little, the colon bacillus grows as rapidly 

 as the typhoid bacillus. It is essential to use a chemically pure compound 

 and the amount to be added to the agar varies very much with the different 

 commercial preparations. A series of experiments should be done to deter- 

 mine the quantity (1 in 4,000 to 1 in 6,000) to be added (Lentz and Tietz, 

 Schindler). These different complications render the method of little practical 

 value, and to make it more efficient Loeffler has recently advised the addition 

 of ox bile to his malachite green media (vide infra). As the result of his own 

 experience Fiirth concludes that methods based upon the use of malachite 

 green are inferior to Conradi-Drigalski's method. 



Growth on malachite green media diminishes the agglutinability of the 

 typhoid bacillus. 



Of the different malachite green methods Leuch's seems to be the best. 



Leuch's technique. Prepare an agar medium with : 



Beef, ... - 500 grams. 



Water, - 1 litre. 



Common salt, - 5 grams. 



Dextrin. - 10 



Agar, - - - 30-40 



Neutralize, using litmus as the indicator. Add 5 c.c. of a normal solution of 

 sodium carbonate and 100 c.c. of a 10 per cent, solution of nutrose. After filtering 

 and sterilizing add 16-18 c.c. of a 1 per cent, solution of malachite green. 



Surface cultures are sown in Petri dishes (p. 407). Colonies of the typhoid bacillus 

 destroy the colour of the medium and a characteristic yellowish zone forms around 

 them. 



