42 METHODS OF CULTIVATION OF BACTERIA 



coagulation of the medium. These media do not coagulate at 

 100 C. and thus can be sterilised in the steam steriliser. 

 They have been extensively used by American workers in 

 studying the fermentative properties of the b. dysenteriae, 

 b. coli, etc. 



Drigalski and Conradi's Medium. This is one of the media used for 

 the study of intestinal bacteria and especially for the isolation of the 

 typhoid group of organisms, (a) Three pounds of meat are treated with 

 two litres of water overnight ; the fluid is separated as usual, boiled 

 for an hour, filtered, and there are added 20 grammes Witte's peptone, 

 20 grammes nutrose, 1 10 grammes sodium chloride ; the mixture is then 

 boiled for an hour, 60 grammes finest agar are added, and it is placed in 

 the autoclave till melted (usually one hour) ; it is then rendered slightly 

 alkaline to litmus, filtered, and boiled for half an hour. (6) 260 c.c. Kubel- 

 Tiemann litmus 2 solution is boiled for ten minutes, 30 grammes milk 

 sugar (chemically pure) are added, and the mixture is boiled for fifteen 

 minutes ; (a) and (b) are then mixed hot, well shaken, and, if necessary, 

 the slightly alkaline reaction restored. There are then added 4 c.c. of 

 a 10 per cent sterile solution of water-free sodium hydrate and 20 c.c. of 

 a freshly prepared solution made by dissolving '1 gramme crystal- violet 

 B, Hoechst, in 100 c.c. hot sterile distilled water. This is the finished 

 medium, and great care must be taken not to overheat it or to heat it too 

 long, as changes in the lactose may be originated. It is convenient to 

 distribute the medium in 80 c.c. flasks. 



The principle of the medium is that while there is a food supply very 

 favourable to the b. typhosus and the b. coli the antiseptic action of the 

 crystal-violet tends to inhibit the growth of other bacteria likely to 

 occur in material which has been subjected to intestinal contamination. 

 In examining faeces a little is rubbed up in from ten to twenty times its 

 volume of sterile normal salt solution ; in the case of urine or water the 

 fluid is centrifugalised and the deposit or lower portion is used for the 

 inoculation procedures. 



For use the medium is distributed in Petri capsules in a rather thicker 

 layer than is customary in an ordinary plate. The sheet of medium 

 must be transparent, but must not be less than 2 mm. in thickness 

 in fact, ought to be about 4 mm. After being poured, the capsules are 

 left with the covers off for an hour or so, to allow the superficial 

 layers of the medium to become set hard. The effect of this is 

 that during incubation no water of condensation forms on the lid 

 of the capsule, and thus the danger of this fluid dropping on to the 

 developing colonies is avoided. The antiseptic nature of the crystal- 

 violet is sufficient to prevent the growth of any aerial organisms falling 



1 Nutrose is an alkaline preparation of casein. 



2 The litmus solution is made as follows. Solid commercial litmus is 

 digested with pure spirit at 30 C. till on adding fresh alcohol the latter 

 becomes only of a light violet colour. A saturated solution of the residue is 

 then made in distilled water and filtered. When this is diluted with a little 

 distilled water it is of a violet colour, which further dilution turns to a pure 

 blue. To such a blue solution very weak sulphuric acid (made by adding 

 two drops of dilute sulphuric acid to 200 c.c. water) is added till the blue 

 colour is turned to a wine-red. Then the saturated solution of the dye is added 

 till the blue colour returns. 



