50 METHODS OF CULTIVATION OF BACTERIA 



Kubel-Tiemann litmus 1 solution is boiled for ton minutes, 30 grros. 

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

 fifteen minutes ; {a) and (ft) 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 carbonate 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. e. flasks. 



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

 favourable to the b. typhosus and the b. eoli, 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 feces, a little is rubbed up in from ten to twenty times its 

 volume of sterile bouillon (a properly made emulsion should just be 

 short of being opaque) ; in the case of urine or water, the fluid is 

 centrifugalised and the deposit or lower portion is used for the inocula- 

 tion procedures. 



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

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

 must be transparent, but must not be less than 2 inm. 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 in- 

 cubation 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 on the agar during 

 its exposure to the air. The plates are usually inoculated by means of 

 a glass spreader made by bending 2 inches of a piece of glass rod at 

 right angles to the rest of the rod. In the case of feces one or two 

 lodpfuls of an emulsion made as described above are placed on the 

 surface of a plate and thoroughly distributed by means of the spreader ; 

 when the material is less rich in bacteria the spreader may be dipped in 

 the infective fluid. In either case two or three further plates are success- 

 ively spread, without any intervening sterilisation of the spreader. The 

 plates are again exposed to the air after inoculation for half an hour, and 

 then incubated for twenty- four hours. At the end of such a period 

 b. coli colonies are 2 to 6 mm. in diameter, stained distinctly red, and are 

 non-transparent. Colonies of the b. typhosus are seldom larger than 

 2 mm., they are blue or bluish-violet in colour, are glassy and dew-like 

 in character, and have a single contour. Sometimes in the plates 

 b. subtilis and its congeners appear, and colonies of these organisms have 

 a blue colour. Their growth is, however, more exuberant than that of 



1 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. 



