48 METHODS OF CULTIVATION OF BACTERIA 



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

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

 Kubel-Tiemann litmus 1 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 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.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 fteces, a little is rubbed up in from ten to twenty times its 

 volume of sterile bouillon ; 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 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 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 spatula made by bending 3 inches of a piece of glass rod at 

 right angles to the rest of the rod. This part is dipped in the infective 

 material, and smeared in all directions over the surfaces of three or four 

 plates successively without any intervening sterilisation. 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 the 

 typhoid bacillus, being often heaped up in the centre, and the 

 contour of the colony is often double. 



J 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 aud 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. 



