44 METHODS OF CULTIVATION OF BACTERIA 



blood may be obtained in several ways, preferably by bleeding 

 from the carotid. For this purpose the vessel is exposed and 

 as long a portion as possible is cleaned. This is ligatured high 

 up, and a ligature is loosely applied round the lower part of 

 the vessel in such a way as not to constrict it, The vessel is 

 clamped above this ligature, and with scissors an oblique 

 opening is made in its side. The clamp being removed, the 

 stream of blood is directed by means of the ligature into the 

 mouth of a stout sterile flask, which ought to contain some 

 fragments of broken glass rod. During the bleeding the flask 

 should be gently agitated, and when filled should be shaken in 

 a bath of water just below blood-heat. We have found that 

 sterile blood can be obtained from the ear vein of the rabbit by 

 the method of bleeding to be subsequently described. The ear 

 is well washed with lysol, the lysol dried off with sterile wool, 

 absolute alcohol dropped on and allowed to evaporate, arid the 

 blood withdrawn. The first c.c. or so is rejected. 



However the blood is obtained, after defibrination it is warmed 

 to 45 C., and added to agar of the same temperature in the 

 proportion of about one-third of blood and two-thirds of agar. 

 Needless to say, such media must be incubated before use to 

 ensure that bacteria have not gained access during preparation. 



Bordet and Gengou's Medium for Bacillus of Whooping-cough. 



An extract of potato is first prepared by adding two parts of water contain- 

 ing 4 per cent, of glycerin to one part of potato chips ; the mixture is 

 then boiled and- the fluid is separated off. An agar medium is then 

 prepared of the following composition : potato extract, 50 c.c.; '6 per cent, 

 solution of sodium chloride, 150 c.c.; and agar, Sgrrns. Of this medium, 

 2-3 c.c. is placed in each of a series of test- tubes, and then to each there 

 is added, by the method described in the preceding paragraph, an equal 

 part of defibrinated rabbit's (or better, human) blood, obtained by aseptic 

 precautions. The mixture is then allowed to solidify in the sloped 

 position. This medium is also very suitable for the growth of the 

 gonococcus, meningococcus, and influenza bacillus. 



t 



Blood- Alkali- Agar (Dieudonne). This medium, introduced 

 for the. culture of the cholera spirillum, for which purpose it has 

 been found extremely suitable, has the property of inhibiting 

 the growth of most of the intestinal bacteria ; for example, the 

 b. coli does not grow on it, or does so very slightly. A blood- 

 alkali solution is prepared by adding equal parts of defibrinated 

 ox blood and of normal caustic soda solution ; the solution may 

 then be sterilised in the steam steriliser. Of this solution three 

 parts are added to seven parts of ordinary peptone-agar rendered 

 neutral to litmus, and the mixture is disposed in test-tubes. 



