44 METHODS OF CULTIVATION OF BACTERIA 



and make faintly alkaline to litmus with 20 per cent, caustic potash 

 solution. Heat this slowly to 75°-80° C. lor 5 minutes. Cool to 37° C, 

 and add 1 per cent, of Liquoi Trypsins; Co. (Allen & Hanbury), and 

 keep it at 37° for 2£ to 3 hours. When trypsinising is finished, test for 

 peptone with copper sulphate and caustic potash as below, then render 

 slightly acid with glacial acetic acid, and bring slowly to the boil for a 

 quarter of an hour. Leave overnight in a cool place, and siphon off the 

 clear liquid in the morning. Make this faintly alkaline to litmus, and 

 sterilise in the autoclave at 118° C. for 1 hour on each of two days (if not 

 to be used at once). The result is trypsinised broth. 



To make Trypagar. — Take a measured quantity of the trypsinised 

 broth, add 2 per cent, of agar fibre (see below for preparation), and '215 

 grm. of calcium chloride per litre! Autoclave at 118° C. for three- 

 quarters of an hour to dissolve the agar. Mix together in an urn or 



saucepan; titrate with' — caustic soda while boiling, using phenol- 



phthalein as the indicator, and add the necessary amount of normal 

 caustic potash to give an absolutely neutral reaction. Cool to 60° C, add 

 white of egg (2 to a litre) beaten up with the crushed shells, autoclave 

 again at 118° C. for 75 minutes (or in the steamer for 2 hours). Filter, 

 add to the filtrate 5 per cent, of the sterile pea extract, and sterilise in 

 the ordinary way. For use, a small quantity of sterile rabbit's blood or 

 serum — 5 c.c. to 200 c.c. of medium — is added to the medium in the 

 melted state at 50° C. before being poured into capsules, or a drop or 

 two of serum may be spread with a glass rod over the surface of the 

 medium after it has solidified. 



Preparation of Fibre Agar. — Weigh out the required quantity, cut up 

 small with scissors, place in a large flask or enamel pail, and wash twice 

 quickly in water. Drain thoroughly ; add water just to cover, and put 

 in glacial acetic acid, 2'5 c.c. per litre of water. Mix thoroughly 

 and leave for a quarter of an hour. Pour off the liquid and wash 

 thoroughly four or five times to make sure that all the acetic acid is 

 washed out. Drain carefully, and use as above. 



Biuret Reaction for Peptone. — Take 5 c.c. of broth, add 1 c.c. of 5 

 per cent, solution of copper sulphate. Mix, and then add 5 c.c. normal 

 caustic potash. A true pink colour indicates that trypsinisation is 

 sufficient ; a bluish-purple shade, that it is incomplete. 



Blood Media. 



Blood-Smeared Agar. — This medium was introduced by 

 Pfeiffer for growing the influenza bacillus, and it has been used 

 for the organisms which do not readily grow on the ordinary 

 media, e.g., the gonococcus and the pneumococcus. Human 

 blood or the blood of animals may be used. " Sloped tubes " 

 (vide p. 54) of agar are employed (glycerin agar is not so 

 suitable). Purify a finger first with 1-1000 corrosive sublimate, 

 dry, and then wash with absolute alcohol to remove the sub- 

 limate. Allow the alcohol to evaporate. Prick with a needle 

 sterilised by heat, and, catching a drop of blood in the loop 

 of a sterile platinum wire, smear it on the surface of the agar. 



