ANIMAL TISSUES AND FLUIDS 33 



3. Then heat to boiling to destroy the excess of pepsin, and pass through a 

 sieve or a thin layer of loosely packed absorbent cotton- wool. 



4. Heat the filtrate to 80 C., and neutralize at this temperature : large 

 flocculent masses are precipitated ; filter the clear supernatant fluid through 

 a Chardin paper. 



5. Autoclave the nitrate for five minutes at 117-120 C., and again 

 filter through a Chardin paper. 



6. Distribute the clear filtrate in tubes, and sterilize for 15 to 20 minutes 

 at 115 C. 



Martin's peptone broth. 



1. Mince 500 grams of lean veal, and macerate in 1000 grams of water 

 for 15 to 20 hours at a temperature of 35 C. to get rid of the sugars. 



2. Filter through a cloth, squeeze out as much of the fluid as possible, and 

 add 5 grams of salt. 



3. Mix this liquid with an equal volume of Martin's peptone solution 

 (see above, Stages 1, 2, 3, 4). 



4. Heat to 70 C. to coagulate the albuminoid compounds, and make 

 exactly neutral. Then add 7 c.c. of normal soda solution per litre. Filter 

 through a Chardin paper. 



5. Sterilize by filtering through a Chamberland bougie (Chap. I.), and 

 distribute in sterile culture vessels. 



Note. A broth prepared in this way is particularly useful for the preparation 

 of diphtheria toxin. 1 For everyday use, it is simpler to sterilize by heat. After 

 .Stage 4 proceed thus : 



5a. Pour the broth into an enamelled vessel or flask, autoclave for 5 minutes 

 at 115-117 C., and filter in the warm through a Chardin paper. 



5b. Distribute the filtered liquid into culture vessels and sterilize for 20 minutes 

 at 110-115C. The broth is often slightly and permanently cloudy. 



Koch's peptone solution. 



1. Dissolve in the warm, 10 grams of peptone (Witte or Chapoteaut) and 

 5 grams of salt in 1000 grams of water. 



It is unnecessary to neutralize : peptone itself is sufficiently alkaline. 



2. Boil. Filter." 



3. Distribute in tubes or flasks. Sterilize at 115 C. 



Metchnikoff's peptone-gelatin medium. 



1. Dissolve in the warm, in 1000 grams of water, 



Peptone (Chapoteaut), - 10 grams. 



Salt, - 5 



Gelatin (extra quality white), - 20 



2. Make very slightly alkaline with normal soda solution. 



3. Autoclave for 5 minutes at 115 C. Filter through Chardin paper. 



4. Distribute in suitable vessels. Sterilize at HO -^ C. 



Miquel's peptone solution. 



1. Dissolve at a moderate heat in a litre of water, 



Peptone (Chapoteaut), - - 20 grams. 



Salt, - 5 



2. Add O'lO gram of wood ash. Boil. Filter through Chardin paper. 



3. The liquid is now generally markedly alkaline. Neutralize very care- 

 fully with st solution of tartaric acid, watching the reaction meanwhile by 

 testing against litmus paper. 



1 Numerous formulse for the preparation of broth suitable for the study of diphtheria 

 toxin have been published (Chap. XV.). 



C 



