42 



SOLID MEDIA 



2. Meat extract, 500 c.c. 



Peptone, - 5 grams. 



Gelatin, 35 



Calcium phosphate, 50 ,, 



Magnesium phosphate, 5 



Calcium carbonate, 10 ., 



Dissolve the gelatin and peptone in the meat extract : heat in the auto- 

 clave and filter in the ordinary way. After filtration and before distributing 

 in tubes add the calcium and magnesium salts. 



2. Agar media. 



Agar-agar is derived from a sea-weed growing in the Indian Ocean, and 

 in commerce occurs as dried fibrous strands. 



When agar is boiled with water it forms a firm jelly which does not melt 

 below 90 C. Agar is therefore substituted for gelatin whenever a solid 

 medium is required for incubation above 25 C. 



The preparation of agar media is tedious because agar readily forms with 

 water a thick jelly which is difficult to filter. This difficulty is overcome by 

 altering the properties of the agar by prolonged boiling or by chemical 

 action, e.g. the addition of acid. 



Another difficulty arises from the fact that agar is always cloudy if not 

 cleared with albumen, and even then it is sometimes opalescent. 



Ordinary Agar. 



An agar medium prepared according to the method now to be described 

 is generally spoken of as agar, and the word will be so used in this book. 



Preparation of Agar. 



[A. Method recommended. 1. Weigh out 30 grams of agar fibre, turn it 

 into a 2-litre flask and fill the flask nearly full of tap water, then add 10 c.c. 

 of a 2 per cent, solution of acetic acid and stir well with a glass rod. 



[2. Leave the agar to soak for 10 minutes, then put a large funnel into the 

 flask, stand the funnel under the cold water tap and wash the agar in running 

 water until the washings are neutral to litmus paper (10 minutes). 



[3. While preparing the agar, stand a flask containing a litre of broth in 

 the steamer and heat to 100 C. 



[4. Add the washed agar to the hot broth. 



[5. Heat the mixture in the steamer at 100 C. until the agar is dissolved 

 (20 minutes). 



[6. The medium is now a little acid. Neutralize with a 10 per cent, solution 

 of caustic soda (about 1 c.c.) and allow the contents of the flask to cool to 

 -50-60 C. 



[7. Beat up the white of two eggs in a beaker, and add to the cooled agar. 

 Mix thoroughly. 



[8. Heat the mixture in the steamer at 100 C. until the egg-albumin is 

 coagulated, and until on holding up the flask to the light the agar is clear (f-1 

 hour). At the same time put into the steamer a Chardin filter paper arranged 

 in a funnel, the latter standing in a sterile flask. 



[9. When the medium is clear there will nearly always be lumps of coagu- 

 lated albumin floating about in the agar pour it, on to the hot filter in the 

 .steamer. The filter must not be taken out of the steamer, and the medium 

 should be poured down the sides of the filter paper. 



[10. Place the lid on the steamer, and maintain the heat until the medium 

 lias all filtered through (15 minutes). 



