SOLID TRANSPARENT MEDIA. 49 



Sterilizing the Gelatine. The gelatine is kept in little flasks 

 or poured at once into sterile test-tubes, careful not to wet the 

 neck where the cotton enters, lest when cool the cotton plug 

 stick to the tube. 



The tubes are then placed in steam-chest for three successive 

 days, 15 minutes each day (or in water-bath 1 hour a day for 

 three days). Then set aside in a temperature of 15 to 20 C., 

 and if no germs develop and the gelatine remains clear, it can 

 be used for cultivation purposes. 



Modifications. The amount of gelatine added to the meat- 

 water can be variously altered, and instead of making gelatine 

 bouillon the gelatine can be mixed with milk, blood, serum, 

 urine, and agar-agar. 



The nutrient gelatine bouillon can also receive additions iu 

 the shape of glycerine (4 per cent, to 6 per cent, being added), 

 or reducing agents to take up the oxygen present. 



Agar-Agar. This agent, which is of vegetable origin, derived 

 from sea-plants gathered on the coasts of India and Japan, has 

 many of the properties of gelatine ; retaining its solidity at a 

 much higher temperature, it becomes liquid at 90 C. and con- 

 geals again at 45 C. Gelatine will liquefy at 35 C. 



It is not affected very much by the peptonizing action of 

 the bacteria. 



Preparation of Agar-Agar Bouillon or Nutrient Agar. The 

 ordinary bouillon is first made, and then the agar cut in small 

 pieces, added to the bouillon (15 grammes of agar to 1000 

 grammes bouillon). 



It is allowed to stand several minutes until the agar swells, 

 and then placed in water-bath or steam-chest for six hours or 

 more. The reaction is taken, very little of the alkali being 

 sufficient to neutralize it. 



A white of an egg added, and boiled for several hours longer, 

 when, even if not perfectly clear, it is filtered. 



The filtering process, very difficult because of the readiness 

 with which the agar solidifies, must be done in steam-chest or 

 with hot-water filter, and very small quantities passed through 

 at a time, changing the filter-paper often. 

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