IT.] PREPARATION OF CULTURE MATERIAL. 15 



to the air ; the proportion of gelatine in the mixture was 2 to 

 3 per cent. But this mixture, although solid at ordinary tem- 

 perature, does not keep solid in the incubator, not even at 20 

 C. I have found that at least 7 '5 per cent, of gelatine must be 

 contained in the mixture to keep it solid at 20 to 25 C. 

 Above this temperature not even 11 per cent, gelatine will 

 keep solid. 



The finest (gold label) gelatine, in thin tablets, is cut up in 

 small strips ; these are soaked in distilled water (1 in 6) over- 

 night, and then dissolved over a water-bath, well neutralised 

 with carbonate of sodium, and filtered hot. If not clear, it is 

 boiled with white of egg, and passed hot through sterilised 

 fine calico. Then this fluid is mixed with half its bulk of 

 broth, peptone solution, or beef-extract solution, so that there 

 is 1 part of gelatine in 9 parts of fluid, or 11 J per cent, of 

 gelatine. This mixture is boiled repeatedly and treated like 

 broth, as described above. The mixture can, when cast solid, 

 be liquefied by melting it on the water-bath, can be easily 

 decanted into sterilised plugged test-tubes (see below), and can 

 then be used as a good solid nourishing material for the 

 cultivation of organisms up to 25 C. 



The above gelatine solution without admixture can be boiled 

 once or twice, and thus made sterile and kept as a stock. 

 This can be used as an addition to blood-serum or hydrocele 

 fluid ; the mixture must be sterilised in the same way as serum 

 or hydrocele fluid alone, i.e. exposed for five to seven days to 

 a temperature of 58 to 62 C. Of course, whatever the propor- 

 tions are in which the two are mixed, the mixture does not keep 

 solid above 25 C. But by exposing it for from several days 

 to several weeks to the heat of the incubator, the mixture 

 can by evaporation be rendered practically solid for higher 

 temperatures also. 



3. More satisfactory, because capable of remaining solid at 

 any temperature, is solid serum of blood, solid hydrocele fluid 

 (G. Makins), and Agar-Agar (Koch). 



The first, i.e. the serum of blood, and the second, i.e. the 

 hydrocele fluid, can be made solid by heating the above sterile 

 serum or hydrocele fluid respectively (see page 13) gradually 

 up to 68 70 C. In the course of an hour or two the 

 material becomes solid, losing slightly its limpidity, but 

 is sufficiently transparent for all practical purposes. By 

 heating it rapidly, or heating it above 70, it becomes solid, 

 granular, and opaque. Of course, once thus made solid it 

 cannot be liquefied again, and therefore must be already 

 contained in the vessels (test-tubes and small flasks^ in which 



