1 84 SPECIAL MEDIA 



3. Inoculate the bouillon from a pure cultivation of the B. 

 lactis aerogenes, and incubate at 37 C. for forty-eight hours. 



4. Heat in the steamer at 100 C. for twenty minutes to destroy 

 the bacilli and some of their products. 



5. Estimate the reaction of the medium and if necessary 

 restore to + 10. 



6. Inoculate the bouillon from a pure cultivation of the B. 

 coli communis and incubate at 37 C. for forty-eight hours. 



7. Heat in the steamer at 100 C. for twenty minutes. 



Now fill two fermentation tubes with the bouillon, tint with 

 litmus solution, and sterilise; inoculate with B. lactis aerogenes. 

 If no acid or gas is formed, the bouillon is in a sugar-free con- 

 dition; but if acid or gas is present, again make the bouillon in 

 the flask + 10, reinoculate with one or other of the above-men- 

 tioned bacteria, and incubate; then test again. Repeat this till 

 neither acid nor gas appears in the medium when used for the 

 cultivation of either of the bacilli referred to above. 



8. After the final heating, stand the flask in a cool place and 

 allow the growth to sediment. Filter the supernatant broth 

 through Swedish filter paper. If the filtrate is cloudy, filter 

 through a porcelain filter candle. 



9. Tube, and sterilise as for bouillon. 



Bouillon prepared in the above-described manner will prove 

 to be absolutely sugar-free; and from it may be prepared nutrient 

 sugar-free gelatine or agar, by dissolving in it the required per- 

 centage of gelatine or agar respectively and completing the medium 

 according to directions given on pages 166 and 167. The most im- 

 portant application of inosite-free bouillon is its use in the prep- 

 aration of sugar bouillons, whether glucose, maltose, lactose, or 

 saccharose, of exact percentage composition. 



Sugar (Dextrose) Bouillon. 



1. Measure out nutrient bouillon, 1000 c.c. (vide page 163, 

 sections i to 6) or sugar-free bouillon (vide svprd). 



2. Weigh out glucose (anhydrous), 20 grammes ( = 2 per cent.), 

 and dissolve in the fluid. 



3. Tube, and sterilise as for bouillon. 



Ordinary commercial glucose serves the purpose equally well, 

 but is not recommended, as during the process of sterilisation 

 it causes the medium to gradually deepen in colour. 



NOTE. In certain cases a corresponding percentage of lactose, 

 maltose, or saccharose is substituted for glucose. 



Sugar Gelatine. 



1. Prepare nutrient gelatine (vide page 164, sections i to 7). 

 Measure out 1000 c.c. 



2. Weigh out glucose, 20 grammes (=2 per cent.), and dissolve 

 in the hot gelatine. 



