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by hand or in a shaking machine) for ten minutes. 4. Liquefy and measure out into a flask Nutrient agar 750 c.c. then cool to 55 deg. C. 5. Mix the oleic acid emulsion with the agar. 6. Add 10 c.c. sterile neutral red, 1 per cent. aqueous solution. 7. Tube in quantities of 10 c.c., slant, and allow to set. 8. Incubate for forty-eight hours at 37 deg. C. and reject any contaminated tubes. Store the sterile tubes for future use. _Coli-typhoid Group._ ~Parietti's Bouillon.~-- 1. Measure out pure hydrochloric acid, 4 c.c., and add to it carbolic acid solution (5 per cent.), 100 c.c. Allow the solution to stand at least a few days before use. 2. This solution is added in quantities of 0.1, 0.2. and 0.3 c.c. (delivered by means of a sterile graduated pipette) to tubes each containing 10 c.c. of previously sterilised nutrient bouillon (_vide_ page 163). 3. Incubate at 37 deg. C. for forty-eight hours to eliminate contaminated tubes. Store the remainder for future use. ~Carbolised Bouillon.~-- 1. Prepare nutrient bouillon (_vide_ page 163, sections 1 to 6). Measure out 1000 c.c. 2. Weigh out carbolic acid, 1 gramme (2.5 or 5 grammes may be needed for special purposes), and dissolve it in the medium. 3. Tube, and sterilise as for bouillon. ~Carbolised Gelatine.~-- 1. Prepare nutrient gelatine (_vide_ page 164, sections 1 to 7). Measure out 1000 c.c. 2. Weigh out carbolic acid, 5 grammes (= 0.5 per cent.), and dissolve it in the gelatine. 3. Filter if necessary through papier Chardin. 4. Tube, and sterilise as for nutrient gelatine. One or 2.5 grammes of carbolic acid (= 0.1 per cent. or 0.25 per cent.) are occasionally used in place of the 5 grammes to meet special requirements. ~Carbolised Agar.~-- 1. Prepare nutrient agar (_vide_ page 167, sections 1 to 8). Measure out 1000 c.c. 2. Weigh out 1 gramme pure phenol and dissolve in the medium. 3. Filter if necessary through papier Chardin. 4. Tube, and sterilise as for nutrient agar. ~Litmus Gelatine.~-- 1. Prepare nutrient gelatine (_vide_ page 164, sections 1 to 8). 2. Add sterile litmus solution, sufficient to tint the medium a deep lavender colour. 3. Tube, and sterilise as for nutrient gelatine. ~Lactose Litmus Bouillon (Lakmus Molke).~-- 1. Weigh out peptone, 4 grammes, and emulsify it with 200 c.c. meat extract (_vide_ page 148), previously heated to 60 deg. C. 2. Weigh
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