NUTROSE AGAR 173 



Blood Serum, page 168, steps i to 5) and add to it 

 450 c.c. sterile distilled water. 



2. Weigh out agar powder, 20 grammes, and emulsify 

 it with 2 50 c.c. of the cold serum water. 



3. Weigh out 



Witte's peptone 10 grammes 



Sodium chloride . 5 grammes 



Nutrose 10 grammes 



and dissolve in 200 c.c. of serum water heated to 80 C. 



4. Mix the agar emulsion and the peptone-nutrose 

 solution in a " tared " flask of 2 -litre capacity and add a 

 further 100 c.c. serum water. 



5. Complete the solution of the various ingredients 

 by bubbling live steam through the flask as in making 

 nutrient agar. 



6. Add further 250 c.c. serum water. 



7. Weigh the flask and its contents: then (1045 

 grammes + weight of flask) minus (weight of flask and 

 its present contents) = weight of fluid required to make 

 up the bulk of the medium to i litre. Add the requisite 

 amount of sterile distilled water. 



8. Titrate and estimate the reaction of the medium 

 mass. Then standardise to reaction of +2.5. 



9. Clarify with egg, and filter as for nutrient agar. 

 (In clarifying, after the addition of the egg white 

 the mixture should be in the steamer for full two 

 hours.) 



10. After filtration is complete measure the filtrate, 

 and to every 150 c.c. of the medium add: 



Litmus solution (Kahlbaum) 20 c.c. 



Krystal violet aqueous solu- 

 tion (1:1000) (B. Hoechst) 1.5 c.c. 



Lactose 1.5 grammes 



11. Tube in quantities of 15 c.c. 



12. Sterilise in the steamer at 100 C. for thirty min- 

 utes on each of three successive days i.e., by the dis- 

 continuous method for three days. 



