ZOOLOGY AND BOTANY, MICROSCOPY, ETC. 471 



tilled water previously heated to 60° C. ; (2) add 10 grm. sodium 

 chloride ; (3) emulsify 40 grm. powdered agar in 400 c.cm. cold dis- 

 tilled water, and mix with the pepton salt solution in a litre flask ; 

 (4) Dissolve the ingredients thoroughly by bubbling live steam through 

 the mixture for half an hour, the flask being suspended in a bath of 

 boiling water during the process. 



B. (1) Measure out 500 c.cm. ox serum into a " tared " 8-litre 

 flask, and add to it 900 c.cm. distilled water ; (2) heat in the steam 

 chamber at 100° C. for half an hour. 



C. (1) Add the agar mass to the diluted ox serum, shake thoroughly 



to mix, and weigh the mixture ; (2) titrate the mixture and add suffi- 



n 

 cient - NaOH to adjust the reaction of the mixture to — 3*5; 



(3) emulsify 20 grm. nutrose in 100 c.cm. distilled water, add the 



emulsion to the mixture in the flask and heat in the steamer for 



30 minutes ; (4) weigh the mixture and make up to 2090 grm. by the 



addition of boiling distilled water ; (5) titrate again, and add sufficient 



n 

 standardised - lactic acid to render the reaction +2*5; (6) cool to 



below 60° C, add the well-beate i whites of four eggs, and heat again 

 in the steam chamber for 80 to 45 minutes ; (7) filter through papier 

 chardin with a tared 2-litre flask, and weigh the filtrate ; about 1900 

 grm. or c.cm. clear agar will be obtained : (8) fill the nutrose agar into 

 small flasks in quantities of 150 c.cm. ; (9) if not needed for immediate 

 use, sterilise in the steamer at 100° C. for 20 minutes on each of three 

 successive days. 



D. (1) Take as many sterilised test-tubes as there are 150 c.cm. 

 flasks of medium, and fill 20 c.cm. Kuhlbaum's litmus solution into 

 each ; (2) heat in the steamer at 100° C. for 20 minutes ; (3) weigh 

 out and dissolve 1*5 grm. lactose in each tube of litmus solution; 



(4) add 1*5 c.cm. of a 0*01 p.c. solution of crystal violet (B. Hochst) 

 to each tube ; (5) sterilise in the steamer at 100° C. on each of three 

 successive days. 



Preparing Plates of Nutrose Agar.* — J. W. H. Eyre gives the 

 following method of making plates of nutrose agar : (1) Liquefy a 

 flask of nutrose agar (for composition and mode of preparation see 

 ants) by immersion in a water-bath at 100° C. As soon as the 

 medium is fluid add the contents from one of the prepared test-tubes 

 and mix thoroughly. (2) Pour the coloured fluid medium into sterile 

 Petri dishes to the depth of 3 or 4 mm. One flask (of 150 c.cm.) will 

 supply sufficient medium for about six Petri dishes of 11 cm. diameter. 

 (3) Open each plate and rest the edge of its cover on the side of the 

 lower half, and so allow the steam to escape freely whilst the agar is 

 solidifying. (4) When the medium has set, open and invert the Petri 

 dishes and place in an incubator at 60° C. for 45 minutes, or at 42° C. for 

 2 hours. At the end of this time the surface of the medium will be 

 firm and dry and ready to inoculate. (5) The material to be plated 

 must first be suspended in either sterile salt solution or sterile broth, 



* Trans. Path. Soc, Iv. (1904) p. 104. 



