462 APPENDIX 



the slide and heated gently over the flame until the steam begins to rise. Allow to 

 steam for from one to two minutes ; wash well in running water and dry carefully. 

 When thoroughly dry, apply a sufficient quantity of the stain, and heat as before, 

 allowing to steam for two minutes. Wash in distilled water, dry, and examine. 



(2) Van Ermengeni's Method. 



Three solutions are required in this method : — 



A. Fixing Solution. 



Osmic acid, 2 per cent, aqueous solution . . . 10 c.c. 



Tannin, 20 per cent, solution . . . .20 c.c. 



To each 100 c.c. of this mixture add 4 to 5 drops of glacial acetic acid. The 

 colour of this solution should be violet, and the solution should be filtered before use. 



B. Sensitising Solution. 



Nitrate of silver . . . . "5 aqueous solution 



This solution should be kept in the dark, and filtered before use. 



C. Reducing Solution. 



Gallic acid ...... 6 grammes 



Tannin . . . . . . 3 ,, 



Fused acetate of soda (or potassium) . . 10 ,, 



Distilled water . . . . . 350 „ 



(a) Cover the film with solution "A," and allow to act for five minutes at 37° C, 

 or one hour at room temperature. Or heat gently until steam rises, and allow the 

 staining fluid to act for five minutes. 



(6) Wash well with distilled water, then in absolute alcohol, and then again in 

 distilled water. 



(c) Treat with solution " B," and allow it to act for thirty seconds, keeping the 

 fluid in movement on the slide. 



(d) Allow the fluid to run off the slide, and without washing treat with " C" for 

 thirty seconds in the same manner. 



(«) Allow fluid to run off, and again treat with " B " until the preparation begins 

 to turn black. 



(/) Wash in distilled water, mount in water, and examine under the microscope. 

 The method is not wholly satisfactory. 



A simple method is as follows : — 



(3) Night-Blue Method (M'Crorie). 



Place 2 or 3 drops of the emulsion on an absolutely clean slide, and dry at room 

 temperature. It is not necessary or desirable to fix by heat. The stain is made by 

 mixing 10 c.c. of night-blue, saturated alcoholic solution, 10 c.c. of a saturated 

 aqueous solution of potash alum, and 10 c.c. of a 10 per cent, aqueous solution of 

 tannin. The stain must be filtered before use. The slides, as prepared above, are 

 stained with this for two minutes in the hot incubator, and then washed gently in 

 running water. It may be found best to change the blue stain several times during 

 the two minutes. A counter-stain may be used if desired, and one of the best is 

 aniline gentian-violet. This should be applied for about a minute, after which a 

 cover-glass may be fixed over the film with Canada balsam. In such a preparation 

 the bacilli will be stained violet and the flagella blue. Better results may sometimes 

 be obtained by staining deeply with the blue (ten minutes),. and then decolorising to 

 the necessary extent in dilute methylated spirit. 



The Staining of Spores 



The following are the methods commonly adopted : — 



(1) Mailer's Method. 



(a) Prepare the film as usual, fix and dry, observing the precautions taken in 

 preparing milk specimens. 



