728 THE SPIROCILETE OF SYPHILIS 



1. Pour about 1 c.c. of the following solution on the dried preparation. 



Marino's blue, 1 - O'lO gram. 



Absolute methyl alcohol, - 50 c.c. 



2. Stain for about 3 minutes. 



3. Without washing, pour a few drops of a O005 per cent, aqueous solution 

 of eosin on the film and leave it to stain for 1 or 2 minutes. 



4. Wash. Dry. Mount in balsam. 



The treponemata will be stained blue and violet. 



If the blue has precipitated on account of the preparation having been left too 

 long in the staining bath, flood the film with the blue again for a few minutes, then 

 pass through eosin for 1 minute and finally wash. 



[The above solution of Marino's blue will keep for about 2 months if the 

 methyl alcohol be pure.] 



Hecht and Wilenko's method. 1. Place a drop of the fluid to be examined 

 on a slide. 



2. Add a drop of China ink, mix carefully, spread, leave to dry (half an 

 hour). 



3. When quite dry, examine with an oil-immersion lens. The treponemata 

 appear as bright structures on a black background. 



Ravaut's method. This method depends upon the use of " largine " 

 (albuminate of silver). 



1. Make the films, which may be thick, on a very clean slide. Fix in methyl 

 alcohol or in osmic acid vapour. 



2. Place the slide in a stoppered bottle containing the following solution, 

 which must have been recently made up and kept in the dark. 



Lillienf eld's largine, - - 2 grams. 



Distilled water, - 100 c.c. 



Keep the slide in the stain at 55 C. for 2 hours. 



3. Without washing, dip the slide into a 5 per cent, solution of pure pyro- 

 gallol. The silver is reduced immediately. 



4. Wash in distilled water, return to the largine bath for half an hour and 

 then pass through the pyrogallol solution again. 



5. Wash. Dry. Examine. The treponemata are stained deep brown. 

 The foregoing is more satisfactory than Stern's silver method. Films on slides 



are dried in the air, placed in the 37 C. incubator for a few hours, then transferred 

 to a white glass bottle containing a 10 per cent, solution of silver nitrate and exposed 

 to diffused light for a few hours. The slides which now have a metallic appearance 

 are washed in running water, dried and examined. The treponemata are stained 

 deep brown or black. 



Borrel and Burnett's method. Recommended. This method is particularly 

 useful for the rapid detection of the treponema and, according to its authors, 

 is more certain than Giemsa's method. 



1. Excise a small portion of the infected tissue and, with the point of a 

 small scalpel, gently scrape a little of it on to a number of slides on each of 

 which a small drop of distilled water has been placed. The dissociated tissue 

 spreads itself out in the water. Dry. 



2. Mordant the tissue with LcefEer's ink as in staining for flagella (p. 150). 



1 [To prepare Marino's blue mix : 



Methylene blue, - .... o*5 gram 



Azur, ... 0-5 



Water, . 100 grams 

 in a 0-5 per cent, aqueous solution of carbonate of soda. Incubate at 37 C. for 24-48 



hours. Add an aqueous solution of eosin. Filter. A powder is thus obtained soluble 

 in water and methyl alcohol. ] 



