SPIROCH^T^ 379 



Probably the most satisfactory stain is that of Fontana.^ In pieces 

 of tissue Ihe spirochete is best stained by the method of Levaditi. 

 For this purpose thin (i mm.) pieces of syphilitic;! tissue are fixed 

 in formalin (lo per cent) for 24 hours or longer and hardened in 

 95 per cent alcohol for a day. The alcohol is then removed by 

 soaking in distilled water and the tissue is transferred to a fresh 

 I to 3 per cent solution of silver nitrate in distilled water. This 

 is placed at 37° C. in the dark for three to five days. The tissue 

 is next washed in distilled water and placed in reducing fluid, 

 consisting of pyrogallic acid 3 grams, formalin (40 per cent for- 

 maldehyde) s c.c. and distilled water 100 c.c, for one to two days. 

 It is then washed in distilled water, dehydrated, embedded in 

 paraffin and sectioned. The spirochetes are stained a dense 

 black by this method. The sections may be stained to show 

 histological structure also, by applying methylene blue or toluidin 

 blue to them after they have been fixed on the slide. 



Cultivation of Sp. pallida has been most successfully practised 

 by Noguchi.^ He has grown the organism in a mixture of serum 

 and water, to which naturally sterile tissue was added, and in 

 ascitic-fluid agar with similar bits of tissue, always under strict 

 anaerobic conditions. The technic of culture is somewhat diffi- 

 cult and the original papers should be consulted in detail. Inocu- 

 lation of the cultures into rabbits and monkeys has caused typical 

 ' syphilitic lesions. 



Noguchi's luetin is prepared by grinding the solid medium 

 rich in spirochetes in a mortar and emulsifying it in a small 

 amount of fluid. This is then heated to 60° C. for an hour and 

 preserved by the addition of 0.5 per cent carbolic acid. The 

 final preparation contains many dead unbroken spirochetes. 



' Three solutions are required (a) Glacial acetic acid i, formalin 20, distilled 

 water 100; (i) Phenol i^ tannic acid 5, water 100; (c) Silver nitrate (25 per cent 

 solution) s c.c, ammonia water i drop. The thin smear is dried in air. Treat with 

 (o) one minute; wash in water; cover with (6) and steam one-half minute; wash 

 in distilled water; cover with (c) and steam one-half minute; wash; blot; dry; mount 

 in balsam. Medical War Manukl No. 6, U. S. Army, 1919, p. 30. 



'Journ. Exp. Med., 1911, Vol. XIV, p. 99; 1912, Vol. XV, p. 90. 



