358 SPECIFIC MICRO-ORGANISMS 



the sole mode of multiplication, although able adherents to the 

 opposite view are not lacking. The refractive index of the 

 filament is not very much greater than that of serum, so that the 

 unstained organism is difficult to see by direct illumination. 

 Dark-field illumination is more satisfactory. Sp. pallida in film 

 preparations stains with difficulty by ordinary methods. Schau- 

 dinn employed Giemsa's modification of the Romanowsky stain. 

 Good results are obtained by staining with solutions of the Roman- 

 owsky staining principles in methyl alcohol provided an excess of 

 methylene-violet be present (see p. 43). Tunnincliff 2 recom- 

 mends staining with a mixture of saturated alcoholic solution of 

 gentian violet, i part, in 5 per cent carbolic acid, 9 parts. Thin 

 films are essential but the staining process requires only a few 

 seconds. In pieces of tissue the spirochete is best stained by the 

 method of Leviditi. For this purpose thin (i mm.) pieces of 

 syphilitic tissue are fixed in formalin (10 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 trans- 

 ferred 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 a re- 

 ducing fluid, consisting of pyrogallic acid 3 grams, formalin (40 

 per cent formaldehyde) 5 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. 3 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 



1 Hoffmann: Centrabl. f. Bakt., I Abt., Orig., 1912, Bd. LXVI, S. 520-523. 



*Journ. A.M. A., 1912, Vol. LVIII, p. 1682. 



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



