Staining 765 



ish metallic in appearance, when they are thoroughly washed in water. The 

 treponemas appear black, the background brownish. 



Burri* has recommended a simple and rapid method of demonstrat- 

 ing the treponema and other similar organisms by the use of India ink. 



A drop of juice is squeezed from a chancre or mucous patch and mixed with a 

 drop of India ink and then spread upon a glass slide as in making a spread of a 

 drop of blood. As the ink dries it leaves a black or dark brown field upon which 

 the spiral organisms stand out as shining, colorless, and hence conspicuous 

 objects. Williams uses Higgins' water-proof ink, and Hiss recommends "chin- 

 chin,'.' Gunther-Wagner liquid pearl ink, for the purpose. 



The method is fairly satisfactory for diagnosis and can be appUed 

 in a few moments. 



II. Section. — Staining the organism in the tissues is a more 

 difficult matter, for the Giemsa stain scarcely shows it at all. Bert- 

 arelli and Volpinof tried a modification of the van Ermengem method 

 for flagella with some success, but there was no real success until 

 LevaditiJ devised his methods of silver impregnation. 



This consists in hardening pieces of tissue about i mm. in thickness in 10 per 

 cent; formol for twenty-four hours, rinsing in water, and immersing in 95 per cent, 

 alcohol for twenty-four hours. The block is then placed in diluted alcohol until 

 it sinks to the bottom of the container, and then transferred to a 1.5 to 3 per cent, 

 aqueoiis solution of nitrate of silver in a blue or amber bottle and kept in a dark 

 incubating oven at 37°C. for from three to five days. Finally, it is washed in 

 water and placed in a solution of pyrogallic acid 2 to 4 grams; formol, s cc; 

 distilled water, 100 cc, and kept in the dark, at room temperature, from twenty- 

 four to seventy-two hours, then washed in distilled water, embedded in parafiin, 

 and cut. The treponemata are intensely black, the tissue yellow brown. The 

 sections are finally stained with — (a) Giemsa's stain for a few minutes, then 

 washed in water, differentiated with absolute alcohol containing a few drops of 

 oil of cloves, cleared with oil of bergamot or xylol, or (J) concentrated solution of 

 toluidin blue, differentiated in alcohol containing a few drops of Unna's glycerin- 

 ether mixture, cleared in oil of bergamot, then in xylol, and mounted in Canada 

 balsam. 



This method was later improved by Levaditi and Manouelian§ 



by the addition of 10 per cent, of pyridin to the silver bath just 



before the block of tissue is put in, and by using for the reducing 



bath a mixture of pyrogaUic acid, acetone, and p3^idin. 



The details are as follows: Fragments of organs or tissues i to 2 mm. in thick- 

 ness are fixed for twenty-four to forty-eight hours in a solution of formalin 10 :ioo, 

 ■ then washed in 96 per cent, alcohol for twelve to sixteen hours, then in distilled 

 water until the blocks fall to the bottom of the container. They are then impreg- 

 nated by immersion in a bath composed of a i per cent, solution of nitrate of 

 silver, to which, at the moment of employment, 10 per cent, of p3Tidin is added. 

 Keep the blocks immersed in this solution at room temperature for two or three 

 hours, and at so°C. for four or six hours, then wash rapidly in a 10 per cent, solu- 

 tion of pyridin, and reduce in a bath composed of 4 per cent, pyrogallic acid, to 

 which, at the moment of using, 10 per cent, of pure acetone and 15 per cent, (total 

 volume) of pyridin are added. The reduction bath must be continued for several 

 hours, after which the tissue goes through 70 per cent, alcohol, xylol, paraffin, and 

 sections are cut. The sections, fastened to 'the slide, are stained with Unna's 

 blue or toluidin blue, differentiated with glycerin-ether, and finally mounted in 

 Canada balsam. 



* "Wiener klin. Wochenschrift," July i, 1909. 



t " Centralbl. f. Bakt. u. Pajasitenk.,"; Orig., 1905, xi, p. 56. 



i " Compt.-rendu de la Soc. de Biol, de Paris," 1905, lix, p. 326. 



§ "Compt.-rendu de la Soc. de Biol, de Paris," 1906, Lvin, p. 134. : 



