Staining 



763 



In 200 cc. of water, 2 grams of lithium carbonate are dissolved and 2 grams of 

 Merck's medicinal, Griibler's BX, or Koch's rectified methylene blue added. 

 This mixture is heated moderately in a rice boiler until a rich polychrome has 

 formed. To determine this a sample is examined in a test-tube every few minutes 

 by holding it against an artificial light. As soon as a distinctly red color is 

 obtained, the desired degree of heating has been reached. After cooling it is 

 filtered through cotton in a funnel. To one-half of this polychrome solution 5 per 

 cent, of acetic acid is gradually added until a strip of litmus-paper shows above 

 the line of demarcation a distinct acid reaction, when the remaining half of the 

 solutionig added, so as to carry the reaction back to a: low degree of alkalinity. A 

 weak eosin solution is now prepared, approximately 0.5 per cent. French eosin, 

 and this is added gradually while the mixture is being stirred until a filtered sam- 

 ple shows the filtrate to be of a pale bluish color with a slight fluorescence. The 

 mixture is allowed to stand for one day and then filtered. The precipitate which 

 has separated is collected on a double piece of filter-paper and dried at room tem- 

 perature (heating spoils it). When completely dried it can easily be removed 

 from the paper and may then be dissolved without further washing in commercial 



4. 



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Fig. 302. — Treponema pallidum in the periosteum near an epiphysis (Bertarelli). 



(not pure) wood alcohol. The solution should be allowed to stand a day, then 

 filtered. The strength of this alcoholic solution is approximately i per cent. To 

 use the stain, one drops upon an unfixed spread enough dye to cover it, permits it 

 to act for three or four seconds, and then pours it off and introduces the glass 

 Slowly, spread side down, into clean water, where it is held for another four or five 

 seconds, after which it is shaken to and fro in the water to wash it. It is next 

 dried and examined at once or after mounting in balsam. The treponemas 

 appear violet in color. 



Ghoreyeb* recommends the following rapid method of staining 

 the organism in smears. A thin spread is to be preferred. No heat 

 fixation is necessary: 



1. Cover the smear with a i per cent, aqueous solution of osmic acid, and per- 

 mit it to act for thirty seconds. This solution acts as a fixative and mordant. 



2. Wash thoroughly in running water. . « t,i 



3. Cover the smear with a i : 100 dilution of Liquor plumbi subacetatis (freshly 



*"Jour. Amer. Med. Assoc," May 7, 1910, Liv, No. 19, p. 1498- 



