DS23.il 



DYE STAINS OF SPECIAL APPLICATION 



461 



use of buffers, and thus gives a clearly 

 reproducible result. Stovall and Black rec- 

 ommend phosphate buffers, of which two 

 will be required, one at pH 3, the other at 

 pH 5.5. It is not known to the writer 

 whether or not other buffers than phos- 

 phate can be substituted in this technique. 

 One per cent of ethyl eosin is then dis- 

 solved in the pH 3 buffer, and 0.25% of 

 methylene blue is dissolved in equal jiarts 

 of the pH 5.5 buffer and absolute alcohol. 

 The only other solution required is 0.4% 

 acetic acid. 



Sections are taken from the distilled 

 water and placed in the buffered eosin 

 solution for two minutes. From this they 

 are passed to distilled water, in which they 

 are rinsed until the whole of the eosin ad- 

 herent to the sUde, but not that in the 

 sections, is removed. They are then dipped 

 up and down in the methjdene blue solu- 



tion for about 30 seconds. Each slide, 

 without washing, is next placed in acetic 

 acid and watched. When it is taken from 

 the blue stain it will be purple-blue, and 

 it will be sufficiently differentiated when 

 it has changed to brownish red. This color 

 change is relatively sharp and is accom- 

 panied by a cessation of the clouds of blue 

 stain, which will be seen leaving the sec- 

 tion when it is first placed in the acetic 

 acid. The slide is then transferred to 

 running water and thoroughly washed to 

 remove the acid as rapidly as possible. 

 It is recommended that the slides be run 

 through the technique up to this point, 

 one at a time, and accumulated in the 

 wash water. They may then be dehy- 

 drated in the regular manner, either in 

 alcohol, or in any other solvent selected 

 by the preparator, before being mounted 

 in balsam. 



23.11 METHODS OF STAINING UNIDENTIFIED ORGANISMS SMALLER THAN BACTERIA 



Little can be said in favor of the methods given under this section, save that they have 

 appeared in the literature and are certified by their discoverers to place in evidence, in 

 sectioned material, certain particles presumed by the writers of the articles to have been 

 living, and which cannot be, with certainty, assigned to any known group or class of organism. 



23.11 Bland and Canti 1935 11431, 40:2,33 



formula: DS 13.13 Gatenby and Cowdry 1928 7, phosphate buffer pll 7 100 

 method: [pieces of infected explant] -^ methanol, 5mins. —* stain, 24hrs. — * acetone, till 

 differentiated — > xylene, till acetone completely removed -^ balsam 



23.11 Gutstein 1937 11431,45:313 



formula: 1% methyl violet 50, 2% sodium bicarbonate 50 

 method: [methanol-fixed smears] -^ stain, 20-30 mins., 37°C. — > wash 



dry 



23.11 Hosokowa 1934 test. 1942 Langeron Langeron 1942, 841 



stock solution: methanol 50, glycerol 50, DS 13.11 Jennor 1899 (dry powder) 0.8, 



azur I 0.2, crystal violet 0.01 

 RsiAGENTS required: A. 1% eosin B; B. stock 100, water 2 

 method: [smears, fixed and dehydrated in F 0000.0101 Hosokowa 1934] -^ A, poured on 



slide and warmed to steaming, 1 min. -^ tliorough wash -+ B, 30-40 mins. — » dry 



23.11 Laidlaw test. 1936 Pappenheimer and Hawthorne G08b, 12:627 



reagents rkquired: A. DS 11.121 Weigert 1903; B. 0.5% hydrochloric acid in 70% 



ale; C. 1% acid fuchsin; D. 1% phosphomolybdic acid; E. 0.25%, orange G in 70% ale. 

 method: [3 M sections of F 3000.0010 Laidlaw (1936) fixed material]-* waters A, 



5 mins. — » B, till nuclei differentiated -^ wash -* C, 5-15 mins. — > rinse — >• D, 30 sees. 



-^ rinse — » E, till inclusion bodies differentiated -^ balsam, via usual reagents 



23.11 Nicolau and Kopciowska 1937 6628,104:1276 



reagents required: A. water 65, methanol 35, glycerol 5, oxalic acid 0.15, methyl blue 



1.5; B. water 100, oxalic acid 0.06, acid fuchsin 1.5 

 method: [thin sections of F 5000.1010 Dubsoscq and Brazil 1905 fixed material] — > 95% 



ale. -^ A, '^ to 1 hr. — > water, rinse -^ abs. ale, rinse — > B, 10-20 mins. -^ abs. ale. 



— » balsam, via usual reagents 



