AZURE OR TOLUIDIN BLUE-EOSIN 27 



BACTERIA 



mixture of Azure A and B. Azure II 

 is an intentional mixture, in equal parts, 

 of Azure I and methylene blue. It is 

 the main constituent of Giemsa's stain. 



1. Azure A is asymmetrical dimethyl 

 thionin and has been Commission Certi- 

 fied for some time. It is considered as 

 the most important nuclear staining 

 component of polychrome methylene 

 blue by MacNeal, W. J., J. Inf. Dis., 

 1925, 36, 538-546. This dye has been 

 used as a nuclear stain following eosin 

 and after phloxine, see Phloxine-Azure 

 (Ilaynes, R., Stain Techn., 1926, 1, 

 68-69, 107-111). 



2. Azure B is the tri -methyl deriva- 

 tive of thionin. It is specified by 

 Jordan, J. H. and Heather, A. H., Stain 

 Techn., 1929, 4, 121-126 as a stain for 

 Negri bodies. Roe, M. A., Lillie, R. D. 

 and Wilcox, A., Pub. Health Reports, 

 1940, 55, 1272-1278 recommend its in- 

 clusion in Giemsa's stain. 



3. Azure C is mono-methyl thionin. 

 French, R. W., Stain Techn., 1926, 1, 

 79 has described a method for its use 

 followed by Eosin Y and orange II in 

 staining sections of formalin fixed mate- 

 rial; but Haynes, R., Stain Techn., 

 1927, 2, 8-16 doubts whether it is sig- 

 nificantly better than Azure A and 

 thionin. 



Azure or Toluidin Blue-Eosin. — Written 

 by Dr. R. D. Lillie, Division of Pathol- 

 ogy, National Institute of Health, 

 Bethesda, Md. May 8, 1950.— Prepare 

 a 1/1000 solution of Azure A, Azure C 

 or Thionin (85-90% dye content) or a 

 0.15% solution of Toluidin Blue (60% 

 dye content) and a 1/1000 solution of 

 Eosin Y or Eosin B (a redder shade). 



Bring paraffin sections to water as 

 usual, including an iodine, thiosulfate 

 sequence for material fixed with mer- 

 curic chloride mixtures. Stain 1 hour in 

 stock azure, Toluidin Blue or Thionin 

 4 cc, stock eosin 4 cc, Mcllvaine buffer 

 of desired pH level 2 cc, acetone 5 cc. 

 and distilled water 25 cc. Rinse, de- 

 hydrate with acetone, clear with 50:50 

 acetone xylene and 2 changes of xylene, 

 mount in synthetic resin (polystyrene, 

 permount, clarite, HSR or the like). 

 The procedure has been used for techni- 

 con staining. The stain mixture is made 

 fresh weekly in this case. 



For neutral formalin or Orth fixa- 

 tions, use pH 4.0-4.5, for acid formalin 

 pH 4.5 is better, for Zenker or Helly 

 pH 5.0, for Bouin pH 5.5-6.0 (less satis- 

 factory than others as the picric acid 

 seems to interfere), for Carnoy, alcohol 

 and similar fluids 4.8-5.5. 



Color values are deep blue for nuclei, 

 bacteria, and rickettsiae, violet to 

 purple for mast cell granules and carti- 



lage matrix, lighter blues for cyto- 

 plasms, varying pinks for muscle, ery- 

 throcytes, fibrin, necrotic cytoplasm 

 and oxyphil inclusion bodies. 



This has been modified somewhat 

 from Histopathologic Technic, Lillie 

 (Blakiston, Phila., 1948), which gives 

 further details. 



Azure II Eosin and Hematoxylin (Maximow, 

 A., J. Inf. Dis., 1924, 34, 549), gives, 

 in addition to coloration of chromatin 

 by hematoxylin, a granule stain some- 

 thing like that provided by Giemsa's 

 method. Make up: (1) azure II eosin: 

 A. eosin water soluble yellowish, 0.5 

 gm.; aq. dest., 500 cc. B. azure II, 

 0.5 gm.; aq. dest., 500 cc. Mix 10 cc. 

 A, 100 cc. aq. dest., and 10 cc. B. (2) 

 hematoxylin (Delafield's) 1-2 drops, 

 aq. dest., 100 cc. to make a pale violet 

 solution. 



Formalin-Zenker fixed tissues (sec- 

 tions, smears, spreads) are stained up- 

 right in hematoxylin washed in aq. dest. 

 and counter-stained with azure II eosin 

 24 hrs. each. Transfer to 95% ale, 

 differentiate and dehydrate in abs. (2 

 changes); clear in xylol and mount in 

 balsam. Care must be taken to use 

 pure aq. dest. The proportions of A 

 and B can be varied slightly to suit the 

 tissue. In order to hold the azure II 

 eosin colors the balsam should be neu- 

 tral or nearly neutral as when Giemsa's 

 stain is employed. 



To appreciate the beauty of this 

 method see numerous colored illustra- 

 tions marked "ZF, Ham, EAz"of agreat 

 many organs and tissues by Maximow, A. 

 Section on Bindegewebe und Blutbil- 

 dende Gemebe in Mollendorff's Handb. 

 d. mikr. Anat. d. Menschen, 1927, 2, 

 (1) 232-583. 



Babes' see Anilin-Safranin. 



Bacillus Typhosus, technique for dark field 

 study of flagella (Pijper, A., J. Path. 

 & Bact., 1938, 47, 1-17). See 9 plates 

 by author. 



Bacteria. Methods employed for the micro- 

 scopic identification of bacteria and to 

 demonstrate their structure are legion. 

 The Committee on Bacteriological Tech- 

 nique of the Society of American Bac- 

 teriologists has prepared a useful leaflet 

 entitled "Staining Procedures" pub- 

 lished in Geneva, N. Y. (Fifth Edition 

 1934) to supplement their "Manual of 

 Methods for the Pure Culture of Bac- 

 teria" (1923). A detailed account of 

 Bacteriological methods by H. J. Conn, 

 F. B. Mallory and Frederic Parker, Jr., 

 is contained in McClung's Microscopical 

 technique to which reference should 

 also be made. Bergey's "Manual of 

 Determinative Bacteriology" (Balti- 

 more: Williams & Wilkins, 1948), which 



