AZURE TOLUIDIN BLUE 



32 



BACTERIA 



Institute of Health, Bethesda, Md., 

 April 22, 1946 — Dissolve 10 gm. Azure 

 A or Azure C (about 85% dye content) 

 or 13 gm. Toluidin blue (about 60% dye 

 content) in 600-800 cc. aq. dest. Or 

 polychrome 10 gm. methylene blue in 

 600 cc. aq. dest. by boiling 20 min. 

 with 5 gm. potassium bichromate and 

 7 cc. concentrated sulfuric acid (95.5% 

 spc. gf. 1.84), cooling to 10°C. and 

 neutralizing by adding gradually 21 gm. 

 sodium bicarbonate. This makes a 

 crude Azure A. Dissolve 8 gm. Eosin 

 Y or Eosin B (a redder shade) in 100 cc. 

 aq. dest. and add to the selected azure 

 or toluidin blue. Filter with vacuum 

 on hard filter paper on a Buchner fun- 

 nel. Just as filtration is completed, 

 add successively 2 washes of 50 cc. aq. 

 dest. and 2 of 25 cc. 95% alcohol. Dry 

 precipitate on filter paper at 37°C. 

 Make 1% stock solution in equal vol- 

 umes of 95-98% C.P. glycerol and C.P. 

 methanol (A.C.S.) (or 100% ethyl alco- 

 hol), shaking at intervals for 2 or 3 

 days. The stock solution is best kept 

 in a cool place, but is quite stable at 

 room temperature. 



Bring paraffin sections to water as 

 usual, pre -stain 5 min. in alum hema- 

 toxylin if desired, wash and stain 1 hr. 

 in stock solution 0.5 cc, Mcllvaine 

 buffer of desired pH level 2 cc, C.P. 

 acetone 5 cc. and distilled water to 

 make 40 cc Rinse, dehydrate in ace- 

 tone, clear with 50:50 acetone xylene 

 and 2 changes of xylene, mount in 

 clarite. 



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 cj^toplasm 

 and oxyphil inclusion bodies. Further 

 details are given in the original: Histo- 

 pathologic Technic, Lillie, 1946 (in 

 press). 

 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 , H am , E Az " of a great 

 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. 



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, 1939), which 

 is a key to identification of bacteria, is 

 often useful. 



Motility, agglutination, lysis under 

 influence of bacteriophage, ingestion by 

 leucocytes and many other phenomena 

 can best be observed by examination of 

 living bacteria by direct illumination or 

 in the darkfield. Smears, usually fixed 

 by heat, are, however, most often used. 

 A choice must be made from many well 

 known stains including: Anilin Gentian 

 Violet, Loeffler's Methylene Blue, 

 Giemsa, Gram and Carbol Fuchsin. 

 Others are best listed under the particu- 

 lar structures to be demonstrated : 

 Spores, Flagella, Capsules. In some 

 cases search for bacteria in Milk, Soil, 

 Cheese, Sputum, etc. is indicated. 



