SUBMICROSCOPIC PARTICLES 



327 



SUDAN BLACK B 



optical methods suggests the orienta- 

 tion of submicroscopic rodlcts parallel 

 to the length of the fibers. The elec- 

 tron microscope is capable of demon- 

 strating the component submicroscopic 

 fibrils of coUagenic fibrils (Schmitt, 

 F. O., Hall, C. E. and Jakus, M. A., 

 Biol. Symposia, 1943, 10, 261-276). 



Submicroscopic Particles. In summarizing 

 work in II. R. Bensley's laboratory, 

 Lazarow, A., Biol. Symposia, 1943, 10, 

 9-26 mentions two of these barely 

 visible as shimmering points of light in 

 the dark field: (1) Lipoprotein complex 

 discovered by Claude at the Rockefeller 

 Institute containing fats, proteins and 

 nucleo-protein and when concentrated 

 en masse by centrifugation of cherry 

 red color. Particle size 0.06-0. 2^. (2) 

 Particulate glycogen discovered by 

 Lazarow containing a little protein but 

 no fat. Water content 75%. See Mi- 

 crosomes. 



Submicrosopicc Structure of cytoplasm, 

 methods and results (Frey-Wyssling, A., 

 J. Roy. Micr. Soc, 1940, 60, 128-139). 



Succinic Dehydrogenase. Semenoif, W. E., 

 Zeit, f. Zellforsch. Micr. Anat., 1935, 

 22: 305-309 as detailed by Click, p. 96. 

 Treat frozen sections of fresh tissue 

 with 2 cc. 0.05 methylene blue plus 2 

 cc. 10% sodium succinate made up to 

 10 cc. with M/15 phosphate buffer, 

 pH 7.6-8.0 for 10-15 min. under cover 

 slip with air bubbles excluded and 

 edges sealed with paraffin. Compare 

 with section in control medium made 

 up without sodium succinate. Fading 

 of dj^e indicates enzymatic activities. 

 Click, p. 96. See Dehydrogenase. 



Sudan, II (CI. 73)— Oil red O. Physical 

 properties, Lillie, R. D., J. Tech. 

 Methods, 1944, 24, 37-45. 



Sudan III (CI, 248) — cerasin red, fat pon- 

 ceau G, oil red AS, O, B or 3B, scarlet 

 B fat soluble, Sudan G, Tony red — A 

 weakly acid dis-azo dye, the most 

 popular of fat stains in alcoholic solu- 

 tion. A sat. sol. in 70% alcohol is used 

 in the same manner as Sudan IV in 

 Herxheimer's solution (see below). 

 Variations in action of Sudan stains 

 depending on character of fat and kind 

 of fixation (Black, C. E., J. Lab. & 

 Clin. Med., 1937-38, 23, 1027-1036). 



Staining in aqueous phase (Dufrenoy, 

 J., Stain Techn., 1937, 12, 71-72). 

 Make concentrated solution of Sudan 

 III in 5 cc. methylal (dimethyloxy- 

 metliane). Add 10-20 cc. aq. dest. 

 The mixture separates into 2 layers : the 

 lower made up of water, methylal and 

 Sudan III and the upper of methylal, 

 Sudan III and water. Whether sections 

 float or sink they take up Sudan III. 

 Another method of staining with Sudan 



III in gelatin solution is given by 

 Telford Govan, A. D., J. Path. & Bact., 

 1944, 56, 262-264. See Bell's Method 



for staining fats mobilized by heat. 

 A promising acetic-carbol-sudan tech- 

 nique for lipids is described by Jackson, 

 C, Onderstepoort, J. Vet. Sci. & Animal 

 Industry, 1944, 19, 169-177. To prepare 

 stock solution heat to simmering 2 gms. 

 finely powdered Sudan III in 450 cc. 

 95% ale. Filter hot. Stopper, leave 

 in refrigerator over night and filter cold. 

 Add to any desired amount stock solu- 

 tion 5% aq. carbolic drop by drop agi- 

 tating vigorously till alcohol content 

 is reduced to 60%. About 2 cc. carbolic 

 to 6 cc. stock solution is required. Let 

 stand few hours well corked. Add 

 glacial acetic drop by drop 2.5 drops per 

 cc. of carbol sudan, or 20 drops to the 

 8 cc. in above instance. 



Cut frozen sections of formol or 

 formol-saline fixed tissue. Place in 

 50% ale. 1 min. Stain in acid-carbol- 

 sudan mixture l^ hrs. in well stoppered 

 container. Differentiate in 50% alco- 

 hol, containing 5% acetic acid, 10-60, 

 sec. Wash in aq. dest., 1 min. Coun- 

 terstain in filtered Delafield's hema- 

 toxylin diluted 1:2 with aq. dest. 

 Differentiate in acid water, 10-20 sec, 

 blue in ammonia water (5 min.) and 

 wash in aq. dest. Finally mount in 

 glycerin-jelly. Method is particularly 

 recommended when existence of so- 

 called "Sudanophobe" lipids is sus- 

 pected. 



Sudan IV (CI, 258) — cerotine ponceau 3B, 

 fat ponceau, fat ponceau R or LB, oil 

 red IV, scarlet red — A weakly acid dis- 

 azo dye also widely used as fat stain 

 sometimes under heading of Scharlach 

 R, especially in Herxheimer's Solution. 

 Place frozen sections of formalin fixed 

 tissue in 70% alcohol for a few sec. 

 Transfer to Herxheimer's solution for 

 2-5 min. in a covered container to re- 

 duce evaporation and precipitation. 

 Rinse in 70% alcohol. Wash quickly in 

 aq. dest. Counterstain with Harris' 

 hematoxylin. Wash in tap water. 

 Mount in Glycerin. Seal with paraffin, 

 or, if permanency is desired, with Duco 

 or Kronig's cement. As a rule these fat 

 stains do not last more tlmn a few months. 

 Physical properties of Sudan IV (Lillie, 

 R.D., J. Tech.Methods, 1944,24, 37-45). 



Sudan Black B. This dye is of English 

 manufacture and is not available in U.S. 

 during the war. Its identity is still 

 uncertain. 



1. For fat. Fix tissues 24 hrs. in 5% 

 formalin in 0.9% saline or in Zweibaum's 

 fluid. The latter is made by adding 

 1 part of 2% aq. osmic acid to 7 parts 

 of a mixture consisting of 3% potas- 



