SUBMICROSCOPIC FIBRILS 



232 



SUDAN BLACK B. 



Submicroscopic Fibrils. These by close 

 association may constitute the neuro- 

 fibrils, spindle and astral fibers, myo- 

 fibrils, and so on. Use of polarization 

 optical methods suggests the orienta- 

 tion of submicroscopic rodlets parallel 

 to the length of the fibers. The elec- 

 tron microscope is capable of demon- 

 strating the component submicroscopic 

 fibrils of collagenic fibrils (Schmitt, 

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

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



Submicroscopic Particles. In summarizing 

 work in R. 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/x. (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). 



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- 

 methane). 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 

 Govan, A.D.T., J. Path. & Bact., 1944, 

 56, 262-264 . See Bell's Method for stain- 

 ing 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 If 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 than 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/a<. 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- 

 sium bichromate 6 cc. ; 2% chromic 

 acid, 3 cc. ; and aq. dest. 5 cc. Wash in 

 running water 24 hrs. In case tissue 

 is delicate and requires support embed 

 in gelatin before cutting frozen sections : 

 12.5% gelatin in 1% aq. phenol filtered, 

 37°C., 24 hrs. 25% solution, same. 

 Embed in fresh 25% aq. gelatin, cool, 

 trim, harden in 5% formalin 24 hrs. 

 Cut frozen sections, whether first em- 



