An improved method of staining lipides : acetic-carbol-sudan

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1. A method is described of using Sudan III, which gives a more complete demonstration of lipide substances, especially those that are refractory to ordinary Sudan III, Scharlach R, or Sudan IV techniques. 2. This improvement does not appear to depend on superior preservation of lipides, but on enhanced staining powers due to the colloidal nature of the solution. 3. Used in this way, the colouration obtained with Sudan III equals that usually obtained with Scharlach R. 4. The method possesses the advantages of constancy, rapidity, and absence of precipitates as compared with the methods of Romeis, and it succeeds with a variety of samples of the dye. 5. The best variation of the technique is as follows: (a) Fix in formol or formol-saline. (b) Frozen sections in distilled water. (c) 50 per cent. alcohol 1 minute. (d) Acetic-carbol-Sudan (60 per cent. carbol-Sudan plus 2·5 drops of glacial acetic acid per c.c., prepared as described) 1½ hours (sometimes longer) in a small well-corked container. (e) Differentiate in 5 per cent. acetic acid in 50 per cent. alcohol (10 to 60 seconds). (f) Wash in distilled water (1 minute). (g) Counterstain in filtered Delafield's haematoxylin diluted with two parts of distilled water. (h) Differentiate in acid water (10 to 20 seconds). (i) Blue in ammonia water (5 minutes). (j) Wash in distilled water. (k) Mount in glycerine-jelly. 6. Positive results have been obtained with two lipides which are not stained by ordinary techniques (so-called "Sudanophobe" lipides) and the method is especially to be recommended wherever the existence of such substances is suspected. 7. No conclusions regarding the absence of lipide substances should be drawn from negative results of ordinary Sudan or Scharlach techniques, until acetic-carbol-Sudan has been used. 8. It is anticipated that acetic-carbol-Sudan technique will supersede all previous Sudan methods, wherever critical demonstration of lipides is required.