FATTY SUBSTANCES 283 



solutions is obviated by the use of diacetin, which dissolves the 

 dye but not the fat. 



660. Technique of Staining. The dye recommended is toluene- 

 azo-toluene-azo-jS-naphthol. A brand of this has been specially 

 prepared by the British Drug Houses Ltd., and is sold as Sudan IV 

 (B.D.H., No. 555722). 



The following solutions are needed (Kay and Whitehead, 1935) : 

 (A) Stock solution of dye. Add 2 g. Sudan IV powder to 1 litre of 

 absolute ethyl alcohol at room temperature, boil gently till all the 

 powder has dissolved, and allow to cool and settle. (B) 45 per 

 cent, alcohol. Mix 4 volumes of absolute ethyl alcohol with 

 5 volumes of distilled water. (C) Staining solution : saturated 

 solution of Sudan IV in 70 per cent, alcohol. To 7 volumes of (A) 

 add slowly, with shaking, 9 volumes of (B). Mix thoroughly, allow 

 to stand for one hour, and filter. 



Formol-fixed frozen sections are placed successively in : — 

 (1) 50 per cent, alcohol, five minutes ; (2) solution (C), thirty 

 minutes at 37° C. ; (3) 50 per cent, alcohol, a few seconds ; 

 (4) distilled water, a few minutes at least ; (5) filtered haemalum ; 

 (6) alkaline tap water, a few minutes at least ; and (7) mounted 

 in glycerin jelly. 



661. Notes on Technique. (1) The alcohol used to prepare 

 solution (A) must be absolute. Any loss by evaporation while 

 the dye is being dissolved must be made good by adding more 

 alcohol while the solution is hot. (2) (A) when hot is transparent ; 

 while cooling it becomes slightly turbid. A small deposit of 

 excess dye forms on the sides and bottom of the bottle and the 

 cold solution is transparent. (3) Within a few minutes after (A) 

 and (B) have been mixed, the mixture, (C), becomes turbid. 

 If (C) is used immediately, even though filtered, a precipitate may 

 form rapidly on the sections. This is avoided or delayed for some 

 weeks by allowing (C) to stand for one hour before filtering. 

 (4) (A) keeps for at least six months without deteriorating, 

 (C) rapidly deteriorates and should be discarded within about 

 four hours after being mixed ; it may, however, be used repeatedly 

 during this period. (5) Sections should be stained on the day after 

 cutting. Sections stained on the day of cutting tend to stick to the 

 glass rod used for manipulation. If staining is delayed for more 

 than a few days after the end of fixation the results are often 

 unsatisfactory, probably owing to the crystallisation of the fatty 

 substances. The finished preparations deteriorate in the course 

 of weeks or months. (6) Precautions to prevent evaporation 

 of (C) are unnecessary. Hollow glass stoppers from wide-mouth 

 2-oz. bottles make convenient staining vessels. (7) If sections 

 overlap, the covered portion is stained less strongly than the 

 uncovered. Sections may be unevenly stained even though they 

 were not overlapped. The risk of uneven staining is minimised 



