STAINING, PRACTICAL AND THEORETICAL 



13. Add 0-5 ml. of acetic aniline blue (solution F) to the 

 phosphomolybdic acid on the slide and mix by gently rocking the 

 slide. Allow this mixture to act for five minutes. 



14. Pour off excess Hquid, 



15. Rinse in distilled water. 



16. Immerse in acridine orange (solution G) for two minutes. 



17. Wash briefly in water. 



18. Mount in Uvak. 



19. Examine in a blue light, with a yellow eyepiece filter. 



Results : 



A yellow fluorescence of remarkable brilliance is observable in 

 the intracellular mucus, while other structures of the mucosa and 

 of the bowel wall are barely distinguishable, in dull greenish 

 colour. 



Notes: 



(a) The authors applied acridine orange solution to an old 

 section, after removing the coverslip and mountant, of bowel 

 already stained by one of the Masson trichrome methods. The 

 result, described above, was totally unexpected, since Masson 

 trichrome stains do not attach themselves to mucus. 



(b) In their previous work (Hicks & Matthaei, 1955), tissues 

 had been treated with acridine orange (without Masson trichrome 

 stain) and the authors had found that connective tissue, muscles, 

 nuclei and other structures fluoresced strongly, but mucin showed 

 an almost insignificant brown. A series of experiments then 

 undertaken pointed to the fact that the presence of iron inhibits 

 or quenches the normal production of fluorescence on staining 

 with acridine orange in practically all tissue components except 

 mucus, and this substance is permitted to develop its normal 

 fluorescence in the more concentrated solution of this dye used 

 in the present technique. 



Note: In their previous work the authors used a considerably 

 weaker (o-oi%) solution of the dye. 



(c) The original paper, which carries six colour photomicro- 

 graphs, should be consulted for further information. 



Reference: Hicks, J. D. & Matthaei, E. (1958). 



106 



