SECTION FIVE 



(c) STAINING METHODS 



Notes on fluorochromic staining technique: 



(a) Fixatives containing salts of heavy metals (with the exception 

 of zinc) ; chlorine, bromine, iodine, and nitro compounds should 

 be avoided as these exert a quenching effect on fluorescence. The 

 most suitable fixatives are 5 to 10% formalin, or Kahle's fluid. 



(b) If tissues are embedded in paraffin wax, all traces of the wax, 

 which is auto-fluorescent, must be removed before sections are 

 stained and examined. 



(c) A special grade of immersion oil known as fluoroil, which 

 is non-fluorescent, should be used for high-power examination, 

 since cedarwood oil and most of the immersion oils available for 

 ordinary microscopy are unsuitable for fluorescence work. 



(d) The usual mounting media, as used for ordinary micro- 

 scopy, contain highly fluorescent materials which render them un- 

 suitable for fluorescence work, and should, therefore, be avoided. 

 For temporary mounts, glycerine may be used, and for permanent 

 mounts there is a satisfactory medium on the market under the 

 name of Fluormount. 



Fluorochromes, of which auramine O, coriphosphine, acridine 

 yellow Hi 07, aesculine, acridine orange, primuline, thiazole yellow 

 are examples, are frequently used in very dilute solutions to pro- 

 duce characteristic fluorescent colours, and when preparations 

 which have been treated with these solutions are examined in trans- 

 mitted light of the visible spectrum, they appear to be unstained 

 or only very faintly tinted. 



Some explanation as to the colour differentiation obtained by the 

 use of general-purpose fluorochrome of which acridine yellow 

 Hi 07 is an example, is explained by the fact that fluorescence 

 colour is effected by hydrogen-ion concentration, and as fluoro- 

 chromes also exhibit differential absorption by various tissues, the 

 production of a great variety of fluorescent colours is brought 

 about by the influence of these two factors. Nuclei can be differ- 

 entiated from cytoplasm by the use of any one of the following 

 general fluorochromes: acridine yellow Hi 07, coriphosphine, 

 phosphine 3R, or acriflavine. 



Titan yellow, rhodamine B, phosphine 3R, methylene blue are 



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