STAINING, PRACTICAL AND THEORETICAL 



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, acridine orange or acriflavine. 



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

 all excellent fluorochromes for fat, while berberine sulphate is 

 much used for protozoal parasites, particularly for malaria. Thio- 

 flavine has been found satisfactory for virus and for bacteria, as 

 well as for general fluorochromic staining, while acriflavine has 

 been used for trypanosomes and as a general stain, and uranin is 

 one of the most suitable fluorochromes for intravital staining. All 

 these fluorochromes are used in very dilute aqueous solutions, 

 that is to say, something to the order of o-i to o-oi per cent. 



Intravital Staining with Fluorochromes 



Dyes used for this purpose must be water soluble, non-diffus- 

 ible in the living body, non-toxic in the workable dilutions re- 

 quired, and highly fluorescent even in greatly diluted solutions. 

 Uranin possesses all these qualifications and is one of the most 

 useful fluorescent dyes for intravital work, as stated earlier in this 

 chapter. Acriflavine is another useful dye for this purpose, al- 

 though it is not so intensely fluorescent as uranin. The fluor- 

 escence of uranin is impaired in basic solution so that it appears 

 most readily in organs of an acid reaction. It is used in o-i% 

 solution in physiological saline, in which form it should be injected 

 into the animal's blood stream or into the organ to be studied. 

 The colour of its fluorescence varies with hydrogen-ion changes 

 and consequently it is of great value as an intravital hydrogen-ion 

 indicator. The colour changes are easily visible in dilutions to the 

 order of one part in ten millions and in dilute solutions the inten- 

 sity of the fluorescence has a definite relation to the concentration 

 of the dye and consequently the intensity of the fluorescence 

 serves as an indicator of the amount of uranin present. Primulin, 

 thioflavine, rhodamine B and berberine sulphate have also been 

 found useful for intravital work. 



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