STAINING, PRACTICAL AND THEORETICAL 



amounts of RNA are present. This specific histochemical reaction, 

 the author states, is a manifestation of the fact that RNA is closely 

 associated with protein synthesis of the cell. In this connection I 

 would suggest that a more recent paper (I. Leslie, 1961), on the 

 biochemistry of heredity, which treats the relationships between 

 DNA, RNA and histones, may be of interest to readers. 



{e) Instead of the alcohol-ether mixture (solution A) absolute 

 alcohol (or 95% alcohol) may be used alone as the fixative. 

 Formaldehyde should be avoided. 



(/) The whole procedure from fixation (step i to step 11) is 

 best carried out in Coplin jars. 



{g) If the pH of the staining solution is shifted too far into 

 the acid range, gradual loss of cytoplasmic fluorescence occurs. 

 A shift into the alkaline range results in overstained cells. This, 

 however, may be desirable, especially for pre-screening by those 

 with little knowledge of cytology. In such cases the buffer 

 solution used may have a pH of 7-0, but no higher, to accentuate 

 cytoplasmic fluorescence of suspicious cells. 



Note: BuflFer tablets having a pH of yo are also available for 

 this purpose. 



{h) The author (F. D. Bertalanffy, i960) emphasizes that the 

 acridine orange should be of good quality. A good indication for 

 this and also for a proper working technique is when after differ- 

 entiating the smears, the nuclei of the leucocytes, superficial 

 squamous cells, etc., show clear, translucent green colour, and 

 the cytoplasm, depending upon the cell type, appears in clear 

 brown, reddish brown, or orange or red fluorescence. Some pre- 

 parations of acridine orange available are considered unsuitable 

 for the fluorescence method. These, due to contained-impurities, 

 stain nuclei in ochre yellow and the cytoplasm in hazy brownish 

 and reddish hues. 



(/) If a very rapid diagnosis is necessary smears may be passed 

 immediately into 80% alcohol and followed by distilled water, 

 then processed in accordance with steps 6-12, described on 

 page 98. 



(7) Smears should not be blotted as this may result in the 

 removal of cells from the specimen. 



{k) Erythrocytes do not appear to fluoresce. This may be due 

 to the iron content of the haemoglobin: heavy metals exert a 

 quenching effect on fluorescence (E. Gurr, 195 iZ>, 1953, 1956). 



100 



