DS 13.1-DS 13.10 



DYE STAINS OF GENERAL APPLICATION 



343 



original formula by Giemsa is'no more than 

 a two-lino footnote to a papor in a sonio- 

 what obscure journal, and both tlie dry 

 powder in commerce and the solutions 

 recommended by subsequent wi'iters vary 

 enormously from the original proportion 

 specified by Cliemsa. A combination of 

 Giemsa 1902 and May-Griinwald 1902 

 was published by Pappenheim 1908 and 

 has become widely disseminated through 

 the hterature as the panoptic method. 

 Two other modifications of the same tech- 

 nique, published by Pappenheim four 

 years later, are still widely referred to in 

 the literature as panoptic, though tlie 

 majority of those using this term refer 

 actually to the 1908 formula. Some of 

 these techniques have been rationahzed, 

 particularly one by MacNeal 1922 which 

 lias become so widespread that the dry 

 stains in the proportion given for Mac- 

 Neal's solution have appeared in com- 

 merce under the name MacNeaVs Tetra- 

 chrome. This is the more unfortunate as 

 there are only three stains involved. The 

 most completel}' rationalized of all these 

 processes is that of Kingsley 1935. He 

 specifies not only the ingredients to be 

 used but also the pH to whicli the solu- 

 tions should be buffered. He has, more- 

 over, provided techniques whereby these 

 same solutions can be applied either to 

 smears, paraffin sections, or frozen sec- 

 tions, and has thus permitted for the first 

 time the complete correlation of the 

 temporary results obtained from a frozen 

 section hurriedly produced in the course 

 of an operation and a permanent paraffin 

 section which may be secured from post- 

 mortem material. 



13.10 TYPICAL EXAMPLES 



Preparation of a blood smear using 

 the methylene blue-azur A-methylene 

 violet-eosin Y stain of Kingsley 1935 



All of the azur-eosin techniques ai'e de- 

 signed primarily to differentiate cell types 

 one from another rather than to differ- 

 entiate gross histological structures. There 

 is, therefore, very little need for detailed 

 instructions to be given. A blood film is as 

 good an object on which to practice as 



any other. In spite of the numerous formu- 

 las which are given in this section it cnn- 

 uot be too strongly recommended that the 

 method of Kingsley (D8 13.13 Kingsley 

 1935) be used exclusively, unless one is 

 endeavoring only to follow a diagnostic 

 method given l)y a previous writer, or 

 endeavoring to understand how some 

 previous author has secured a result which 

 one is unable to dui)li('ate by a more 

 rational metliod. The oidy diliiculty in the 

 method of Kingsley here given is the 

 preparation of the necessary solutions: 

 these tnust be made with chemically pure 

 reagents. Tlie two stock formulas are 

 quite stable, but they must be prepared 

 accurately. Tlie figures for the dye con- 

 tents of the solutions, which are given in 

 terms of milligrams, should be adhered to 

 and should be weighed on an accurate 

 balance. Both the formulas are, however, 

 liable to a certain degree of biological 

 degradation from molds, and it is, there- 

 fore, not desirable to prepare very large 

 quantities at one time. The reference to a 

 buffer at pH 6.9 does not specify the 

 buffer salts to be used. Kingsley {loc. cit.) 

 specifies phosphate buffers, but the writer 

 has used phthalate buffers with ecjual 

 success. The ver}' small quantity of the 

 l)uffer required and the necessity for 

 having it at an accurate pH suggests that 

 (unless very considerable facilities are at 

 the disposal of the preparator) he purchase 

 this buffer solution ready-prepared rather 

 than prepare it himself. The methanol, 

 acetone, and glycerol specified as staining 

 ingredients should be of a reagent grade. 

 Since the working solution C requires only 

 half a milligram of eosin Y to be added 

 to it, and since it is improbable that the 

 majority of people have the facilities for 

 weighing this accurately, 1 % solution of 

 eosin Y in acetone can be prepared as a 

 stock solution and a 1 % dilution of acetic 

 acid in acetone may be prepared as 

 another stock solution. A moment of 

 calculation will illustrate how these can 

 be diluted with acetone to give the re- 

 quired staining reagent. 



Blood films are simple to make, pro- 

 vided one has chemically clean glassware 

 and remembers, if he has never made such 

 a preparation before, tliat the smear must 



