354 



METHODS AND FORMULAS 



DS 13.30 



formula of Pappenheim 1901 has been 

 adapted for use with bacteria by Saathof 

 1905, and is now widely used for this pur- 

 pose. The combination of methyl green 

 with other dyes is best known from the 

 Ehrhch 1898 (or Heidenhein 1888) "tri- 

 acid" mixtures. These mixtures and their 

 variations continue to occur from time to 

 time in the literature, but it is difficult to 

 justify their employment today. Far better 

 methyl green-acid fuchsin combinations 

 are the two triple stains of Foley, 1930 

 and 1931, which will give all the staining 

 reactions of the earlier mixtures without 

 their manifest disadvantages. Most of the 

 other formulas in this class are various 

 modifications of the "triacid" mixtures. 



13.30 TYPICAL EXAMPLES 



Staining a section of the suprarenal 



body in the methyl green-acid 

 fuchsin-orange G stain of Foley 1939 



The method of staining here described 

 is the most rational of all the variants of 

 the old Ehrhch's "triacid" stain. These 

 stains are largely designed to differentiate 

 cell types, and should be employed only on 

 organs in which so many cell types are 

 present that many varieties of shade are 

 desired to differentiate between them. 

 The suprarenal bodies fulfill this require- 

 ment and are best obtained from a young 

 mammal. A rabbit is excellent for class 

 demonstration; a two-third-grown male 

 should be selected, killed in the customary 

 manner, eviscerated, and the suprarenal 

 bodies removed from under the peri- 

 toneum before being rinsed in physio- 

 logical sahne solution. 



There is considerable discussion in the 

 literature as to the best fixative to select 

 for these materials, but a combination of 

 dichromate and osmic acid will provide 

 excellent fixation of the chromaffin mate- 

 rial (which will be retained in paraffin sec- 

 tions) and will thus differentiate at least 

 this cellular material with a reasonable 

 degree of certainty. Numerous fixatives of 

 this general composition have been sug- 

 gested; that of Altmann 1890 (Chapter 

 18, F 1700.0000 Altmann 1890) is well 

 suited for this particular preparation. No 

 fixative containing osmic acid has much 



penetrating power, so that the suprarenal 

 body, having been rinsed in physiological 

 saline, should be cut with a sharp scalpel 

 into at least three pieces before being im- 

 mersed in about 50 milliliters of the 

 selected fixative. Fixation is permitted to 

 continue in the dark for about 24 hours 

 before the specimen is removed and 

 washed in running water for at least a 

 further 12 hours. If, after this washing, 

 the outer surface of the specimens are un- 

 duly blackened (they will in any case be 

 a liglit brown color), a few drops of hy- 

 drogen peroxide may be applied in dis- 

 tilled water; but the pieces should not be 

 left in this mixture for longer than it takes 

 the black stain to disappear. They should 

 then be washed in several changes of dis- 

 tilled water before being rewashed in 

 running water for at least three or four 

 hours. The hydrogen peroxide does not 

 destroy the osmic hydroxides precipitated 

 on the surface, but only causes them to 

 revert to the water soluble tetraoxide. 

 The soluble tetraoxide must therefore be 

 removed by prolonged washing unless the 

 blackening is to recur. 



Dehydrating, embedding, and section- 

 ing should present no difficulty whatever 

 with an object of this type, though it is 

 recommended that sections no thicker 

 than 5 microns should be employed. These 

 sections are attached to a slide in the 

 customary manner, dewaxed, and brought 

 down through the customary dehydrating 

 agents to distilled water, in which the 

 slides may be accumulated until they are 

 required for staining. 



The only difficulty lies in the prepara- 

 tion of the stain itsejf (DS 13.32 Foley 

 1930) which, if prepared imperfectly, will 

 contain a precipitate. The addition of the 

 acid fuchsin and the orange G solutions to 

 the glycerol occasions no difficulty; but 

 the greatest care should be taken to have 

 these completely mixed together before 

 the methyl green is added. The methyl 

 green should be added as slowly as possi- 

 ble in small drops and each drop should 

 be thoroughly mixed in before the next is 

 added. The stain should then be allowed 

 to remain for at least 12 hours at room 

 temperature before being filtered im- 

 mediately before each use. 



