DS 12.30 



DYE STAINS OF GENERAL APPLICATION 



333 



men. On removal from the phospliomo- 

 lybdic acid it must be emphasized that no 

 water rinse may be given. Each slide must 

 be taken separately, drained, and the very 

 deep blue "C" solution poured onto the 

 surface of the slide where it may remain 

 from two to five minutes. It may be men- 

 tioned in parenthesis that this deep-blue 

 stain has just as great an affinity for the 

 human skin as for any other protein ma- 

 terial, and that it is far more difficult to 

 remove from the skin than it is from any 

 section. Rubber gloves or forceps are most 

 warmly recommended to the operator. 

 The D solution (1% acetic acid) should 

 be available in two vessels, one containing 

 a fairly large volume (a 500-ml. beaker is 

 admirable) and the other a standard 

 coplin jar or whatever vessel is customarily 

 employed for slide staining. The section is 

 rinsed rapidly in the large container to 

 remove the adherent blue, and then is 

 placed in the smaller container until such 

 time as no further color is seen to leave it. 

 One of the best features of this triple stain 

 of Masson is that one does not require to 

 control differentiation, for if the technique 

 has been followed properly to this stage, 

 the blue will not leave the connective 

 tissues in the acid solution. Modifications 

 in which it is recommended to differenti- 

 ated in alcohol should be avoided, since 

 this reagent can remove the whole of the 

 blue color. Even when the 1% aqueous 

 acetic acid is used at this stage of the 

 proceedings, it is undesirable to leave the 

 slide for too long a period after the blue 

 color has ceased to be liberated from it, 

 because there is a tendency to remove the 

 hematoxylin from the nuclei, and it is for 

 this reason that the slides cannot be 

 accumulated in this reagent. Tlie only 

 delicate portion of the preparation now 

 follows: tlie dehydration of the sections 

 without the loss of the blue. Even after 

 mordanting. in phosphomolybdic acid, the 

 blue is hkely to be removed from the 

 tissues with alcohol, and dehydration 

 should be undertaken directly in absolute 

 alcohol to which has been added 1 % acetic 

 acid. Either amyl alcohol with 1 % acetic 

 acid, or acetone with 1 % acetic acid, may 

 be substituted for absolute alcohol and 

 will render the dehydration of the speci- 



men much more easy without loss of the 

 blue. There seems to be, liowever, a 

 tendency to adhere to absolute alcohol on 

 the part of histologists; and the alterna- 

 tive reagents are only mentioned for the 

 benefit of those who might be prepared 

 to break with tradition. The least possible 

 time should be employed in dehydration; 

 preferably the slide should be dipped up 

 and down in tlie alcohol and transferred 

 to xylene at intervals to see whether ov 

 not it is sufficiently dehydrated to clear. 

 After they have been cleared in xylene, 

 the shdes may be accumulated in this 

 reagent until the whole of the batch is 

 ready for mounting. 



Many workers have specified that sali- 

 cylic acid be dissolved in the xylene to 

 render it acid, but this is a precaution 

 really only necessary if large quantities 

 of alcohol are carried over into it. The 

 normal provision of two or three changes 

 of xylene will render the addition of sali- 

 cylic acid unnecessary. Each slide is now 

 mounted; the most strongly acid balsam 

 which can be obtained is used ; or one may 

 employ salicylic balsam. This does not 

 necessitate making up a special mounting 

 medium, since the easiest method of 

 adding the acid to the balsam is from the 

 coverslip. A saturated solution of salicylic 

 acid is made in xylene and each coverslip 

 is dipped in this and permitted to dry in 

 air so as to become coated with a thin 

 film of salicyhc acid. Ordinary xylene 

 balsam, or any other resin dissolved in 

 xylene, is then placed on the sections and 

 one of the salicylic-acid-treated coverslips 

 is placed on the surface. Within a moment 

 or two the salicylic acid will have dis- 

 solved in and dispersed through the 

 mounting medium; that remaining on the 

 upper surface can easily be wiped off 

 after the slide is dry. 



Preparations prepared in this manner, 

 with due attention to the maintenance of 

 an acid environment, will be found to be 

 quite permanent enough for use for many 

 years in class, and will be such a great 

 improvement over the sections of earth- 

 worm usually supplied for class teaching 

 purposes as more than to warrant the 

 slight additional trouble which is required 

 for their preparation. 



