DS 12.20 



DYE STAINS OF GENERAL APPLICATION 



323 



have been fixed, if the term can properly 

 be used in this case, in formaldehyde for a 

 considerable period. Each embryo, after 

 removal from the oviduct, should be 

 thoroughly washed, using soap if neces- 

 sary to remove the mucus from the out- 

 side, rinsed thoroughly to remove the last 

 traces of soap, and then thrown into a 

 gallon jar of picro-carmine prepared by 

 the method given under DS 11.24 Ranvier 

 1899. The quantity of picric acid in this 

 solution causes it to act as fixative and 

 preservative, as well as a nuclear stain, 

 and it has the additional advantage that it 

 is quite impossible to overstain in it. Em- 

 bryos may therefore be thrown into such a 

 jar of picro-carmine during the course of 

 the first half of the year's work and uti- 

 lized during the second half of the year 

 when microtechnique is usually taught. 



When it is desired to cut sections, the 

 embryos are removed from the large jar 

 of picro-carmine and washed in running 

 water until no further color comes away 

 from them. Though it is immaterial how 

 long they have remained in stain, it must 

 be added that some weeks are usually 

 required to secure a sufficient impregna- 

 tion. The picric acid in a properly pre- 

 pared Ranvier solution is usually suffi- 

 cient to have decalcified the placoid scales 

 of a young dogfish. These should be tested 

 by rubbing a steel needle against the direc- 

 tion of the skin. A hard irregularity will 

 indicate that the calcium of the placoid 

 scales has not been removed. In this case 

 recourse must be had to one of the 

 methods for decalcification given in Chap- 

 ter 19 under AF 20. 



The specimens are then embedded and 

 sectioned in the customary manner, it 

 being perfectly possible for even a moder- 

 ately skilled operator to cut a reasonable 

 hand section from the paraffin block con- 

 taining embryos of this size. It is, of 

 course, preferable to use a microtome if 

 one is available. The sections are then ac- 

 cumulated and attached to the slide in the 

 ordinary manner, deparafinized, and run 

 down through the customary reagents 

 until they reach 70% alcohol where the 

 slides may be accumulated. A brief check 

 under the low power of the microscope 

 vUl now establish whether or not the 



nuclei have been satisfactorily stained 

 scarlet throughout the body of the prepa- 

 ration. If they have not, it is no use pro- 

 ceeding further, for one must devote some 

 other staining technique to these sections. 

 Nine times out of 10, however, it will be 

 found that the nuclei are clearly stained 

 and quite reasonably differentiated. If 

 the tissues other than the nuclei are 

 stained a dark red rather than the light 

 pink which is ine\dtable, each section 

 should then be briefly differentiated in a 

 0.1 % solution of hydrochloric acid in 70% 

 alcohol. This, however, will very rarely 

 be necessary. 



The sections are now run down to 

 water before being stained in picro- 

 indigo-carmine (DS 12.211 Cajal 1895), 

 which is the easiest, simplest, and most 

 foolproof of all double staining methods. 

 The shdes bearing the sections are placed 

 in the solution from three to five minutes, 

 withdrawn from the stain, rinsed briefly 

 in water, and then placed directly in ab- 

 solute alcohol, and left until a naked-eye 

 examination shows that the connective 

 tissues are clear blue. This clear blue is in 

 distinction to the bright green of the 

 muscles or the red of such other tissues as 

 have retained the carmine. The moment 

 that this change from green to blue, which 

 is clearly and sharply seen, takes place the 

 slide is placed in xylene which stops the 

 differentiation and permits subsequent 

 mounting in balsam. 



The results obtained by this method 

 are not, of course, to be compared for 

 brilliance with the more complex methods 

 using acid fuchsin and some of the phos- 

 photungstic reactions. But this is undoubt- 

 edly the first double-staining method 

 which should be tried by any beginner — 

 to whom encouragement is probablj' of 

 more value than the actual colors to be 

 obtained. 



Preparation of a transverse section of 

 the tongue of a rat using coelestin 

 blue B followed by picro-acid fuchsin 



The chief difficulty in preparing a trans- 

 verse section of the tongue is to avoid 

 hardening of the muscle which tends to 

 become brittle, either if imperfectly fixed 



