138 



THE ART OF MAKING MICROSCOPE SLIDES 



S. S. mouse 



very difficult to overharden or overfix a 

 specimen of this kind, and at least a 

 month should be allowed to make sure 

 that there is perfect fixation throughout 

 while an exposure of six months will cause 

 no damage. 



After fixation is complete, the mouse 

 (or mice) is removed from the fixative, 

 and without removing from the cheese- 

 cloth bags, hung in a jar through which 

 running water flows from a tube reaching 

 to the bottom. It (or they) should be 

 washed for at least three days in running 

 water before being removed and hung in a 

 large jar of 20% alcohol or other de- 

 hydrant. For those who find alcohol diffi- 

 cult to olitain, either acetone, isopropanol, 

 or methanol are equally good for dehydra- 

 tion, but must in each instance be used in 

 a fairly close series. Small objects may, as 

 has been stated elsewhere, be passed with- 

 out danger from water into absolute al- 

 cohol, but as large an object as this mouse 

 will have to be dehydrated very slowly. 

 The mouse should be left in 20% alcohol 

 for about five days and subsequently for 

 about five days each in 50%, 70% and 

 90%. The experienced reader will have 

 noticed that we have so far said nothing 

 of decalcification which must obviously 

 take place before the sections can be 

 made. On the basis of the writer's experi- 

 ence, born out by the earlier workers but 

 apparently nowadays ignored, it would ap- 

 pear that hardening in alcohol after fixa- 

 tion yields a specimen which behaves very 

 much better under the knife than does one 

 which has been fixed only without the sub- 

 sequent alcohol hardening. It is for this 

 reason that he recommends that it be 

 taken up in the manner described to 90 % 

 alcohol, left there for a week or two, and 

 then brought down through the same 

 series to water, where it is left until 

 the whole of the alcohol has been re- 

 moved. The specimen is now ready for 

 decalcification. 



For as large an object as this, particu- 

 larly one which has been fixed in a di- 

 chromate mixture, the method of von 

 Ebner would be desirable. This solution, 

 the formula for which is given in Chapter 

 19 under the heading AF 21.1 von Ebner 

 (1891), employs a strong solution of 

 sodium chloride to diminish the swelling 



of the tissues caused by the nitric acid 

 used. It is also possible to use phloroglucin 

 for the same purpose of diminishing the 

 swelhng; a typical formula is given in the 

 same chapter as AF 21.1 Ferreri (1895). 

 It has, however, been the writer's experi- 

 ence that these phloroglucin formulas 

 work better on smaller objects and he 

 strongly recommends the formula of von 

 Ebner in the present case. 



Following this formula, the specimen is 

 hung in a large volume of the solution and 

 left for three or four days. At the end of 

 this time a further 1 % of nitric acid is 

 added and the whole stirred up. This proc- 

 ess of adding a milliliter of nitric acid per 

 100 milliliters is continued every third or 

 fourth day until decalcification is com- 

 plete. It is as undesirable to decalcify for 

 too long a period as it is to leave patches 

 of hard bone to wreck the knife, hence the 

 worker will often find himself in a quan- 

 dary as to how to determine when de- 

 calcification is complete. The only way 

 this can be done with complete success is 

 by x-ray examination, for the least trace 

 of undissolved calcium remaining will 

 show clearly upon the x-ray plate or 

 fluorescent screen. It is often possible to 

 find some friendly dentist who will prepare 

 an x-ray of the mouse at intervals, but if 

 this is impossible one must judge on deU- 

 cate probing with a needle. The two 

 places which are usuallj^, but by no means 

 always, the last to decalicify are the 

 inner ear and the molar teeth, and it is a 

 reasonably safe assumption that if a fine 

 needle can be passed through these with- 

 out meeting more resistance than would 

 be occasioned by tough leather, it is safe 

 to continue. It is not safe to probe in the 

 direction of the vertebrae, which are often 

 slow in decalcification, because they are 

 too close to the central area which will 

 subsequently be sectioned. In the ab- 

 sence of x-ray information it is much safer 

 to decalcify too long than too short a 

 time, and it will be suggested later that a 

 solution be used to mordant the sections; 

 this will undo, to a certain extent, any 

 excessive hydrolysis which has taken 

 place. 



The decalcified mouse must now be 

 thoroughly washed to remove all traces of 

 acid, but water should not be used for this 



