724 A GUIDE FOE STUDENTS OF MICROTOMY 



both these stains should be used depends almost entirely upon the 

 accessibility of the cells of the object to the stain. Daphnids are 

 covered by a chitinous shell, which though delicate tends to prevent 

 instant penetration. It is a good thing to leave the animals in the 

 stain for about five hours at least, and overnight preferably. 



Take two clean capsules, pour into one about 10 c.c. of borax carmine, 

 into the other a similar quantity of the haemalum. With a camel-hair 

 brush or a pipette transfer some of the organisms to the stains and leave 

 as directed above. See that the capsules are securely covered. 



After some hours in the stain, the latter is poured back, and the 

 process of differentiation (§ 240) is begim. The object of differentiation 

 is to wash away superfluous stain from certain organs or parts of organs, 

 in order that a contrast in depth of colour may be obtained in the 

 various other organs and tissues. Both borax carmine and Mayer's 

 acid hsemalum may be differentiated in acid alcohol (4 to 6 drops 

 of HCl to 100 c.c. of 70 per cent, alcohol), which should generally 

 be allowed to act at least as long as the stain has been used, and, 

 if necessary, longer. In both cases when differentiation has reached 

 the right stage, the objects examined under a microscope have a trans- 

 parent appearance, and such parts as the viscera and muscles should be 

 ivell contrasted. 



The borax carmine specimens are washed out for several hours in 

 neutral 70 per cent, alcohol. They are then upgraded to 90 per cent, 

 and absolute alcohol, two hours in each, or overnight in absolute alcohol, 

 and cleared in methyl benzoate, cedar- wood or clove oil for at least two 

 hours, and then mounted in xylol balsam. 



The haemalum specimens have to be brought to an alkaline solution 

 in order to " blue " the stain, and to get rid of all acid. Some workers 

 " blue " the stain in 70 per cent, alcohol made slightly alkaline with 

 ammonia or bicarbonate of soda, but the best results are obtained by 

 downgrading the objects to tap-water, which is allowed to run over 

 them gently till they go quite blue, which should occur for small objects 

 within an hour. The animals are then gradually upgraded through 30, 

 50, 70 and 90 per cent., to absolute alcohol and cleared as above 

 described for borax carmine specimens. 



In order to obviate the differentiation stage, one may dilute both the 

 borax carmine and the acid haemalum till they are about one-third or 

 one-half as strong ; dilution of the borax carmine may be carried out 

 with 50 per cent, alcohol (not methylated spirit) and with distilled 

 water in the case of haemalum. In these solutions the animals remain 

 till sufficiently stained. But the best results are got by the overstaining 

 and differentiation method. 



1421. Example II. From a frog remove a large leg or thigh muscle, 

 and cut it into two pieces about as big as the nail of the little 

 finger. If desired, the liver, a halved testis, or a kidney may also be 

 used. 



Transfer the material to a capsule containing at least 20 c.c. of 

 Susa (wash out in 90 per cent., see § 68), Zenker's or Helly's fluid 

 (§ 78). Leave till next morning, and wash in running water under 

 the tap for at least three hours, preferably overnight, then transfer to 

 50 per cent, alcohol for an hour ; then to 70 per cent, alcohol, containing 

 enough tincture of iodine to give the solution a light port-wine shade. 

 Add more iodine as the colour disappears, prolonging the treatment 

 overnight for large pieces. Pour away the alcohol, and add pure 70 per 

 cent., in which the material is washed at least three hours. Transfer to 

 90 per cent, for several hours and leave in absolute alcohol overnight. 

 Next morning it is safest to give the material another hour in a fresh 

 change of alcohol absolute. Pour away a good deal of the alcohol and 



