CHAPTER . XXX VIII. 557 



should be used until it no longer loses its colour, which indicates excess 

 of iodine. The whole process should last several hours and may be 

 carried on overnight. 



The iodine and 70 per cent, alcohol are poured away, and the animals 

 washed for several hours (a minimum of two) in at least two changes of 

 70 per cent, alcohol to remove as much of the iodine as possible. The 

 objects are then transferred to 50 per cent, alcohol for one half- 

 hour, then into 30 per cent., for the same time. They are brought 

 down these grades in order that shrinkage may not occur when 

 they are being transferred to stains containing little alcohol, or none 

 at all. 



Two stains may be tried, Mayer's acid hsemalum ( 248 and 249), and 

 Grenadier's alcoholic borax carmine ( 213 and 233). The time that 

 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 away, and the 

 process of differentiation ( 203) is begun. 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 HC1 to 100 c.c. of 70 per cent, alcohol), which should generally 

 be allowed to act at least for 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 

 well 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 cedar wood or clove oil for at least two hours, and then 

 mounted in xylol balsam. 



The hsemalum 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. 



