18 EXPERIMENTAL EQUIPMENT AND PROCEDURES 



b. Javelle water : Slow tut satisfactory bleaching agent. 



0. Bichromate bleach : Following bichromate fixation such as Smith's tissues may 

 be bleached in 10 cc. of 2$ sodium bisulphite to which Is added (just before 

 using) 2-h drops of concentrated HCl. Acts over 6-12 hours. 



d. gydrop;en peroxide : Used as 2^ solution but will macerate tissues if used for 

 long period. 



DEHYDRATION 



The standard method of dehydration la to run the tissues up through a graded series 

 of alcohols (55^ to absolute alcohol) with 15-50 minute stops for tissues and 2-5 minute 

 stops for sections or small eggs. To conserve alcohol, dehydration may be accomplished 

 in small vials by decanting off and changing the alcohols. If 5^ glycerine or triethana- 

 lamine is added to the dehydrants, the tissues will not become so brittle and will be 

 easier to section. 



It has been found that dioxan is an excellent substitute for alcoholic dehydration. 

 Following the washing (or even without washing) the tissues are put directly into dioxan 

 for 2 two-hour changes. Larger tissues may be left in dioxan for a month without any 

 deleterious effects. If the dioxan Is kept in covered Jars and over copper sulphate, it 

 should last a long time. The dioxan fumes are known to be poisonous to humans. 



Amphibian eggs and yolk-laden embryos should not be completely dehydrated. It la 

 thought that the dioxan method does not give complete dehydration and that this la one of 

 the reaaona that dioxan givea better reaults with amphibian egga. 



CLEARING 



Clearing followe dehydration and must be accomplished before embedding. In any case, 

 the absolute alcohol should be mixed with the clearing agent so that the transition la 

 gradual. Several steps of 15-50 minutes each are best. The dehydrant dioxan acta as its 

 own clearing agent. 



Xylol - this agent tends to make yolk-laden egga and embryos rather brittle and sec- 

 tions are apt to crack. This tendency can be somewhat compensated by the addition of 5^ 

 lanolin (Sheep fat) to the xylol. Xylol will become cloudy if the embryos were not suf- 

 ficiently dehydrated. 



Benzole - same aa xylol. 



Dioxan - this is both a dehydrating and clearing agent, and can be mixed with fixa- 

 tives. The transfer to paraffin is generally made by way of a paraff In-xylol mash. Uaed 

 for both cytological and histological preparations. 



Chloroform - tissues are transferred from absolute alcohol to a mixture of equal 

 parts of alcohol and chloroform until they sink to the bottom of the container, thence to 

 pure chloroform for final clearing. Keep the vial covered. 



Wintergreen oil - excellent for yolk masses and glandular tissue, used as chloroform. 

 Tissues become translucent and may thus be photographed. 



Clove oil - uaed same aa wintergreen oil. 



Aniline oil - aame aa wintergreen oil except that it will mix with lower alcohols 

 down to 80^. Stained sections are dehydrated to 95^, then to equal parts of 95^ and 

 aniline oil for 10 minutes; finally to 2 ten-minute changes in pure aniline oil. Mount 

 In anillno-balBam. Good for mitotic figures. 



