EXPERIMENTAL EQUIPMENT AND PROCEDURES 23 



(7) Dehydrate further in 95% alcohol - 2 thirty minute changes. 



(8) Carboxylol - 1 hour. (This may be too drastic for some eggs. ) 



(9) Infiltrate with tissue mat; 56-58° M. P. paraffin for 1 hour. . 



(10) Imbed, section and mount. 



(11) Preliminary to staining, remove paraffin with xylol; rinse in absolute alcohol 

 and dip (or flood with pipette) quickly into a solution of equal parts of absolute 

 alcohol, ether, and 10% celloid in solution. Place in 70% alcohol to harden 

 the very thin coating of celloidin on the sections. This coating is a precaution 

 against loss of an occasional section during staining, etc. It does not inter- 

 fere with staining and need not be removed. 



(12) Use any stain desired. Neither Feulgen nor acid haematoxylin will stain yolk. 



WASHING 



Most tissues in aqueous fixatives should be washed in running tap water for 6-24 hours, 

 the time depending upon the size of the tissue. The function of washing is to remove 

 salts, crystalline substances that may have been added with the fixative, and any extra- 

 neous materials within the tissues. The completion of washing cannot be determined by 

 loss of color in the tissues. In Bouin-dioxan fixation, the washing process is generally 

 omitted until the sectioned material is passed down through the alcohols. Alcoholic 

 iodine is used to remove excess corrosive sublimate. 



BLEACHING 



Two processes are included under this title. First, the removal of color added in the 

 fixation process. This may be accomplished by 12 hour changes in saturated aqueous 

 solutions of lithium carbonate when picric acid has been used. A more satisfactory 

 bleaching agent, because it is more rapid and leaves no residue, is a 2% solution of am- 

 inonium hydroxide (NH^OH) in 70% alcohol used in half-hour changes until no more of 

 the yellow picric color is visible. 



The second process has to do with the actual bleaching of tissue pigments, and this may 

 be accomplished with any of the following: 



a. Mayer's chlorine method : Transfer specimens from 70% alcohol to freshly made 

 Mayer's solution, cover, and leave for period of from several minutes to several 

 days, depending upon the degree of bleaching desired. 



b. Javelle water : Slow but satisfactory bleaching agent. 



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

 be bleached in 1 cc. of 2% sodium bisulphite to which is added (just before using) 

 2-4 drops of concentrated HCl. Acts over 6-12 hours. 



d. Hydrogen peroxide : Used as 2% solution but will macerate tissues if used for 

 long period. 



DEHYDRATION 



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

 alcohols (35% to absolute alcohol) with 15-30 minute stops for tissues and 2-5 minute 

 stops for sections or small eggs, longer stops for sizeable tissues. To conserve alcohol, 

 dehydration may be accomplished in small vials by decanting off and changing the alcohols. 

 If 5% glycerine or triethanalamine is added to the dehydrants, the tissues will not become 

 so brittle and will be easier to section. 



