EXPERIMENTAL EQUIPMENT AND PROCEDURES 



17 



2. 



3. 



k. 



5. 

 6, 

 7. 

 8, 



9. 

 10. 

 Il- 

 ls. 

 13. 



If eggs are left in fixative 2l4-i^8 houra, the Jelly will be removed.. 



Wash 2k hours in running water. 



Carry to 70^ alcohol through gradual changes. 



Change to 1 part aniline oil, and 2 parts 70^ alcohol for 2 to 6 hours. 



Change to 2 parts aniline oil, and 1 part 95^ alcohol for 2 to 6 hours. 



Change to pure aniline oil luitil clear - 1 or more hours. 



Change to 50^ aniline oil plus 50^ toluene for 1 to 6 hoiu-a. 



Change to 100^ toluene, for 1 to 5 hours. 



Place in 100^ toluene for 1 to 5 hours. 



Place in saturated 53° paraffin in toluene for 1 to *+ houra. 



Place in 53° paraffin for 5 to l^ houra, at 55°C. 



Embed in 53° paraffin. 



C. L. 



The following procedure has been used with considerable success by Dr. 

 Parmenter in studying the cytology of the amphibian egg. 



1. Fix for 2k hours in Smith's fluid. 



2. Preserve egga in Jelly in 5^ formalin. 



3. To remove Jelly: Uae 20^ solution chlorox in distilled water for 3 to 



k minutes. Watch and atop before the cortex is injured. 

 k. Binse thoroughly in distilled water, several changes. 



5. Dehydrate to 70^ alcohol with 5 minute changes in ascending. 



6. Dehydrate further: (Leave in this overnight) 



8Cff> alcohol 96 cc. 



Phenol k cc. 



(See King & Slifer, 1935; Science 78.) 



7. Dehydrate further in 95^ alcohol - 2 thirty minute changes. 



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



9. Infiltrate with tisaue mat; '56-58° M.P. paraffin for 1 hour. 



10. Imbed, aection and mount. 



11. Preliminary to staining, remo.ve paraffin with xylol; rinae 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 aections. Thie coating ia a precaution againat loss of an occa- 

 sional section during staining, etc. It does not Interfere with 

 staining and need not be removed. 

 11. Use any stain deaired. Neither Feulgen nor acid haematoxylin will 

 atain yolk. 



WASHING 



Moat tissues in aqueous fixatives should be waahed in running water for 6-21+ 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 extrane- 

 ous materiala within the tissues. The completion of washing cannot be determined by loss 

 of color in the tissues. In Bouln-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 proceaaea are included under thla title. Firat, the removal of color added in 

 the fixation procees. This may be acconplished 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- 

 monium hydroxide (NB1).0H) 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 acconjiliahed 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 aeveral minutes to 

 several daya, depending upon the degree of bleaching desired. 



