EXPERIMENTAL EQUIPMENT AND PROCEDURES 19 



EMBEDDING 



This la accompli shed by Infiltration with paraffin, wax, rubber, or mixtiires of 

 these. Whatever clearing agent Is used, the process of embedding shoiild be gradual and 

 at a temperature slightly above the melting point of the mixtures. To the vial contain- 

 ing the clearing fluid and cleared tissue, add shavings of the embedding mixture and bring 

 to about 1+0°C. for several hours. The paraffin will become gradually more concentrated 

 with the evaporation of the clearing agent. Then transfer to embedding substance for 2 

 half -hour changes. 



If dloxan Is used for dehydration and clearing, wann a mixture of 25 cc. dloxan, 

 5 cc. xylol and 20 cc. of 50°C. paraffin and transfer the tissues to this mixture for 50 

 minutes. Then transfer to pure 50°C. paraffin for 15 minutes and finally to 2 changes of 

 55-55°2. paraffin. For eggs, a lower melting point paraffin Is better. Prolonged em- 

 bedding tends to make the eggs brittle. 



Various embedding eubatancea may be used, starting with paraffin of different melting 

 points, the softer paraffin being best for the yolk eggs and glandular tissue and the hsird 

 paraffin for tissues In general. 



a. Paraffin alone often crystallizes when cooled, or It may even flake, particular- 

 ly If the xylol has not been entirely removed. In order to prevent crystalliza- 

 tion and to facilitate ribbon formation, a mixture has been devised which allows 

 sections down to 5 microns even during the summer, without the use of Ice. 



Paraffin M.P. l+8°-50°C. - 90 gms. 

 Beeswax, white - 5 gms. (for ribboning) 



Bayberry wax, pale green - 5 gms. (for hardening) 

 The same mixture can be used for tissues rather than eggs but the higher melt- 

 ing point paraffins would be advised. 



b. Tissue mat - a commercial mixture probably very similar to the above excellent. 



c. Rubber - small amount of white rubber may be melted into the paraffin to give 

 better ribbons. Particularly good for large sections or semi-hard (i.e., car- 

 tilage) tissue. 



The conventional method of embedding Involves paper boxes made in appropriate sizes. 

 Syracuse dishes lined with glycerine or white vaseline may be used for large tissues or 

 large numbers of tissues. Paraffin buttons made by pipetting a small amount of melted 

 paraffin onto a clear slide will prove satisfactory for small tissues. 



The most satisfactory method is to use Plaster of Paris (see Solberg) embedding boxes. 

 These are made by cutting out several blocks of soft paraffin, cut into the shapes and 

 sizes desired, and making certain that the sides of the blocks all slant outward from the 

 bases. Place these blocks with larger surface down on glazed paper and cover, carefully 

 with wet Plaster of Paris. When dry, tear off the paper and dig out the soft paraffin 

 with a scalpel. Finally shave off the excess Plaster of Paris until a thin-walled embed- 

 ding box is made. To use, first submerge the box in cool water, pour out all of the 

 water; add melted paraffin; add tissue and orient it with hot needle; bring box into ice 

 water but do not submerge it until there is a surface film. When the film entirely covers 

 the paraffin, plunge the whole box beneath the surface of the water and the paraffin block 

 will pop out and come to the surface. 



SECTIONING 



Amphibian ceils are among the largest known so that sections should rarely be less 

 than 10 microns. When organ systems are to be studied, sections may be as much as 25 

 microns. 



The standard rules to be followed are: to use a clean, sharp knife at a fair angle; 

 to clean the knife blade frequently with xylol; and to section yolk-laden material veiy 

 slowly . Examine the knife under binocular magnification for knlcks. If yolk-eggs are 

 embedded so that the knife cuts from vegetal toward the animal pole, cracks will be 

 eliminated. 



