EXPERIMENTAL EQUIPMENT AND PROCEDURES 25 



Then transfer to pure 50°C. paraffin for 15 minutes and finally to 2 changes of 53-55°C. 

 paraffin. For eggs, a lower melting point paraffin is better. Prolonged embedding tends 

 to make the eggs brittle. 



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

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

 paraffin for tissues in general. 



a. Paraffin alone often crystallizes when cooled, or it may even flake, particularly 

 if the xylol has not been entirely removed. In order to prevent crystallization 

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

 tions down to 2 microns even during the summer, without the use of ice. 



Paraffin M. P. 48°-50OC. - 90 gms. 

 Beeswax, white - 5 gms. (for ribboning) 



Bayberry wax, pale green - 5 grms. (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 miost satisfactory method is to use Plaster of Paris (see Solberg '39) 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 care- 

 fully 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 embedding 

 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 cells 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 mi- 

 crons. 



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 very slowly . 

 Examine the knife under binocular magnification for knicks. If yolk-eggs are embedded 

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



Some investigators expose the amphibian egg by cutting off one or two sections and then 

 soaking the entire block in water, overnight. Such an egg will expand beyond the cut sur- 

 face and several sections will be lost, but frequently very nice serial sections of the 

 remaining portion of the egg can be acquired. A second soaking, half-way through the 



