Embedding 



PARAFFIN SECTIONS 



97 



solvent for emheddiiig media and as a 

 clearing agent before them. These three 

 hydrocarbons are so cheap, and are ob- 

 tainable in such a pure form, that there 

 seems no necessity to use any other clear- 

 ing agent, unless one prefers the reagents 

 which are supposed to combine the func- 

 tions of both dehydration and clearing. 



It is still occasionally recommended that 

 essential oils, such as cedar oil, be used for 

 clearing objects for embedding. There is 

 no justification for this unless it is vital 

 that the object be rendered transparent 

 (rather than alcohol-free) in order that 

 some feature of its internal anatomy may 

 be oriented in relation to tlie knife. Essen- 

 tial oils are excellent for wholemounts, 

 l)ut they are not readily removed from the 

 specimen by molten wax; therefore, if they 

 must be used, they should always be 

 washed out with a hydrocarbon before the 

 wax bath. Relatively small traces of any 

 essential oil will destroy the cutting prop- 

 erties of any wax mixture and, as they are 

 nonvolatile, there is no chance of getting 

 rid of them in the embedding oven. 



Choice of an Embedding Medium 



Formulas for the various wax mixtures 

 used in the preparation of ribbons of sec- 

 tions will be found in Chapter 27 (E 21.1). 

 It is to be presumed at the present time 

 that no one will endeavor to use a plain 

 paraffin but will use one of these mixtures. 

 If, for some strange reason, a pure paraffin 

 is preferred, then it is necessary to buy (in 

 the United States by importation) a care- 

 fully fractionated and very expensive wax. 

 Ordinar}' cheap paraffin is a mixture of a 

 great variety of compounds of slightly dif- 

 ferent melting points, and it is essential in 

 the use of jnire wax that a wax of a veiy 

 sharp melting point should be obtained. 



The choice of an embedding medium 

 should be dictated less by the nature of the 

 specimen than by the conditions under 

 which it should be cut. If pure paraffin is 

 to be employed, it sliould be seloct('(l with 

 sucli a melting point, that tlie hardened 

 wax will give a crisp section at the re- 

 quired room temperature. In the Europe 

 of twenty years ago, when many writers 

 were recommending a wax with a melting 

 point of 52°C., the average laboratory 



temperature in winter was between 50° 

 and GO^F. A wax of 52°C. melting point, 

 in an American laboratory kept between 

 70° and SO°F., is far too soft to cut any 

 but the thickest sections. The use of waxes 

 of 58°C., which are quite hard enough for 

 cutting sections in an American labora- 

 tory, is unfortunate, since such use re- 

 quires an oven temperature of at least 

 60°C. which results in many tissues be- 

 coming hard and brittle. As the introduc- 

 tion of any foreign substance automati- 

 cally lowers the melting point of the wax, 

 it is obviously desirable to use mixtures 

 rather than the pure material. The writer's 

 preference is naturally for his own compo- 

 sition (E 21.1 Gray 1944). The advantage 

 of the mixtures there specified is that they 

 have a relatively low melting point but 

 soften ver}' little before reaching the melt- 

 ing point. The degree of hardness (that is 

 the thinness of the section which may be 

 cut) may be controlled accurately by the 

 proportion of resin added; and the writer 

 has once secured, on a demonstration, 

 a paraflftn ribbon more than 20 feet 

 long of 1 -micron-thick sections. Media of 

 this hardness, however, impregnate very 

 slowly and should only be used for mi- 

 nute objects. For ordinary routine prepa- 

 rations the writer's jircference is for any 

 of the paraffin-rubl)er-bayberry-wax mix- 

 tures. The introduction of rubber un- 

 doubtedly increases the stickiness of the 

 wax and makes it easier to secure continu- 

 ous ribbons, while the bayberry wax not 

 only prevents the crystalUzation of the 

 paraffin but also lowers its melting point. 

 The beginner is strongly recommended to 

 experiment with several of the rubber- 

 bayberry-wax compositions and to select 

 after exj)eriment that which gives him uni- 

 formly successful results in his own 

 la])oratory. 



Technique of Dehydrating, Clearing, and 

 Embedding 



Before passing to tlic choice of a micro- 

 tome and tiie method of using it, it is 

 necessary to discuss briefly tlie actual op- 

 erations which are involved in using the 

 dehydrating, clearing, and embedding me- 

 dia selected. The techniques of dehydra- 

 tion and dc-alcoholization do not differ 



