70 



Journal of Applied Microscopy. 



Notes on Microscopical Technique. 



G. Carl Huber, M. D. 



Second Paper. 



IMBEDDING TISSUES FOR CUTTING SECTIONS. 



In order to cut even relatively thin sec- 

 tions of hardened tissues for micro- 

 scopical examination, it is usually neces- 

 sary to imbed them in a substance.which, 

 in a fluid state, may be caused to per- 

 meate the tissue thoroughly, and which 

 may then be hardened to a consistency 

 which admits of cutting readily; and this 

 without detriment to the tissues im- 

 bedded. Experience has shown that two 

 methods, viz., imbedding in paraffin and 

 imbedding in celloidin, meet these re- 

 quirements. 



Paraffin Imbedding. — In describing this 

 method, it is assumed that the tissues 

 are hardened, and are in alcohol, ready 

 for imbedding. From the hardened tis- 

 sue, small, square, rectangular or trian- 

 gular pieces, the surfaces of which do 

 not exceed one-half square inch, and 

 the thickness one-twelfth to one-eighth 

 of an inch, are cut with a sharp knife 

 from several places of the tissue to be 

 studied and placed in absolute alcohol. 

 (For this and several succeeding steps, I 

 have found tube vials about two inches 

 high and three-fourths to one inch in 

 diameter very satisfactory). In the ab- 

 solute alcohol the tissues remain for 

 twelve to twenty-four hours. They are 

 then transferred to a solution which 

 mixes readily with alcohol and at the 

 san\e time dissolves paraffin. A num- 

 ber of reagents may be used. I have 

 found xylol the most convenient and 

 most satLsfactory of all. Toluol, chloro- 

 form, turpentine, cedar oil, etc., may, 

 however, be used in place of the xylol. In 

 the xylol — or in the other reagents men- 

 tioned — the tissues remain four to eight 

 hours, the time depending somewhat 

 upon the size and the density of the 

 pieces to be imbedded. When thoroughly 

 permeated with xylol, the tissues are 

 perfectly transparent. If this is not the 

 case, the xylol should be renewed and 

 the tissues allowed to remain in it until 

 they are transparent. If, when the tis- 

 sues are trtmsferred from the absolute 

 alcohol to the xylol, this becomes cloudy, 

 they should bo returned to absolute 

 alcohol for further dehydration. From 

 the xylol the tissues are placed in melted 

 paraffin. Two kinds of paraffin are used: 

 a paraffin having a melting point of 

 about 40° C. — so-called " soft paraffin " — 

 and "hard paraffin" with a melting point 

 of 50° C. It is obvious that, in order to 

 have the paraffin permeate the tissue, the 

 paraffin must be in the fluid state while 

 the tissue is in It. It is essential, how- 

 ever, that the melted paraffin have a 

 fairly constant temperature, two to three 



degrees above its melting point. Other- 

 wise, the tissues will become hard and 

 brittle, difficult to cut, and usually suffer 

 structural change. A fairly constant 

 temperature may be attained in several 

 ways. The best way is by means of a 

 paraffin bath, the temperature of which 

 is regulated by means of a thermostat. 

 A simple paraffin bath, one that answers 

 every purpose, is shown in Fig I. 



Fig. I. 



The paraffin bath consists of a rectan- 

 gular copper box, ten inches long, seven 

 wide, and five inches deep. The top is 

 provided with two cups, four Inches in 

 diameter and three and one-half inches 

 deep, each fitted with a cover, one of 

 which is provided with an opening about 

 one-half inch in diameter, into which a 

 thermometer should be fastened. The 

 top of the glass should further be pro- 

 vided with two openings, three-fourths 

 of an inch in diameter, one for the ther- 

 mostat, the other for filling the bath with 

 water. The bath rests on iron supports 

 eight inches high. It should be provided 

 with a false bottom of sheet iron. It 

 may be heated with a bunsen burner and 

 should be regulated to a temperature of 

 52° to 53' centigrade. The paraffin is 

 kept in small glass beakers, the soft 

 paraflin in a beaker in one compartment 

 of the bath, the hard paraffin in another 

 beaker in the other compartment, ready 

 for use. It Is essential, however, that 

 both the hard and soft paraffin be filtered 

 before they are used for imbedding. This 

 may be done by melting the paraffin in 

 a dish and pouring it into a glass funnel, 

 which has beer warmed over a flame just 

 before using. The paraffin may at once 



