PARAFFIN IMBEDDING 



186 



PARAFFIN SECTIONS 



ter. First smear a little glycerin evenly 

 over the bottom and sides of the dish. 

 Then pour in a little paraffin, a tliin 

 layer of which will harden so that when 

 the tissue is placed in the dish, it will 

 not come in contact with the bottom. 

 It is customary to orient the tissue so 

 that the surface to be cut first is next 

 to the bottom of the dish. Quickly 

 pour in more paraffin until the tissue is 

 covered to a depth of say 6 mm. Hold 

 the dish in ice water until the surface 

 of the paraffin has hardened just to the 

 point when on immersion in the iced 

 water the surface will hold its shape and 

 not run. However too rapid cooling 

 of paraffins of high melting point may 

 cause cracks in the surface and even in 

 the depth of the blocks. After a few 

 minutes the paraffin block slips out 

 easily because the glycerin prevented it 

 from sticking. When several different 

 specimens are imbedded in the same 

 dish identify each by partly imbedding 

 near it a small strip of paper bearing its 

 number. Finally some of the paraffin 

 is cut away from each tissue so that it 

 can be conveniently filed away but it is 

 important not to remove too much 

 paraffin . 

 Paraffin Sections. 1. Blocking. If the 

 specimen is a slice of tissue it was 

 trimmed at the time of fixation into a 

 quadrangular form with each edge and 

 surface parallel to the opposite one. If 

 the specimen is a cross section of a 

 tubular structure the cutting will be 

 more difficult. Heat the metal holder 

 of the microtome, gently press the sur- 

 face of the paraffin block against it and 

 harden in iced v/ater. The surface of 

 tissue, protected by the most paraffin 

 (which is the upper surface, remote 

 from bottom of the dish, as it was im- 

 bedded), should be next to the holder 

 and as far as possible evenly equidistant 

 from the surface of the holder. Unless 

 there is plenty of paraffin between the 

 tissue and the holder, difficulties will 

 be encountered if it becomes necessary 

 to remount the block on subsequent oc- 

 casions to cut more sections. Since the 

 slice of tissue is of even thickness its 

 outer surface will be evenly parallel 

 to the sweep of the knife so that the 

 tissue included in a given section will 

 be approximately the same distance 

 from the surface of the block and equally 

 subjected to fixation and subsequent 

 technique. 



2. Cutting. The knife should cut 

 from long side to opposite long side. 

 Trim the edges of the paraffin block so 

 that it will have to pass through an even 

 layer of paraffin at least 5 mm. wide both 

 before and after it enters and leaves the 

 tissue. When more paraffin is cut away 



it may be later needed if more trimming 

 is required to make the sections into 

 straight ribbons. The sides of the 

 tissue should also be protected by layers 

 of paraffin which are parallel and of even 

 thickness. The object of all this is for 

 the knife to cut through the paraffin and 

 tissue squarely and for it to encounter 

 as nearly as possible equal resistance. 

 The resistance of the paraffin at the sides 

 will, however, always be less than that 

 of the paraffin plus the tissue at the cen- 

 ter. For this reason it may be necessary 

 to cut away most of the paraffin from the 

 sides. 



But all specimens are not rectangular 

 slices of tissue of uniform thickness. 

 Spherical bodies are easy to cut but the 

 sections obtained are very difficult to 

 flatten. Specimens containing large 

 cavities are troublesome because the 

 paraffin in the cavities offers so little 

 resistance. In such cases celloidin im- 

 bedding is advised. When a part of the 

 tissue is brittle and the rest soft it is 

 best to orient the tissue so that the knife 

 passes through the soft part first. In 

 orientation of fairly large objects a 

 beam of light passed through the paraffin 

 block from an arc lamp or other powerful 

 source is of great assistance. For very 

 minute objects a method described by 

 Fry (H. J., Anat. Rec, 1927, 34, 245- 

 252) is suggested. For refractory tis- 

 sues, like yolk laden eggs, McClung (p. 

 40) suggests hydration. The block is 

 trimmed until the imbedded tissue is 

 exposed when it is soaked in water for 

 several hours. This reduces friability 

 and brittleness and good sections may 

 often be obtained. 



Temperature and humidity are factors 

 in securing a good ribbon by making one 

 section stick evenly to the next in series. 

 Sometimes a little boiling water near at 

 hand will help but it should not be 

 necessary if the tissue has been properly 

 infiltrated with paraffin of the right 

 melting point which set firmly when 

 cooled. Static electricity, causing the 

 ribbon to adhere in a troublesome way 

 to surfaces, is partly dependent upon 

 difference in density of tissue and paraf- 

 fin. But the most important factor in 

 obtaining excellent sections is have the 

 microtome in good working order and 

 the knife sharp (see Sharpening). For 

 ordinary purposes sections should be cut 

 6 microns thick. To mount them on 

 slides first smear carefully cleaned 

 slides (see Slides) with Albumen- 

 Glycerin, cover with aq. dest. and 

 gently heat over an alcohol lamp if a 

 slide warmer is not available. Then 

 mark the slides with a diamond point 

 pencil and leave for about 6 hrs. in a 

 drying oven at 40-45°C. 



