160 



THE ART OF MAKING MICROSCOPE SLIDES 



T.S. Fat 



tempt should be made to cut sections 

 thinner than 20 microns by this method; 

 sections of 30 microns are usually good 

 enough for diagnostic purposes. 



The sections must be observed closely 

 as they come from the block. If they 

 crumble under the action of the knife, 

 while the gum melts instantly on contact 

 with it, it may be presumed that the block 

 has not been frozen sufficiently hard, and 

 the carbon dioxide should again be turned 

 on for a few moments. It will only take a 

 moment or two to establish the optimum 

 condition under which only slightly curled 

 sections appear on the blade of the knife. 

 These prehminary cuts will, however, have 

 soiled the blade which must now be 

 washed with a drop of warm water to re- 

 move the gum and then used to cut as 

 manj^ sections as are required. As each 

 section is cut it is removed from the blade 

 of the knife to 70% alcohol. Most people 

 working under pressure use the little 



finger (Fig. 86) for the removal of the sec- 

 tion, though a number of very competent 

 technicians prefer to use a brush. The gum 

 quickly dissolves from the sections in 70% 

 alcohol. The sections may then either be 

 handled with a section lifter or attached to 

 a slide with one of the adhesives given in 

 Chapter 28 under the heading V 21.3. 



Staining and Mounting Sections 



Every pathological laboratory has its 

 own well-established routine for staining 

 frozen sections obtained from biopsy. 

 Without wishing to suggest that this rou- 

 tine be altered, the writer would like to 

 draw attention to the existence of the 

 techniques of Kingsley (Chapter 20, 

 DS 13.13 Kingsley 1935) which, standard- 

 ized as a routine, permit the comparison of 

 diagnostic frozen sections, if necessary, 

 with permanent paraffin sections stained 

 in an identical manner. 



Typical Example 



Peeparation of a Section of Fatty Material Embedded in Gelatin by the 



Method of Clark 1947 



The discussion of general principles 

 given above was centered around the as- 

 sumption that a freezing microtome is to 

 be used for the production of diagnostic 

 sections under pressure of time. The pres- 

 ent description is of a method whereby a 

 section of research quality may be ob- 

 tained of material which cannot be sec- 

 tioned by any other means. Let us suppose 

 that it is necessary to section some fatty 

 bodies, either from vertebrate or inverte- 

 brate material, with a view to making 

 permanent slides in order to record such 

 changes as may have taken place under 

 varying conditions. 



The fatty bodies are dissected out from 

 wherever it is decided to secure them, and 

 are placed in 4% formaldehyde until re- 

 quired. They may be left indefinitely but 

 they should be hardened for at least a 

 week. 



Before embedding tlie pieces are washed 

 in running water overniglit. The technique 

 of Clark 1947 (11431, 59:337) requires a 

 25% dispersion of gelatin in water (made 

 by soaking 25 grams of gelatin in water 



until it is thoroughly swollen, melted, and 

 chluted to 100) and a 12.5% gelatin (which 

 may either be prepared fresh or by the 

 dilution of the 25% gelatin stock). 



The tissue, after having been washed 

 for at least 24 hours in running water, is 

 placed in 12.5% gelatin in an oven at 

 37.5°C. A block of tissue of about 3'^-inch 

 cube requires 24 hours in this solution 

 before being transferred to 25% gelatin 

 maintained at the same temperature. It 

 should spend from 24 to 36 hours in this 

 thick gelatin bath and should then be em- 

 bedded in 25% gelatin exactly as though 

 it were a paraffin block. That is, a paper 

 box should be prepared in the manner 

 described in Chapter 12. Molten gelatin is 

 poured onto the bottom of the box and, as 

 soon as it has sohdified, the specimen is 

 inserted and surrounded with further 

 gelatin. The block is now placed in a re- 

 frigerator to harden and left there until 

 required. When it is taken out for cutting, 

 the paper is stripped from the outside, and 

 the block trimmed until the object lies in 

 the required orientation. This trimmed 



