Experiments on Staining Thin-Sections 



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l-ig. 3. Locust primary spermatocyte fixed in 45 "„ isotonic acetic acid and section-stained in lanthanum nitrate. The chro- 

 matin masses in the unstained area on the right of the section appear only as diffuse blobs, while those in the rest of the 

 section (which is stained) show a complicated form of structure. 



comparison of this type has the great advantage of 

 eliminating the effect of variations in section thicl<- 

 ness, focussing, and photographic processing. Exper- 

 ience has shown, however, that it is necessary to clear 

 the methacrylate in the stained areas of section at 

 the same beam intensity as that used in the initial 

 irradiation of the reference areas because the amount 

 of clearing obtained is dependent on the beam inten- 

 sity used. This method of obtaining unstained ref- 

 erence areas is best applied in an electron microscope 

 with a double condenser lens system. With the sec- 

 ond condenser focussed to give beam cross-over in 

 the object plane, one irradiates only a fairly sharp 

 spot about 5 // in diameter, it has been found most 



convenient to move the section in such a way that 

 this spot follows a zig-zag path across the section. 

 In this way one obtains a reference area in all regions 

 of the section. 



Staining solutions so far tried have been 2 "« un- 

 buffered osmium tetroxide and saturated phospho- 

 tungstic acid as genera! protein stains, and saturated 

 ferric alum of 2 "„ lanthanum nitrate and of 2 "„ 

 thorium nitrate as stains for chromatin. In each case 

 positive results have been obtained. By way of control 

 a section has been floated on distilled water instead 

 of stain. 



This method of staining is particularly applicable 

 to tissues fixed other than with osmium tetroxide. 



Kig. I. Locust testis fixed in 45 "o isotonic acetic acid and section-stained with osmium tetroxide. («) Stained (left) and 

 unstained (right) regions of a section in which tlic sperm tails are cut approximately transversely. Kach sperm tail contains 

 the 9 + 2 fibre bundle plus 2 mitochondrial strands, (h) Higher magnification micrograph of longitudinally cut sperm tails 

 in a stained area of section, (r) High magnification micrograph of transversely cut sperm tail in a stained area of section. 



Fig. 2. Locust primary spermatocyte fixed in 5 "„ buffered formaldehyde and section-stained with osmium tetroxide. The 

 light area on the right side was pro-irradiated and has not stained; the dark area on the left was not irradiated and has 

 stained. At the junction of the stained and unstained areas is a narrow region, about 1 /< wide, which is much darker and 

 so, presumably, has stained more intensely than the rest of the stained area. 



