952 THE ANIMAL VIRUSES 



conditions of examination it is impossible to resolve particles less than 0-2 /x in 

 diameter. Resolution, it will be remembered, is limited by the numerical aperture 

 of the objective and the wave-length of the light used. Since there are serious 

 difficulties in increasing the numerical aperture of the objective, it follows that 

 the only way to resolve very small bodies is to use a wave-length shorter than any 

 present in the visible spectrum. Resolution, however, is not always required, 

 and considerable attention has been devoted of recent years to methods for rendering 

 small bodies visible. Though certain filtrable viruses may be demonstrated in 

 sections or smear preparations, their study is greatly facilitated by obtaining them 

 in a suspension relatively free from tissue cells and other gross matter. Usually 

 this is done by differential centrifugation, sometimes accompanied by filtration. 

 The suspension can then be examined by one of the following methods. 



I. Fixing and staining with a suitable dye. — Numerous workers, among whom 

 Ledingham (1931) has been one of the foremost, have used this method. The 

 dyes chosen are most frequently Giemsa's stain, or one of its modifications. By 

 this means minute particles — the so-called elementary bodies — may be rendered 

 visible in appropriate preparations. Since, however, it is impossible to demon- 

 strate very small particles by transmitted light, even when they are deeply stained, 

 it follows that this method is limited to the larger viruses. Its most conspicuous 

 success has, in fact, been achieved hitherto with the virus of vaccinia, the diameter 

 of which is about 0-15 ju. It has proved of particular value in the microscopical 

 observation of agglutination, where, of course, visibility, and not resolution, of 

 the aggregating particles is alone required. 



II. DarJc-ground examination using visible light. — Provided the particles under 

 examination can scatter enough light, and there is a sufficient difference of refractive 

 index between them and the medium in which the)' are suspended, this method 

 enables very small particles to be rendered visible, even though they are incapable 

 of resolution. It provides a useful means of direct microscopic observation of 

 virus particles. 



III. Photography in ultra-violet light. — Barnard (1925) has been the chief 

 exponent of this method. After preliminary examination by method II, photo- 

 graphs are taken at particular wave-lengths in the ultra-violet spectrum (see 

 Chapter 2). Either transmitted or dark-ground illumination may be used. The 

 former suffers from the disadvantage that the ability of viruses to absorb light 

 is very low, and the image so obtained is smaller than it otherwise would be. With 

 dark-ground illumination there is strong contrast, and sharply defined images are 

 obtainable, though their size tends to be slightly too large. With a wave-length of 

 257 m/i, particles as small as 75 m.fx can be actually resolved. Their approximate 

 size can then be determined from the mean of the images given by transmitted and 

 dark-groxmd illumination. Theoretically, this method is open to almost unlimited 

 extension, but in practice great technical difficulties are encountered. 



Fluorescence microscopy. — This makes use of the ability of certain bodies to 

 transmute the short invisible waves of ultra-violet light to longer visible waves 

 (see Clauberg 1939). 



V. Annular oblique incident illumination. — In this method a relatively opaque 

 body, like the chorio-allantoic membrane, can be examined by oblique illumination 

 from above (see Himmelweit 1938). 



VI. Electron microscope. — More recently this has provided a tool that bids 

 fair to outstrip any method hitherto known. Though technical defects prevent 



