184 THE TECHNICS OF PHASE MICROSCOPY 



3.5. Adding color contrast to phase contrast 



In the previous section the gain in contrast by phase microscopy with 

 shghtly absorbing specimens was considered in general terms. When the 

 specimen is sufficiently transparent and colored, color filters may enhance 

 the visibility, as with brightfield microscopy. A filter of complementary 

 color between the lamp and the microscope will increase contrast, and 

 one of nearly the same color will lessen contrast and often reveal greater 

 detail. Further results are possible with photographic recording, be- 

 cause the availability of emulsions of different color sensitivities makes 

 possible effective color combinations that the specialized color sensitivity 

 of the eye would not recognize (Evans, 1948). 



Reversing the contrast of the specimen from dark to bright may make 

 color contrast more effective. Red stained wheat chromosomes (Fig. 

 IV. 4), examined in light from a Wratten A or an equivalent red filter, 

 stand out in better contrast than with brightfield alone (Figs. V.2A- 

 Y.2D). The same technic is useful wath Ziehl-Neelsen stained acid-fast 

 bacteria, which then appear as shining bright red rods against a dark 

 background, the contrast almost as enhanced as in fluorescence micros- 

 copy. Unfortunately any bacteria stained blue are also reversed in 

 contrast and, w^hen the staining is not dense, also appear in a translucent 

 red. The method is useful for locating the bacteria in the preparation, 

 and further examination with dark-contrast phase, or with brightfield 

 after swinging the annulus out of the phase system, will re^'eal whether 

 or not they are acid-fast in nature. This method of increasing contrast 

 from reversing the image to bright contrast and examining it with light 

 of the same, or nearly the same, color as the specimen may be used to 

 advantage with other colored specimens, and the more transparent 

 detail is particularly helpful in stereoscopic methods. 



Optical dispersion staining may be combined with phase microscopy 

 and may be more effective with diffraction plates giving bright or B — 

 dark contrast. The light-bending ability of transparent specimens is 

 usually different for long and short wavelengths of light, and this 

 difference in dispersion may be expressed as j^ = (no — l)/(^f — nc), 

 where no, np, and ric are the refractive indices for yellow, blue, and red 

 light of those solar spectral lines. When the specimen and its surround 

 have nearly the same dispersion little color difference will be noticed; 

 if their index is closely the same and their dispersions different the 

 specimen will appear in colors. In 1946 Richards found that dust 

 mounted in nitrobenzene could be used for the approximation of its 

 silica content as the quartz particles had enough difference in dispersion 

 to appear violet when examined with the phase microscope in bright 

 contrast with a 0.2A-|-X/3 diffraction plate. The color is helpful in 



