198 PHASE MICROSCOPY IX BIOLOGY AND MEDICINE 



stained preparations with the brightfield microscope. The B — diffrac- 

 tion plates are useful with stained organisms and often provide better 

 contrast for fine detail with unstained organisms than the harsher 

 A— type for dark contrast (Fig. \.1B). 



With the long-focus condenser equipment it is possible to examine 

 bacteria growing on agar in Petri dish cultures when the agar is not too 

 thick, even with the oil immersion lens. A cover glass should be placed 

 on the agar to prevent moisture from the preparation fogging the 

 objective. The phase microscope shows more detail within organisms 

 than the darkfield microscope, which reveals outlines or large inclusions. 

 However, in the examination of exudates from sores the phase microscope 

 makes the pus cells and other material likewise more visible, and the 

 darkfield microscope is preferable for most observations of this type, 

 e.g., finding spirochetes in smear preparations from syphilitic sores. 

 Sputum smears with considerable amounts of mucus should be examined 

 by fluorescence or staining methods rather than with the phase micro- 

 scope, because the enhanced visibility of the mucus obscures any 

 bacteria present. When the surrounding medium is not too full of 

 extraneous material the phase microscope is useful for locating bacteria. 

 Perry (1948) illustrated a spirochete in a tissue culture. 



When the shape and optical path of a bacterium and material near 

 it are the same, it may not be possible to tell them apart by phase 

 microscopy. A single coccus often cannot be distinguished from a 

 similar-sized fat globule in milk without other methods, but bacteria of 

 other forms may be found and in some cases identified from their shape. 

 Some organisms are too small to be seen, as unstained Leptospira in 

 kidney tissue sections. The special stains add enough material (e.g., 

 silver) to the organism to make it of size to be visible, and with these 

 stains B— diffraction plates give still better contrast. For these 

 the further development of phase microscopy may show that the 

 0.07A-I-0X or 0.07A+0.4-0.5X diffraction plates are appropriate. 



Although one of the advantages of phase microscopy is the saving of 

 time and materials involved in staining procedures, the use of the phase 

 microscope with stained materials is often advantageous, Table V.2 

 (see also Section 3.5 of Chapter IV). Eisenstark and McMahon (1949) 

 have combined nigrosin negative staining with phase for study of the 

 capsules of Azotobader. 



Phase and electronic flash have been used by Richards (19486) to make 

 very sharp pictures for accurate measurement of the size of bacteria 

 (Fig. V.l, P and Q). A pure strain of B. megatherium had an average 

 width of 1.0 M (o" = 0.06 ju). For B. cercus an average width of 1.10 n 

 was obtained with bright-contrast phase and 1.05 /x with dark-contrast 



