Monochromatic Ultraviolet Microscopy of Microorganisms: Preliminary Observations on Bacterial Spores

An ultraviolet source may be used in light mi-croscopy to excite visible fluorescence, increase resolution, or differentiate absorbing materials. The last two aims require ultraviolet-transmitting optics, usually of quartz, and have infrequently been sought by bacteriologists, especially since electron microscopy with its vastly increased resolving power became available. However, ultraviolet light has the special advantage of permitting the examination of aqueously mounted bacteria for cell structures that are strongly absorbing. If used in conjunction with a precision mono-chromator, the ultraviolet source can be resolved to narrow wave bands that may allow further qualitative or even quantitative identification of structural components. We have sought to use this potential in conjunction with studies on the structure and function of bacterial spore membranes (1-3). A point of particular concern was the localization in the spore of dipicolinic acid (DPA), which is uniquely present in large amounts (4) and is believed to be associated with heat resistance (3, 5). Dipicolinic acid has absorption peaks at 263, 270, and 277 m# and has been thought (6) to account for the uniformly dark appearance of spores in ultraviolet light, as reported in 1931 by Wyckoff and Ter Louw (7) for Bacillus subtilis and in 1938 by Piekarski (8) for an unidentified bacillus. Malmgren and Hed~n (9) photographed Bacillus cereus at 257 m/z and noted that "there were present in the otherwise pale bacterial plasma more strongly absorbing grains," which apparently were spores. A similar picture of Bacillus megaterium spores may be found among the extraordinary but little known photomicrographs of microorganisms taken with filtered (275 m/z) ultraviolet light by Kruis in 1913 (10). The seemingly uniform absorption of ultraviolet light by bacterial spores and the inference that DPA is distributed throughout their structure seemed worthy of reexamination. Materials and Methods Bacillus megaterium was chosen for study because spores of this species characteristically have a high DPA content (4), which was confirmed for this strain by colorimetric analysis (11). It also possesses a prominent cortex in the multilayered wall (12). Virtually complete sporulation was attained by growing the culture in trypticase-soy broth on the shaker at 30°C. for 24 hours. After vegetative autolysis, the spores were separated and repeatedly washed in distilled water by means of differential centrifugation. The ultraviolet source was a Sylvania type SH high pressure mercury vapor arc lamp, light from which passed through a Bausch and Lomb grating mono-chromator with both slit widths …