Interference Contrast and Phase Contrast Microscopy of Sporulation and Germination of Bacillus megaterium

Abstract

The techniques of Nomarski interference contrast microscopy and phase-contrast microscopy were compared for their utility in monitoring sporulation and germination in Bacillus megaterium . The Nomarski technique permitted rapid and easy delineation of septation and engulfment during sporulation, whereas with phase contrast microscopy these stages were not detected at all. The later stages of sporulation were easily seen by either technique. Thus, of the seven stages of sporulation as recognized by the electron microscopy of thin sections, five can now be routinely detected quantitatively by optical microscopy: septation (stage II), engulfment (stage III), phase-dark forespore (corresponding to cortex formation, stage IV), phase-bright spore in a sporangium (corresponding to coat formation, stage V), and the free spore (stage VII). This means that now only stage I (axial filament) and stage VI (maturation of the refractile spore) require electron microscopy for routine detection. There was no advantage in using Nomarski optics for germination studies.

Polarized relationship of bacterial spore loci to the "old" and "new" ends of sporangia

“…A strain derived from B. megaterium ATCC 19213 originally obtained from the laboratory of R. A. Slepecky was used in this study. General culture techniques have been de-on October 8, 2020 by guest http://jb.asm.org/ Downloaded from POLARIZED RELATIONSHIP OF SPORE LOCI scribed previously (4,5,6). Modifications are mentioned in the footnotes to the Tables.…”

“…Microscopy. Phase-dark forespores (prespores), phase-bright spores, and cross wall types were detected by phase contrast microscopy (4).…”

Antibiotic Inhibition of the Septation Stage in Sporulation of Bacillus megaterium

“…,O-phenethyl alcohol inhibition of septation, the second stage of sporulation. We are able to report here the effects of eight antibiotics and nalidixic acid on septation in Bacillus megaterium ATCC 19213, because use of Nomarski interference contrast optics has simplified the study of septation (5). Thus, sporulating cells can now be scored easily for the presence or absence of polar sporulation septa.…”

“…a Penicillin G (Eli Lilly) was used. All experiments (Tables 1 to 3) were carried out as described previously (5), except that the inoculum for the sporulation medium was 1 or 2 ml of a 10hr nutrient broth (Difco) culture (2 X 108 to 4 X 108 cells per ml). Unheated stock spores were used as inoculum for broth cultures.…”

Variable multimodal light microscopy with interference contrast and phase contrast; dark or bright field

“…Such comparisons have already been carried out several years and decades ago. Thus, for instance, interference and phase contrast have been compared in examination of bacterial cells (Hitchins et al, 1968), spermatozoa (Cortner et al, 1978) and organs of corti (Kuhn et al, 1971). An extensive comparison of phase contrast and DIC microscopy can be obtained from the web (Murphy et al, 2005).…”