Motility Tracks: Technique for Quantitative Study of Bacterial Movement
A method for recording movements of bacteria in time and space on a single photograph is described. Quantitative information on the behavior of various motile organisms may easily be obtained for comparative studies. The method possesses certain advantages over cinematography, and illustrations of applications of the technique are presented. Aside from cinematographic and television techniques, the study of bacterial movement is notorious for its paucity of readily available, simple,
... simple, reproducible methods (1). As a consequence, results of work on bacterial motile behavior are reported in vague subjective terms such as "fast," "somewhat slow," "erratic," and the like, which convey scant information concerning the events observed. It is probable that lack of interest in this area of bacteriology is due in large measure to the poorly developed state of relevant techniques. Accordingly, the work described here represents an attempt to rectify this situation by dealing with the problem quantitatively. MATERIALS AND METHODS Cultures. The following bacteria were used: Escherichia coli (ATCC 13070), peritrichous flagella; Pseudomonas aeruginosa (University of Maryland collection), single polar monotrichous flagellum; Bacillus licheniformis (9945-A), peritrichous flagella, from F. J. Tyeryar; Sarcina ureae (ATCC 13881), peritrichous flagella; Thiospirillum jenense and Chromatium okenii, lophotrichous flagella, both from R. L. Gherna. The latter two organisms were cultured and observed in a synthetic medium specifically developed for large photosynthetic purple sulfur bacteria by Pfennig and Lippert (4). The other bacteria were grown in 5-ml amounts of Trypticase Soy Broth (BBL) in screw-top tubes (18 by 125 mm) for 18 hr at 30 C on an oscillating shaker (Lab-Line Instruments, Melrose Park, Ill.). Quantities of these cultures (0.1 ml) were transferred to 5-ml amounts of fresh broth; after 1 hr of incubation at 30 C, their motility tracks were studied. One drop (ca. 0.05 ml) of culture was placed on a glass slide (0.96 to 1.06 mm thickness) and immediately covered with a cover slip (0.13 to 0.16 mm thickness); 1 min was then allowed for equilibration at 20 C before photography was started. The cultures were not sealed under the cover slip and, over the period of observation (generally less than 5 min), no diminution of motile activity was noted. Microscopic methods. A Zeiss photomicroscope with the following arrangement was employed. A 1oX planachromat (NA 0.22) objective was used in conjunction with the condenser set at "phase 3." The substage auxiliary lens was always kept out, but a green interference filter occasionally was introduced. Photography was done with Kodak Tri-X Pan or Panatomic-X film, and exposures of 1 to 15 sec were made by means of a Time-O-Lite interval timer (Master Model, Industrial Timer Corp., Parsuppany, N.J.) introduced in the line. Illumination was provided by either 12 v, 60 w or 6 v, 15 w lamps. This arrangement provided an extremely bright image on a black background similar to that seen in dark-field microscopy.