Motility, Swarming, and Growth of Proteus

Kopp, RIUDOLF (Hygiene-Institut der Universitat, Freiburg, Germany), AND JOHANNES MULLER. Effects of related anionic detergents on flagellation, motility, swarming, and growth of Proteus. Appl. Microbiol. 13:950-955. 1965.-The effects of a series of sodium alkyl sulfates (C4 to C16) on flagellation, motility, swarming, and growth of Proteus were examined. The concentrations of the various sodium alkyl sulfates completely inhibiting the swarming phenomenon (on solid medium) and motility (in liquid medium) were in the same order of magnitude. The inhibiting effect of the detergents examined increased from sodium hexyl sulfate (inhibitory concentration, 20 to 30 mmoles per liter) to sodium tetradecyl sulfate (inhibitory concentration, 0.1 to 0.5 mmoles per liter). Flagella were produced neither in liquid nor on solid medium at these concentrations as could be observed by electron microscopy. At concentrations where motility was not impaired, intact flagellation could be observed. At a concentration of 0.1 mmole per liter, sodium tetradecyl sulfate completely inhibited the motility of Proteus in the liquid medium employed without impairing growth. The biological activities of surface-active agents and of the proposed underlying reaction mechanism were recently reviewed by Bernfeld (1963). A strong dependence of bacteriostatic and bactericidal power on chain length is observed within a given homologous series. The effect reaches a maximum at C12 to C16 according to the chemical nature of the chain and other substituents. According to Lominski and Lendrum (1942), surface-active agents like the sodium alkyl sulfates strongly impair the swarming phenomenon of Proteus. This paper reports on a study of the effect of a homologous series of sodium alkyl sulfates on swarming and flagella formation of P. mirabilis on solid medium, and on motility, flagella formation, and growth in liquid medium. MATERIALS AND METHODS Two strains of P. mirabilis (21 and 94) isolated from human urine and kept on agar slants stored at 4 C were transferred to a liquid medium and then streaked on a solid medium before they were used in the experiments. Solid medium. The basal medium used contained: agar (Difco), 20.0 g; NaCl, 5.0 g; meatpeptone (Merck), 10.0 g; in 1,000 ml of distilled water; pH 6.8. Sodium alkyl sulfates of different chain length with an even number of carbon atoms, subsequently referred to as C4 to C16, obtained through the courtesy of Prof. Blaser, Henkel AG, Di)sseldorf, Germany, were added aseptically in the form of a concentrated solution to final concentrations of 0.03 to 30 mmoles per liter. Conventional petri dishes with a diameter of 70 mm were employed. The swarming distance (maximal distance between the edge of the parent colony and the peripheral ring of growth) was usually read after 12 hr at 37 C, and was compared with controls not containing detergents. The procedure is described in detail elsewhere (Kopp, 1962). In one of the experiments (strain 94), the swarming distances were read also at 1-hr intervals. For studies by electron microscopy, a sample of the culture broth was spread on agar containing detergents at concentrations which had prevented swarming completely (swarming-distance = 0) in the experiments described above. Grids covered with Formvar films were then placed on the agar surface. Media free from detergents were employed as controls. After 3 hr of incubation at 37 C, the grids were removed and fixed for 3 min in the gaseous phase of osmium tetroxide. Electron micrographs were taken with the Siemens Elmiskop I electron microscope at 60 kv. Liquid medium. A 5.0-g amount of casein-peptone (Merck) in 1,000 ml of phosphate-buffered saline (pH 7.2) was used as basal medium in most experiments in which C6 to C14 sodium alkyl sulfates were dissolved to final concentrations of 0.01 to 50 mmoles per liter. For the electron microscopic investigations, meat-infusion broth with 0.5% glucose was used. The motility and growth studies were performed in 200-ml Erlenmeyer flasks containing 50 ml of 950 on A uust 6, 2017 by gest ht://aem .sm .rg/ D ow nladed fom EFFECTS OF ANIONIC DETERGlENTS ON PRUO7E9tS1 iedium iinoculated with 5 ml of a culture of P. tulirabilis. The bacteria were grown at a temperature of 23 to 25 C, which is about the optimum for motility according to Bergey's Manual. Increased aeratioin by agitation was avoided to prevent damage to flagella. Gentle intermittent shaking was employed. Motility was studied with the "hainginig-drop technique" unider direct illumination after 4 to 6 hr of inctubation, at which time motility was optimal. Motility was recorded as the percentage of bacteria being motile. Three criteria were used: (i) motility clearly impaired (about 50%1-1 motile), (ii) most bacteria immotile (fewer than 10%c motile), and (iii) complete inhibitioin of motility (0% motile). Growth of the organisms was followed turbidimetrically at 515 m,u in a Zeiss-PMQ II spectrophotometer in a 0.5-cm cell. An initial study (viable count) was carried out under idenitical conditions to establish that the observed inicrease in absorbancy corresponds to an aetual inicrease in the ntumber of viable cells. Uninioculated flasks served as sterility control, and were used to zero the photometer. At 1, 2, 4, 8, and 12 hr after inoculation, samples were taken to eheck on motility. For each level of concentration of the detergents, the growth curves for all detergents examined were taken in one series of simtultaneous experiments, with the same culture used for inoculationi. When growth was inhibited by the detergents, the bacteria were transferred to detergent-free broth 48 hr after inoculation. For electron microscopic studies, the bacteria were grown in a meat-infusioni broth containing 0.5%,7 glucose unider otherwise identical conditions. The bacteria were harvested after 12 hr and suspended in distilled water. Amounts of 0.01 ml of the diluted suspension were transferred to grids. After drying, the preparations were investigated without fixation in the electroni-optical dark field for bacterial flagella. The control observations on motility were performed by the hanging-drop technique as described above.