printed microgermination of bacillus cereus spores .nation of bacillus cereus t and b. megaterium

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JOURNAL OF BACTERIOLOGY, Dec. 1969, p. 1385-1392Copyright 0 1969 American Society for Microbiology

Vol. 100, No. 3Printed In U.S.A.

Microgermination of Bacillus cereus SporesTADAYO HASHIMOTO, W. R. FRIEBEN, AND S. F. CONTI

Department of Microbiology, Thomas Hunt Morgan School of Biological Sciences, University of Kentucky,Lexington, Kentucky 40506

Received for publication 2 September 1969

The biphasic nature of germination curves of individual Bacillus cereus T sporeswas further characterized by assessing the effects of temperature, concentration ofgerminants, and some inorganic cations on microgermination. Temperature wasshown to affect both phases of microgermination as well as the microlag period,whereas the concentration of L-alanine and supplementation with adenosineexerted a significant effect only on the microlag period. The germination curves ofindividual spores induced by inosine were also biphasic and resembled those ofspores induced by L-alanine. High concentrations (0.1 M or higher) of calcium andother inorganic cations prolonged both phases of microgermination, particularlythe second phase, and had a less pronounced effect on the microlag period. Thesecond phase of microgermination was completely inhibited when spores weregerminated either in the presence of 0.3 M CaC12 or at a temperature of 43 C; thisinhibition was reversible. Observations on the germination of spore suspensions(kinetics of the release of dipicolinic acid and mucopeptides, loss of heat resistance,increase in stainability, decrease in turbidity and refractility) were interpreted on thebasis of the biphasic nature of microgermination. Dye uptake by individual sporesduring germination appeared also to be a biphasic process.

Ideally, the kinetics of germination of bacterialspores should be investigated in individual spores,because germination curves obtained by use ofspore suspensions represent the summation ofevents occurring in the individual members ofthat population. Although loss of refractilityunder a phase-contrast microscope is frequentlyused for following germination, the lack ofreliable methods to quantitate phase-darkeninghas limited its use for following the germinationof individual spores (17; H. Riemann, Ph.D.Thesis, Univ. of Copenhagen, Copenhagen,Denmark, 1963).The successful application of microscope

photometry to quantitate phase-darkening hasprovided a new tool for exploring the kinetics ofgermination of individual spores (15). Hashimotoet al. (2) recently demonstrated that microgermi-nation of Bacillus cereus T and B. megateriumQM B1551 spores is a biphasic process, and it wassuggested that each phase of microgermination isaffected differently by certain environmentalfactors.Although the effects of environmental factors

on microlag and microgermination have beenpreviously studied by direct phase-contrastmicroscopy and by employing statistical analysis(18, 19), the development of a more precise tech-

nique for analyzing microgermination has led usto reinvestigate the kinetics of microgermination.

This communication deals with the effects oftemperature, type and concentration of germi-nants, and some divalent cations, particularlycalcium, on the kinetics of microgermination ofB. cereus T spores.

Events occurring during each phase of micro-germination are correlated further with data ongermination obtained by use of spore suspen-sions.

MATERIALS AND METHODS

Spores. Spores of B. cereus strain T were produced,harvested, and stored as previously described (2).Heat activation and temperature effects. Spores were

activated by heating at 65 C for 4 hr and were ger-minated in 0.05 M tris(hydroxymethyl)aminomethane(Tris) buffer (pH 8.3) under the conditions specified.The effect of temperature on germination of single

spores was studied in an adjustable constant-tem-perature room (range of 16 to 33 C); similar studieson spore suspensions were carried out in the range of10 to 45 C with the desired temperature maintainedby use of a variable-temperature water bath.

Heat sensitivity. Loss of heat resistance in sporesuspensions during germination was followed byremoving 1-ml samples at intervals and transferringthem to 9.0-ml distilled water blanks equilibrated to

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HASHIMOTO, FRIEBEN, AND CONTI

80 C. Samples were heat-challenged at 80 C for 10min and chilled in an ice bath; then 0.1-ml amountsof appropriate dilutions were plated on TrypticaseSoy Agar. After 24 hr of incubation at 30 C, thecolonies were counted with a New Brunswick colonycounter.

Optical density. The decrease in optical density ofspore suspensions was measured at 420 nm in aBauch & Lomb Spectronic-20 colorimeter.

Refractility and stainability. Germinating sporesuspensions were stabilized by removing samples atvarious time intervals and transferring them to apredetermined amount of sulfuric acid to lower thepH to 2.0 (1). Duplicate smears were prepared fromthis mixture and quickly dried over gentle heat. Toone smear, a drop of 1% crystal violet was addedand mounted with a cover slip; spores were examinedfor stainability while wet. To a duplicate slide, a dropof sulfuric acid (0.05 M) was added, and spores wereexamined for loss of refractility (phase-darkening)by use of dark phase-contrast optics. Preparationsexamined 2 hr after the addition of sulfuric acidshowed no more stainability or phase darkening thanspecimens prepared immediately after stabilizationwith acid.

Loss of spore components. Loss of dipicolinic acid(DPA) and mucopeptides during germination wasfollowed by measuring the DPA or mucopeptidesremaining in spore pellets. Appropriate volumes ofgerminating spore suspension were removed atvarious intervals and immediately transferred tosufficient sulfuric acid to lower the pH to 2.0. Sulfuricacid effectively stabilized germination with respectto DPA and loss of mucopeptides, for spore suspen-sions to which sulfuric acid had been added at variousintervals showed no subsequent excretion of DPAor mucopeptides over a 60-min period. Spore pelletswere obtained by centrifuging at 6,000 X g for 15 minat 4 C. DPA was estimated colorimetrically by themethod of Janssen et al. (4) with purified DPA(Matheson, Coleman and Bell, Norwood, Ohio) as astandard, and hexosamine was estimated after hy-drolysis by the method of Rondle and Morgan (16)with glucosamine as a standard. Hydrolysis for 5 hrat 100 C in 6 N HCI was carried out in sealed glassampoules.

Normalization of data. To compare the time se-quence of the various germination parameters, eachevent was plotted relative to completion of that eventin 30 min. Such normalization of the data permitscomparison of the rate of change among all of theparameters (7).

Measurement of single spore germination. Exceptfor the few minor modifications described below, thetechniques employed for measuring germination ofsingle bacterial spores were essentially the same asreported earlier (2). To keep the number of spores perslide relatively constant, the following procedure wasregularly employed: approximately 2.5 ,liters ofaqueous spore suspension (optical density of 0.8 at420 nm) was spread evenly over the entire surface of acover glass (# 1 thickness) by use of a 5-/Aliter capillarypipette. After drying, without prior fixation, the coverglass was mounted over a microscope slide on which

approximately 2.5 ,liters of the desired concentrationof germinant had been placed. The edges of the coverglass were sealed immediately with melted vaspar,and the preparation was placed under a Zeiss Uni-versal microscope equipped with an MPM micro-scope photometer. The instrument was modified inthis study by using a 3.2-mm rather than a 2.0-mmphotometer stop. The 3.2-mm photostop was usedbecause we could include the entire spore in thephotosensitive area, thus enabling us to measureabsorbancy or refractility changes more accuratelyduring germination. The initial contact of germinantsolution with the dried spore film was taken as timezero in calculating the microlag period. Germinationof individual spores was followed for up to 30 minwith the use of monochromatic illumination (540nm).

Measurement of dry uptake by single bacterialspores during germination. A cover glass smearedwith a thin film of spores was mounted over a micro-scope slide on which a germinant solution containing0.3 to 0.7% crystal violet had been placed; mostexperiments were run at a dye concentration of 0.5%.For measuring dye uptake, the phase-contrast opticalsystem of the microscope was replaced with a bright-field objective (Planapo 100/1.30, Zeiss) and con-denser for optical density measurements. An areaadjacent to a selected ungerminated spore was broughtinto the photosensitive area, and the instrument wasadjusted to give an optical density reading of zero.The spore was carefully brought into the photosensi-tive area, and the optical density reading caused bythis interpositioning was recorded as the initialoptical density. Uptake of crystal violet by individualspores during germination was recorded as a gradualincrease of optical density of the spore at 590 nm.

Phase-contrast microscopy. A Zeiss UniversalMicroscope equipped with a 100 X objective lens(Neofluar phase 100/1.30, Zeiss) was used for routinedark phase-contrast microscopy. Photomicrographswere taken on panchromatic film (Kodak plus X).

RESULTSEffect of temperature. Temperature appears to

be by far one of the most significant factors affect-ing bacterial spore germination. As shown inTable 1, both the microlag period and duration ofthe first and second phases of microgerminationwere significantly affected by temperature. It isevident that there was a wide fl

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