Water vapor, aqueous ethyl alcohol, and heat activation of Bacillus megaterium spore germination

Journal of Bacteriology (Impact Factor: 2.81). 07/1968; 95(6):2090-101.
Source: PubMed


Dormant spores of Bacillus megaterium were activated for germination on glucose by heating them in aqueous suspension (but not if heated dry), by treating them with aqueous ethyl alcohol at 30 C, or by exposing them to water vapor at room temperature. The degree of water vapor activation depended upon the relative humidity, the time, and the temperature of exposure. Activation increased the extent and rate of glucose-induced germination and decreased the average microlag. Extended water vapor treatment also activated spores for germination induced by KI and by l-alanine. Spores activated by any of the three treatments were deactivated by treatment at 66 C, either for 18 hr in 100% ethyl alcohol or for 40 hr over P(2)O(5). Deactivated spores were reactivated by heat, by 5 m ethyl alcohol, or by water vapor. It is postulated that heating and ethyl alcohol may change the structure of liquid water, so that it is more like water vapor and can more readily penetrate to and hydrate a critical (enzymatic?) spore site, leading to activation.

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    ABSTRACT: Theactivation properties ofClostridium perfringens NCTC 8679sporeswere demonstrated byincreases in CFUafter heating inwateror aqueousalcohols. Thetemperature rangeformaximumactivation, whichwas 70to80°Cinwater, was lowered bytheaddition ofalcohols. Theresponseata giventemperature was dependent onthetimeofexposure andthealcohol concentration. Themonohydric alcohols andsome, butnot all, ofthepolyhydric alcohols could activate sporesat37C.Theconcentration ofa monohydric alcohol that produced optimal sporeactivation was inversely related toitslipophilic character. Sporeinjury, whichwas manifested as a dependence on lysozyme forgermination andcolony formation, occurred undersome conditions ofalcohol treatment thatexceeded those foroptimal sporeactivation. Treatment withaqueous solutions ofmonohydric alcohols effectively activated C.perfringenssporesandsuggests a hydrophobic site for sporeactivation. Clostridium perfringens isoneofthemostcomnmon bac- terial agents infoodbome-disease outbreaks. Thevehicle of infection isfrequently acooked, high-protein foodinwhich spores ofthis organism will survive cooking temperatures andgerminate sothat multiplication occurs dunngpost-cook handling (36). Sublethal heatwill activate spores ofmany, butnotall, strains ofC.perfringens (28)andpromote germination underconditions notpermissive forthegermi- nation ofdormant spores. Barnes etal.(8)reported that heat treatment activated spores ofC.perfringens inmeatsothat colony formation increased 25times onaplating medium. Roberts (33)reported thatonly0.13to3.6%ofspores of "food-poisoning" strains grewwithout heat. Heattreatment ofaninoculum containing C.perfringens spores resulted in aculture withanenhanced ability toproduce enterotoxin during asubsequent sporulation event (39). Heatactivation isroutinely usedasa prerequisite for studies ofbacterial spore germination and,byuseofplating procedures, fortheenumeration ofspores fromfoodprod- uctsandotherenvironmental sources. Time-temperature conditions formaximumactivation mayvarywithstrains of C.perfringens (17). Treatment withethanol (ET)hasbeenreported toincrease thegermination rateofBacillus megaterium spores (21, 22) andtobeaneffective technique fortheselective isolation of sporeforming bacteria frommixedcultures (24). Inthis study, we compared theefficiency andkinetics ofheat activation withthose ofalcohol activation ofC.perfringens spores andreport ontheconditions ofalcohol exposure that injure spores.
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    ABSTRACT: QM B1551 spores were activated by heating in aqueous suspension. Activation was measured by increase in germination rate (on glucose at 30 C) over that of dormant spores. At any one temperature, activation increased linearly with time of exposure. Spores were activated throughout the range of test temperatures (50–75 C). The exposure time required to achieve a given germination rate increased with decreasing temperature. The logarithm of the activation velocity (increase in germination rate/sec of heating) was a linear function of the temperature of heating. The apparent average thermodynamic constants for the system were: ΔH‡ = 72.4 kcal/mol; ΔF‡ = 22.8 kcal/mol; ΔS‡ = 147.4 cal/mol/deg.
    Biochemical and Biophysical Research Communications 12/1969; 37(6):909-916. DOI:10.1016/0006-291X(69)90217-4 · 2.30 Impact Factor
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    ABSTRACT: The effects of temperature on the activation, glucose-induced germination, and outgrowth of Bacillus megaterium QM B1551 spores were investigated. There was no evidence for discontinuities in the response of spores to temperature in these processes reflecting reported thermal anomalies in the physical structure of water. Increasing the temperature of heat activation (aqueous suspensions, 5 min) increased the germinability of spores. Activation, as measured by extent of germination, was optimal after heating at 62 to 78 C, and the rate of spore germination was maximal after heat activation at 64 to 68 C. Increasing the temperature of activation above 68 C depressed the germination rate and increased the time lag before this rate was reached. Germination occurred over a wide range of temperatures, but was optimal between 28 and 38 C. The highest rate of germination was at 38 C; at lower incubation temperatures, the maximum attained rate was lower and the lag in attaining this rate was extended. Outgrowth (postgerminative development through the first cell division) of the germinated spores in Brain Heart Infusion (BHI) occurred in at least two phases-a temperature-dependent lag phase followed by a relatively temperature-independent phase of maximum outgrowth rate, during which increase in optical density was a linear function of time. Outgrowth time (time required for doubling of the initial optical density), essentially dependent on the time for completion of the lag phase, was shortest at temperatures between 34 and 40 C. The temperature-dependent lag phase was completed in a rich medium (e.g., BHI) but not in the glucose germination medium, suggesting that the endogenous reserves of the germinated spore were inadequate to support the metabolic synthetic events occurring during this period.
    Journal of Bacteriology 02/1970; 101(1):58-64. · 2.81 Impact Factor
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