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BACTERIAL SPORES
II. A STUDY OF BACTERIAL SPORE GERMINATION IN RELA-TION TO ENVIRONMENT1
EDWARD W. MORRISON AND LEO F. RETTGERFrom the Division of Bacteriology, Yale University
Received for publication June 20, 1930
Perty (1852) was probably the first to observe and describebacterial spores. Pasteur (1870), incidental to his work on dis-eases of the silk worm, confirmed the observations of Perty,finding similar elliptical, light-refracting bodies in bacteria. Heextended the knowledge concerning the characteristics of thesebodies by observing that they possess much greater resistance toinjurious agents than do the bacterial cells themselves. Cohn(1877) was the first to establish the true nature of these bodiesas spores, since Perty and Pasteur both failed of proof, not havingobserved the actual processes of sporulation and germination.Cohn saw the appearance of the light-refracting spore within thecell and observed the process of germination of spores of B. subtilisdirectly under the microscope.Ever since this earlier work bacterial spores have been the
basis of controversies as to their formation and function. Thatthere are spore forms in bacteria other than the classical endosporehas been claimed by numerous investigators, particularly in con-nection with the hypothesis that spores function as reproductivebodies. Hueppe (1885) described swollen, irregular elements inold cultures of several organisms, which he termed "arthro-spores." Conidia formation was observed by Lister (1873) incultures of B. lactis, and later by others in various organisms.These forms often were regarded as spores, and in fact Enderlein
1 This communication is based on a portion of a thesis submitted to the Grad-uate School of Yale University by the senior author in partial fulfillment of therequirements for the degree of Doctor of Philosophy.
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314 EDWARD W. MORRISON AND LEO F. RETTGER
(1925), who has reported them in many bacterial species, con-siders them as analogous to the ascospores of the fungi, and indeedas the true bacterial spores. Mellon (1925) described formsoccurring in bacteria of the colon-typhoid and diphtheria groups,which he called "zygospores" because of their complete parallelto isogamic conjugation or zygospore formation in yeasts. Noc-ard and Roux (1887), Metschnikoff (1888), Wherry (1913) andothers have observed bodies within the tubercle bacillus whichresembled ordinary spores.Numerous theories have been evolved concerning the causes
and conditions, both cellular and environmental, leading to sporeformation. One of the first was that of Buchner (1890), whichdesignated exhaustion of nutrient material as the primary factor.Preisz (1904) considered sporulation as a definite stage in thedevelopment of bacilli, and held that the optimum conditions forthe initiation of the process are identical with those favoringmaximum vegetative development. Matzuschita (1902) recog-nized lack of nutrient material as a factor, but attached moresignificance to oxygen accessibility in sporulation. He statedthat aerobic bacilli under hydrogen or vacuum of less than 30 mm.never form spores. Hall (1922) found that sporulation of theanaerobic bacilli was most abundant in mediums characterized bya low content of fermentable carbohydrates, such as deep brain,alkaline egg broth and blood agar. Henrici (1924) showed thatspore formation is initiated at the end of the active growth stage ofa culture and that, due to this fact, sporulation proceeds morerapidly in quarter strength medium because of the earlier cessa-tion of active growth. Churchman (1925) attacked the concep-tion that sporulation is merely a protective mechanism broughtabout by the stimulus of adversity in the environment, and pre-sented a certain amount of evidence from the standpoint of dyeinhibition of sporulation in support of his contention. He statedthat certain members of the tri-phenyl-methane series of dyes,among other effects, have the property of inhibiting rather thaninducing sporulation. The complexity of the phenomenon ofsporulation was further indicated in the work of Daranyi (1927),who attributed the principal r6le to colloidal influences. His
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BACTERIAL SPORES
explanation was that upon aging a shrinking of the cell colloidsoccurs which brings about the stimulation necessary for the initia-tion of spore formation. He drew an analogy between bacterialcells and the body cells of animals, pointing out that age in thelatter is characterized by a diminution of the water content and aconcentration of the colloids. In support of this contention hepresented evidence that sporulation could be induced in young,actively growing bacteria by diminishing the cell water content byartificial means.Any review of the literature concerning the germination of
bacterial spores must embody material of diversified nature, forthis property of spores is intricately involved in studies onlag, dormancy, heat relationships, single spore isolation, disinfec-tion, bacterial nutrition, and the r6le of bacterial spores in disease.Preisz (1918) contributed a critical discussion on the finer struc-ture of bacterial spores and changes incident to germination. Bymeans of a delicate, intra-vital staining technic, using dilutefuchsin solutions, he was able to observe the cytological changesoccurring in the process of germination, which he described andillustrated in detail. Meyer (1909) brought out the relationshipof spore germination to oxygen. His tables evaluate the mini-mum, optimum and maximum concentrations of oxygen for thegermination of spores of numerous species. For example, ex-pressed in amounts of oxygen per liter, he found that with B.subtilis 4.3, 400 and 4317 mgm. represented respectively theminimum, optimum and maximum. Eijkman (1918) reportedthat the germination of spores of B. anthracis was hindered bythe presence of salts that raise the osmotic pressure of the culturemedium. He found that sodium chloride, sodium nitrate andlevulose prevented germination at approximately the sameosmotic pressure, as determined by the freezing point method,although the respective concentrations were greatly different.
Direct observation of the germination time of bacterial sporeswas undertaken by Swann (1924). There had been a dearth ofinvestigation on this subject, in spite of its obvious practicalapplicability, probably due to the fact, pointed out by Swann,that it had been taken for granted generally that spores placed in
JOURNAL OF BACTERIOLOGY, VOL. XX, NO. 5
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EDWARD W. MORRISON AND LEO F. RETTGER
a favorable environment would germinate promptly. In hisdirect microscopic observations he made distinction between twotypes of spores to be found under different cultural conditions,the "young type" and the "old type." He found that thegermination time of the "young type" of anthrax spores wasshort (1 hour and 30 minutes), constant, and independent of theage of the spore, while that of the "old typ-e" was variable(minimum two hours and maximum seven hours). Allen (1923),in work concerned with the attenuation of bacteria due to tem-pQrature shock, brought out a point that was contradictory tothe general trend of opinion in regard to the r6le of injury in latentgermination or dormancy. He found that with spores of B.subtilis more rapid germination and growth were exhibited after aheat shock of 145°1. for thirty minutes than before.There has been some confusion in the usage of the terms "lag"
and "dormancy" or delayed germination. However, Burke andhis collaborators (1925) evaluated the terms satisfactorily, desig-nating "lag" as the latent period of a few hours shown by trans-planted cultures, while "dormancy" expresses the prolongedquiescence of a few cells in the transplant after the majority havemultiplied. Although dormancy or delayed germination ofbacterial spores has been observed repeatedly and encounteredas a disturbing factor in various bacteriological studies, there hasbeen no concerted effort to get at the underlying causes of thisphenomenon until comparatively recent years, perhaps for thereason that dormancy has been more or less universally acceptedas an unavoidable natural phenomenon.The classical example of dormancy, of course, is that so fre-
quently observed with spores of Cl. botulinum. Burke (1919,1923), Dickson et al. (1925), Dickson (1927) and numerous othershave reported extensive observations and investigations on thespores of this organism. The prevalent note in this work hasbeen that dormancy is inherent or normal in the spores of Cl.botulinum, as contended by Burke (1923) in her work withunheated spores, and that dormancy is accentuated in heatedspores, due to the injury and shock caused by the heat.
Burke, Sprague and Barnes (1925) initiated a further develop-
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BACTERIAL SPORES
ment in the study of dormancy. They presented evidence toshow that the phenomenon of dormancy, usually associated withthe spores of Cl. botulinum, is exhibited by the spores of commonaerobic organisms, and indeed by non-sporing bacteria. Underconditions stated to be optimal for growth, they found that cells ofE. coli remained dormant for as long as sixteen days and thatunheated spores of B. subtilis and B. megatherium were dormantfor thirty-nine and ninety days, respectively. This they spokeof as "normal dormancy."That dormancy may be encountered as a disturbing factor in
various researches is well illustrated in the work of Barber (1920)on the single spore isolation technic. Isolated spores showedvariable germination times and Barber was inclined to attributethe instances of delayed germination to slight but significantdifferences in tubes of medium from the same batch. Staxin(1924) also encountered dormancy following single spore isola-tions, finding that, although the majority of the positive culturesappeared within a week, a small percentage of the single sporesexhibited prolonged periods of dormancy, reaching ninety days.
In the r6le of dormancy in disease, emphasis has been placedon the question of latent tetanus infections. Canfora (1907),Koser and McClelland (1917), and others have recognized thepossibility of latent infection being caused by spores lying dor-mant in the blood or organs, particularly in case of traumaticinjury or secondary invasion by other organisms in the immedi-ate vicinity of these spores, creating conditions favorable togermation.
EXPERIMENTAL
In a previous communication (1930) the authors made certainobservations pertaining to conditions under which spores of agiven thermo-resistant aerobic organism exhibited properties ofdormancy. The bacillus in question was isolated from spoiledcans of evaporated milk, the spoilage being due to understeriliza-tion. In the course of heat-resistance determinations with thisorganism, it was found that spores heated in buffered distilledwater and subcultured in standard nutrient broth showed marked
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EDWARD W. MORRISON AND LEO F. RETTGER
and variable periods of dormancy. In parallel tests on sporesfrom the same suspension heated in evaporated milk, with orwithout subcultivation on agar, the dormancy was completelyeliminated. This observation suggested that the dormancy inthe first instance was neither "heat induced" or "normal"dormancy, but rather an expression of the inadaptability of thespores to the particular environment supplied by the standardnutrient broth. The variability of these spores in their germina-tion time after heating, depending upon the nutritional or otherstimuli supplied in the environment, led to the following investiga-tions which were designed to shed some light on the extent towhich various environmental factors influence the germination,and hence the dormancy, of washed unheated as well as heatedbacterial spores.
1. The germination time of aerobic spores as influenced by thenutritive value of the culture medium
In preliminary attempts to determine the effect of various cul-ture mediums on germination two criteria of germination wereadopted; the one was a macroscopic study of the extent of growthin a given time, and the other a determination of the increase ofviable cells, by the interval plate counting method. Throughoutthis work washed spore suspensions were used, and efforts weremade to'keep inoculums uniform in all comparative tests.
The culture mediums employed in this work were made up as follows:a. The plain nutrient broth was prepared from Difco Bacto meat
extract and peptone according to standard methods of the AmericanPublic Health Association Committee.
b. The tomato-extract peptone medium was prepared according to themethod of Kulp (1927) and had the following composition:
1 part filtered juice of canned tomatoes2 parts distilled water1 per cent peptoneReaction adjusted to pH 6.8
c. Various forms of yeast autolysate or yeast extract mediums havebeen in use since the time of Pasteur (1858). As prepared in this labora-tory the medium consists of an extract (autolysate) of yeast (Fleisch-
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BACTERIAL SPORES
mann's) obtained by incubating 1 pound of yeast in 5 liters of distilledwater for 3 days, with a small amount of ptyalin, and sufficient chloro-form to prevent bacterial growth. This concentrated extract is steamed,filtered, clarified and sterilized by autoclaving. The finished mediumconsists of:
1 part concentrated extract of yeast2 parts distilled water1 per cent peptoneReaction adjusted to pH 7.0(Optimal amounts of glucose may be added)
d. Glucose broth was prepared from the standard nutrient broth baseby the addition of desired amounts of glucose.
e. Sugar-free broth was obtained from the standard nutrient broth bythe removal of traces of fermentable sugars by E. coli.
f. The synthetic medium consisted of the following ingredients:
(NH4)2S04...............................................0.3 gramMgSO4................................................. 0.07 gramKH2PO4................................................ 0.1 gramK2HPO4................................................ 0.016 gramNaCl................................................... 0.05 gramCa(NO3).2g...............................................0.04 gramH20 (distilled)......................................... 100 cc.Glucose................................................0.5 per cent
As shown in table 1, the tomato-extract and yeast-extractmediums had a definite stimulating influence on the germinationof washed unheated spores of the aerobic organisms, as comparedwith the plain, sugar-free, and to a certain extent with the glucosebroth. Spores of the various organisms exhibited marked differ-ences in their responses to the deficient mediums. A "lag" periodwas evident in all instances, but this was so pronounced with themilk spoilage organisms as to warrant the term dormancy.Although it is not shown in the table, this variability of responsewas not confined to certain organisms, for various tests on a singlestrain often resulted in very different germination times in thedeficient mediums. The spores of strains I and II of the milkorganism were particularly susceptible to the stimulating influ-ence of the tomato and yeast mediums. B. cereus was the leastexacting in its nutritional requirements. The spores of B. cereus
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EDWARD W. MORRISON AND LEO F. RETTGER
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BACTERIAL SPORES
apparently germinate as rapidly in plain broth as in tomato-extract medium, as determined by the interval plate countingmethod (see table 2), although in the macroscopic tests growth inthe tomato medium seemed to be more luxuriant than in plainbroth.
2. The influence of temperature on the germination of washed sporesIt is commonly supposed that non-thermophilic, aerobic spore-
forming bacteria, except B. anthracis, have, as their optimum for
TABLE 2
Influence of medium on the germination of washed aerobic spores
GERMINATION INDICATED BY INTERVAL PLATE
ORGANISM MEDIUM
0 hours 1 hour 3 hours 5 hours 7 hours 9 hours
Milk strain I . TfPlain broth 9,600 9,000 6,800 9,400 9,100 9,000MilkstranI..Tomato broth 6,200 12,500 13,800 32,000 51,000 *
Milk strain I f Plain broth 700 600 800Tomato broth 1,100 2,900 9,000
f Plain broth 13,000 18,000 56,000 * * *B. anthracis . Tomato broth 16,00026,00096,000 * * *
B. -subtili&...... Plain broth 36,000 30,000 32,000 42,000 120,000 *B.8Ubtili8 . Tomato broth 38,000 62,000 86,000 166,000 * *
f Plain broth 9,000 12,000 36,000 * * *B. cereu .. Tomato broth 7,200 10,00032,000 * * *
f Plain broth 1,400 1,700 1,800 6,300 19,600 *B. vulgatue . lTomato broth 1,600 1,800 3,300 9,700 *
* Countless in the dilutions plated.
growth, temperatures ranging from 240 to 3000. In table 3 thetemperature ranges for the germn ation of washed spores ofseveral representative species are presented; they show that, in sofar as rapidity of germination is concerned, temperatures of 3700.,and in most instances, 4800., are much more effective than thelower temperatures. Although all but one of the test organismswere capable of germination and growth at 1200. there was a
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TABLE 3Influence of temperature on the germination of washed spores
GERMNATION OF SPORES (APPEARANCE OF COLONIES)INCUBATION TIME OF INCUBA-
TERA- TION B. anthr- B. subtilis B. cereus B. vulgatus Milk strain IIcis
"C.
12 hours 0 0 0 0 06 24 hours 0 0 0 0 0
60 hours 0 0 0 0 016 days 0 0 0 0 0
12 hours 0 0 0 0 012 24 hours + 0 + 0 060 hours + 0 + 0 0
Delayed + 7 days + 7 days 0 16 days
12 hours 0 0 0 0 024 hours + 0 + 0 016 60 hours + + 0
Delayed + 10 days
12 hours 0 0 0 0 020 J 24 hours + 0 + 0 0
60 hours + + +Delayed
12 hours + 0 + 0 0
24 J 24 hours + + 02 60 hours +
Delayed
12 hours + + + + 0
30 24 hours +60 hoursDelayed
12 hours +* + +* + +24 hours60 hoursDelayed
12 hours 0 +* 0 +* +*
48 124 hours 0 060 hours 0 0Delayed 0 0
12 hours 0 + 0 0 060 24 hours 0 0 0 0
60 hours 0 0 0 0
322
Standard nutrient agar.* Optimum in time of germination; greater number of colonies appearing.
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BACTERIAL SPORES
variable lag at this temperature. With B. subtilis and B. vulgatusthe first visible colonies appeared in seven days, which would indi-cate an actual delay in germination, rather than simply a slowrate of growth after germination had taken place. Furtherevidence in this direction was obtained from a numerical deter-mination of the effect of temperature on germination. Platecoulnts on washed spore suspension inoculum showed that, withinthe temperature range for germination, the number of coloniesappearing became progressively larger as the temperature in-creased. Thus, at 4800. the average count was approximatelyfour times as great as the average count at 12°C. and in most in-stances as that at 160C., within the maximum incubation time of16 days. Thus, it seems that the germination of the majority ofthe spores in a given amount of suspension is indefinitely delayedat the lower temperatures.The conclusion that low temperature is an environmental factor
to which these spores must become adapted before germinationcan ensue, seems justifiable.
S. The influence of gaseous environment on the germination ofwashed aerobic spores
In preliminary experiments with washed aerobic spores it wasfound that the substitution of atmospheres containing variableamounts of oxygen in mixture with carbon dioxide, ethylene andother gases, in place of ordinary air, exerted little or no influenceon the germination of the spores.
It is generally supposed that certain minimal amounts ofoxygen are required for the germination of aerobic spores, butthis seems to hold only for some of the organisms employed in thepresent work. Meyer (1909) and others have considered oxygenavailability as one of the cardinal factors in spore germination.Table 4 gives the results of the effect of vacuum2 on the germina-tion of several representative species of aerobic organisms on thesurface of agar medium. The data reported are based on numer-ous experiments in which complete anaerobiosis was obtained as
2The term "vacuum" is used here in a relative, and not absolute, sense. Thesame applies to anaerobiosis."
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EDWARD W. MORRISON AND LEO F. REITGER
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BACTERIAL SPORES
shown by the manometric readings and by the sensitive methyleneblue indicator used in all of the vacuum jars. In some of theexperiments in which these same results were obtained, oxygenabsorption was resorted to in the vacuum jars, supplementary toevacuation. For this purpose sodium hydrosulphite in alkalinesolution was placed in the bottom of the jars. A Cenco Hyvacpump was used in this work. According to the results obtained,no measurable minimum oxygen requirement can be establishedfor the germination of spores of the strains of B. anthracis, B.subtilts and B. vulgatus (I) used. It is significant that Cl. sporo-genes and various strains of Cl. botulinum developed on the surfaceof the standard nutrient agar under the same conditions (in thesame or separate vacuum jars), and in approximately the sametime as did the three so-called aerobic species mentioned above.
The influence of ethylene gas on the germination of aerobic andanaerobic spores. Gaseous environments in which unsaturatedgases like acetylene and ethylene are incorporated have not beenstudied in connection with the germination of bacterial spores, inso far as the writers have been able to determine. Several con-siderations, particularly the striking physiological effects exertedby ethylene on the cells of growing plants (Harvey, 1915), thestimulation of the germination of seeds and the sprouting ofdormant plant organs by this gas (Rosa, 1925), and the accelera-tion of the ripening of fruits and vegetables by ethylene, probablydue to stimulation of the enzymic function and other vital cellactivities, prompted the following investigation of the effectof various concentrations of ethylene gas on spores. Since thisgas is stimulative to various biological processes, an analogousacceleration of spore germination was considered possible.
Since no stimulation was detectable in the preliminary experi-ments, in which broth and agar mediums inoculated with washedaerobic spores were incubated in bell jars containing atmospheresmade up of different mixtures of ethylene with oxygen or air,experiments were undertaken to determine the effect of ethyleneon the germination of aerobic and anaerobic spores in vacuo. Itwill be seen from table 4 that, compared with vacuum only, ethy-lene does apparently stimulate the germination of both aerobic
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EDWARD W. MORRISON AND LEO F. RETTGER
and anaerobic spores under identical conditions. This suggeststhat the effect involves a direct stimulation of vital forces, perhapsof the enzymic function of the spores. It is noteworthy, also,that even in the highest concentration attainable ethylene isnon-toxic to the sporulating bacteria. Although maximumgrowth of the aerobic organisms, following germination from thespore, does not take place in vacuo or in any concentration ofethylene displacing the vacuum, this effect is not due to toxicity ofthe gas, since growth takes place about equally well in all concen-trations, and since uniform, maximum growth ensues when agarslant cultures are removed from the vacuum jars and incubatedunder atmospheric conditions, though the exposure to ethylenehas been continued for ten days or more.
4. Nutritional factors influencing the dormancy of heated washedspores
Since the germination of washed unheated spores of the milkorganism was markedly stimulated by the nutritional factorssupplied in tomato or yeast-extract mediums, as compared withstandard nutrient broth and other mediums, parallel heatingexperiments were conducted to determine whether or not thesesame media exert the samefavorable influence on spores that havebeen heated, keeping in mind the fact that the spores of the milkorganism exhibited pronounced and variable dormancy in all pre-vious instances when heated in water and subcultured in standardnutrient broth. In these experiments a single tube series of sporesuspensions, all from the same stock, was heated in buffereddistilled water at 105°C. At the end of each time interval asingle tube was removed trom the oil bath, and equal portionsof its contents were subcultured in the different mediums. Theresults are shown in table 5.The striking points in these comparative tests in which equal
portions of single heated suspensions were subcultured in differentmediums are: (a) That the same pronounced "dormancy" ordelayed germination occurred in the standard nutrient broth sub-cultures in all of the individual tests on heated spores; (b) thatin the tomato and yeast-extract subculture mediums dormancy
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BACTERIAL SPORES 327
and skips were entirely eliminated, growth occurring promptly(within twelve to twenty-four hours) in.every tube of the singletube series up to the critical time; and (c) that heat shock or in-jury can play no part in the dormancy of this organism, sincespores receiving the maximum heat germinate as promptly asothers when a favorable environment is supplied in the sub-culture medium.
TABLE 5
Influence of nutritional factors on the dormancy of spores after heating
TEMPERA-TURZHEATED
"c.
105105105105105105105105105105105105105105105
TIMEHEATED
minutes
1530405060708090100115130145160180200
1E STRAIN I (STBCUTUEtZ | MILK STRAIN II (SUBCULTURZS AT 3rC.)
Growth in 12to 24 hours
Tomato Plainme- brothdium
+
+
+++
Delayedgrowth inplain broth
+ 16 days+ 16 days+ 22 days+ 18 days+ 24 days+ 22 days+ 22 days+ 30 days- 50 days- 50 days- 50 days- 50 days- 50 days- 50 days- 50 days
Growth in 12 to 24 hours
Tomatome-dium
++++++
Yeastme-dium
+
+
+
Plainbroth
Delayedgrowth inplin broth
+ 2 days+ 2 days+ 2 days+ 13 days+ 30 days+ 10 days+ 16 days- 50 days- 50 days- 50 days- 50 days- 50 days- 50 days- 50 days- 50 days
5. Factors contributing to or determining the dormancy of washedunheated bacterial spores
The method employed in this work for the determination ofdormancy follows, with certain important modifications, the pro-cedures worked out by G. S. Burke (1923) and by Victor Burke andhis associates (1925) for the study of "normal dormancy" inspores of Cl. botulinum and in common aerobic spores.
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EDWARD W. MORRISON AND LEO F. RETTGER
Technic. Approximately single spores of the various organismsstudied were inoculated into deep tubes of different mediums containing0.5 per cent agar. To this end, washed spore suspensions were dilutedand subjected to the plate count method to determine the amount ofinocul.um necessary for the desired distribution of spores. After select-ing a definite dilution, trial tubes of soft agar and of liquid mediums wereinoculated with the determined amounts, incubated for twenty-fourhours and examined before the regular series of tubes were inoculated.During this interval the dilutions were held in the refrigerator. Ininoculating parallel sets of tubes with the same suspension in order todetermine the relative stimulation of the germination of the singlespores in different mediums, the tubes were seeded alternately from thesame pipette. Furthermore, the various mediums were always pre-pared at the same time and, as far as possible, from the same materials.Other conditions, such as temperature of the medium when inoculated,incubation temperatures, the time of sealing the tubes, etc., were keptconstant in parallel tests. The theoretical total of spores in the amountof inoculum used for all of the tubes in a given test was calculated froman average of multiple plate counts of the dilution used. Tubes showingno growth in twei,ty-four hours were sealed with paraffin and observeddaily during the first ten days and at less frequent intervals thereafter.
The results of these experiments on the dormancy of individualwashed spores of common aerobic organisms are presented intables 6 and 7, but a summary of these results seems to be desir-able here in order to emphasize certain of the observations.B. subtilis. It will be seen in table 6 that in an extensive series
of tubes distributed through three different mediums there is noevidence of dormancy or delayed germination of the spores of B.8ubtilis within the maximum incubation period of fifty days; allof the tubes showing growth did so within twenty-four hours.The yeast-extract and glucose infusion agars have apparently noadvantage over the plain agar for this organism, although thepercentage of the theoretical total spores developing in the yeastmedium is considerably higher than in the plain or glucose in-fusion agars. Since no further spores develop in any of themediums between the first and fiftieth days, the variation inpercentages is assumed to be due to an experimental error in thedistribution of the spores. This is in accord with the observations
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TABLE 6
Results of experiment. on the dormancy of individual-wahed 8pores
PLAIN 0.5 PUr CNT AGAR; YEAST-EXTRACT 0.5 PRt CENT GLUCOSE4NPUMON 0.5 PER213 TUB3E INOCULATED AGAR; 110 TUBES INOCULATED CENT AGAR; 40 TUBES
INOCULATED
Theoretical total spores in Theoretical total spores in Theoretical total spores inINCUBATION inoculum: 213 inoculum: 110 inoculum: 40
TIME
Per cent Per cent Per oentTubes Total of theo- Tubes Total of theo- Tube Total of theo-positive lonies retical positive colonies reticl positive colonies reticalaltbs total all tubes ptottal all tubes total
spores spores spores
A. B. subtilisdcya1 143 206 96.7 70 115 104.5 26 34 85.023
50
143 206 96.7 70 115 104.5 26 34 85.0
B. B. megatherium
50 TUBES INOCULATED 50 TUBES INOCULATED 50 TUBBES INOCULATED
Theoretical total spores in Theoretical total spores in Theoretical total spores ininoculum: 38 inoculum: 38 inoculum: 38
1 23 27 71.0 26 36 94.7 1 1 2.62 4 5 13.2 1 1 2.63 1 1 2.64 1 2.65 2 5.3
11 1 1 2.645
29 37 97.3 27 37 97.3 1 1 2.6
C. B. cereus
25 TUBES INOCULATED 25 TUBEs INOCULATED
Theoretical total spores in Theoretical total spores ininoculum: 22 inoculum: 22
1 14 20 90.9 14 16 72.7245
14 20 90.9 14 16 72.7
D.- B. vulgatus
50 TUBES INOCULALTED 50 TUBES INOCULATED
Theoretical total spores in Theoretical total spores in
inoculumn: 28 inoculum: 28
1 22 24 86.0 18 24 86.02 2 2 7.145
22 24 86.0 20 26 93.1
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EDWARD W. MORRISON AND LEO F. RETTGER
made previously that this strain of B. subtilis is not at all exactingin its nutritional requirements, growth occurring about as wellin plain broth as in glucose broth or in any other special medium.The fact that this organism was the only one of those testedwhich developed from the spore in the synthetic medium furtherconfirms this assertion.
B. megatherium. In the experiments with B. megatherium aninexplicable idiosyncrasy was displayed by the spores. Withinthe maximum period of incubation only one spore out of atheoretical 38 germinated in the glucose infusionO.5 per cent agar,although the three mediums indicated in table 6, B, were inocu-lated alternately from the same pipette with the same amount ofinoculum. It is apparent from the table that this effort to insureeven distribution of the spores was successful in so far as thefirst two mediums are concerned. The glucose infusion mediumemployed was part of the same batch which was used in the experi-ment with B. subtilis and, as has been shown, the spores of B.subtilis germinated as well in this as in the other mediums. Sinceconditions were kept constant in all other details of this paralleltest it must be assumed that these results are due to subtlepeculiarities of these spores in their nutritional requirements forgermination. It will be noted in this experiment also that theperiod of dormancy extends to the eleventh day in the plain agar,with 26.3 per cent of the total spores developing after 24 hours.In the yeast-extract medium dormancy is practically eliminated,extending only to the second day, with one spore or 2.6 per cent.This further indicates special requirements for germination of thespores of this organism.
B. cereus and B. vulgatus. Although no very definite conclu-sions can be drawn from the limited number of tubes inoculatedwith single spores of these organisms, as shown in table 6, C andD, the indication is that they do not exhibit particular tendenciestoward dormancy under the environmental conditions supplied.Milk strain I. The most marked peculiarities in requirements
for germination observed in these experiments, and the most pro-longed periods of dormancy, were exhibited by the spores ofstrain I of the milk organism. As was expected, on the basis of
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BACTERIAL SPORES 331
the earlier experiences with this organism and the dormancydisplayed by it in plain broth subcultures after heating, the indi-
TABLE 7
Results of experiments on the dormancy of individual washed spores. Milk Strain I
PLAIN 0.5 PEE CCNT AGA3; YEAST-EXTRACT 0.5 PER CENT GLUCOSE-INFUSION 0.5 PER100 TUBES INOCULATED AGAB; 100 TUBES INOCULATED CzNT AGAR; 45 TUBES
INOCULATED
Theoretical total spores in Theoretical total spores in Theoretical total spores ininoculum: 100 inoculum: 100 inoculum: 45
Tubespositive
669
1*
1*
22
2
322
Totalcoloniesall tubes
7891*
1*22
2322
90 102
Per centof theo-reticaltotalspores
78.09.01.0
1.02.02.0
2.03.02.02.0
102.0
pTubes |eTotalpstubes coloniesipoiieall tubes
Surface growth.
vidual washed unheated spores showed pronounced dormancyin the plain deep 0.5 per cent agar (table 7). In glucose infusion
JOURNAL OF BACTERIOLGY, VOL. XX, NO. 5
INCUBATIONTIME
days12345689101214161719212224263035404550
Tubespositive
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
2
1
2
5
4
11
6
33
Totalcoloniesall tubes
1121254116
33
Per centof theo-reticaltotalspores
1.01.02.01.02.05.04.011.06.0
33.0
Per centof theo-reticaltotalspores
17.78.82.22.24.42.24.42.22.24.44.46.64.42.22.2
4.4
2.22.2
80.0
84
11
2121122321
1
2
11
36
g41121211223211
2
11
36
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EDWARD W. MORRISON AND LEO F. RETTGER
agar variable periods of dormancy were encountered; yet, unlikethe action in plain agar, germination began on the first day, show-ing that the spores were supplied with better, though not optimal,environmental conditions. Carrying this observation further,it is seen that conditions still more favorable, and yet not optimal,are supplied in the yeast-extract agar, as evidenced by the fact that78 per cent of the theoretical total number of spores developedwithin 24 hours in this medium, whereas only 17.7 per cent of thespores in glucose infusion agar showed growth in the same time.The fact that 78 per cent of the individual spores of this organ-
ism developed promptly in the yeast-extract agar, while there wasa prolonged quiescence in the plain agar medium, offers an explan-ation of the elimination of dormancy in heat resistance tests onthis organism by substituting yeast-extract medium for plainbroth in the subcultures. The objection is of course raised thatthe final criterion as to whether or not an organism has tendenciestoward dormancy, according to definition, is the prolonged quies-cence of a few spores out of a transplant, and that since only 78per cent of the total single spores showed prompt germination, thisorganism has inherent tendencies toward dormancy. This mayor may not be the case, and in the discussion that is to followseveral factors will be considered which should enable us to format least an hypothetical conception of the fActors which deter-mine dormancy.
It may be said here, however, that the graduated results obtained with this organism in three different mediums-pro-nounced dormancy in plain agar, less marked dormancy in glucoseinfusion agar and still less in yeast-extract medium,-suggestthat the dormancy is simply an index of the degree of unfavorable-ness of the environment or medium rather than a normal orinherent quality of the spores.
6. Discussion
The present work has shown that bacterial spores may revealmarked variability in their response to environmental influences.It seems justifiable to assuime that spores in the process of ger-mination are active metabolizing bodies, having exacting nutri-
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tional and other requirements for germination. In the absenceof particularly favorable conditions, spores undergo a period ofadaptation just as growing vegetative cells do under changes ofenvironment (Lag phase). When, however, unfavorable environ-mental conditions are encountered by spores, the lag period isusually so extended that it acquires the aspect of what is termeddormancy.Of the various nutritional factors influencing spore germination,
and therefore spore dormancy, the availability of nitrogen isperhaps of outstanding importance. From the work of Sperryand Rettger (1915), Rettger, Berman and Sturges (1916) andBerman and Rettger (1918) it is evident that the nutritional valueof a culture medium is largely dependent upon the nature of thenitrogenous material present, and that amino acids and other sub-stances which readily give up their nitrogen are of particularsignificance. Although their work was not directly concernedwith spore germination, it is readily applicable to this process onthe basis of the foregoing assumption that spores are not inert,but active bodies having the same or more exacting requirementsfor their metabolism than do vegetative cells. Stickel and Meyer(1918) suggested the general use of peptic or tryptic digestionproducts of protein material in bacteriological work, particularlyin the cultivation of the sporulating anaerobes, due to the highamino acid content and nitrogen availability of such mediums ascompared with the usual standard mediu;ms. Among thenumerous investigators suggesting the use of various forms ofyeast autolysate or extract mediums, either because of theirgrowth-stimulating properties or as economical substitutes formeat extracts, are Kressler (1918), Kligler (1919), Eberson (1920)and Ayers and Rupp (1920). Thj6tta (1921) and Thjotta andAvery (1921) made important contributions to the subject ofbacterial stimulation by growth accessory substances in certainculture mediums. In the latter communication they reportedmarked stimulation of bacterial growth by small amounts ofextracts of yeast cells, and of certain vegetables such as greenpeas, string beans and tomatoes. Wagner, Dozier and Meyer(1924) emphasized the high "biologic value" of mediums contain-
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334 EDWARD W. MORRISON AN] LEO F. RETTGER
ing beef heart infusion, and the superiority of these mediums overothers in the original isolation of the anaerobic bacteria fromspores.That incubation temperature and heat stimuli may play im-
portant roles in bacterial spore germination is evidenced by theresults of the experiments on the temperature ranges for germina-tion reported herein, and by the work of previous investigators.Weyer and Rettger (1927) showed that marked stimulation of thegermination and subsequent vital activity of spores of Cl. aceto-butylicum followed a brief exposure of the spores to boiling tem-perature. Also, Allen (1923) in his work wvith spores of B. subtilisshowed that more rapid germination and multiplication tookplace after heat treatment or shock at 145°F. for 30 minutes thanin the unheated material.Ludwig (1918) contributed the only direct work on the effect
of ethylene gas on bacteria that has come to the attention of thewriters. His work was concerned with the influence of illuminat-ing gas and its constituents on bacteria and fungi, particularlyvarieties pathogenic for plants. He found that the developmentof bacteria in the presence of ethylene was not materially checkeduntil high concentrations were reached. In spite of the variousstimulative properties of ethylene, it has been reported as havngextreme toxicity for certain plants and plant organs. Crockerand Knight (1908) reported on the toxic effect of ethylene uponflowering carnations. Knight and Crocker (1913) noted extremetoxicity for sweet pea epicotyls. Harvey (1915), and Harvey andRose (1915) have shown that the extreme toxicity of ethyleneholds for numerous different species of plants. Ludwig, in thework jlust cited, was seeking an analogous toxicity for bacteriaand fungi that are pathogenic for plants.
Other than to suggest that gaseous environment may enter asone of the factors determining germination and hence dormancy,and especially that the unsaturated hydrocarbon gases such asethylene may act as stimulants of germination, the present workon gases is not sufficiently complete to be conclusive. If, how-ever, the writers' hypothesis relative to the facilitation of sporegermination by means of pre-germination enzymic function be
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taken into consideration, an hypothetical explanation of ethylenestimulation may be suggested; namely, that ethylene acceleratesthis enzymic or other metabolic function of the spores. Therecent work of Nord and Franke (1928) tends to support thisexplanation. Their paper dealt with the protective and stimula-tive effect of ethylene on various enzymes, either in solutions (cellextracts) or in living cells (zymase of yeast) or in plant tissues(catalase of tobacco leaves).In the experiments on the dormancy of heated spores of the
milk organism it was conchisively shown that the dormancyexhibited by these spores in standard nutrient broth subcuWturescould not have been "normal" dormancy, or due to factors of"heat inhibition" or "heat shock," to which previous investi-gators have usually attributed prolonged dormancy after heating.Spores of single heated suspensions showed prompt germinationwhen supplied with adequate nutritional stimuli in the subculturemedium, that is by simple substitution of tomato or yeast-extractmedium for standard nutrient broth. This observation seemssignificant, since standard nutrient broth is commonly used as thesubculture medium in heat resistance determinations on commonaerobic spores and in sterility tests of various kinds.
In the experiments on the dormancy of individual washedspores of aerobic bacilli there is further evidence that the dor-mancy of unheated spores, usually called "normal dormancy,"is in reality a function of the environment or an index to the degreeof unfavorableness of the environment of the spores, since theextent of dormancy varied markedly according to the nutritionalstimuli supplied in the medium. It was observed that B. subtilis,B. cereus and B. vulgatus did not show any tendency toward so-called "normal" dormancy when single washed spores were inocu-lated into plain, glucose infusion or yeast-extract soft agarmediums. This observation is at variance with the findings ofBurke et al. (1925), who reported that unheated spores of B.8ubtilis remain dormant for as long as 39 days in deep agar culture.Differences in the strains of B. subtilis used, or in the technic,might well account for this discrepancy in results. In determin-ing dormancy, Burke inoculated approximately single, unwashed,
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unheated spores, or rather growth from an agar surface of suffi-cient age to insure the presence of spores, into standard 1.5 percent deep nutrient agar containing 0.5 per cent glucose. WithB. megatherium and strain I of the milk organism, however, therewere marked and variable periods of dormancy in the plain soft(0.5 per cent) agar medium employed in the present work, asshown in tables 6 B and 7.In discussing the factors contributing to, or determining, dor-
mancy utnder experimental conditions, it will be perhaps moreconvenient to present the additional observations and commentsunder headings denoting the principal factors thought to be ofsignificance:
a. Consistency of the medium. Preliminary tests with the B.subtilis strain employed here showed that deep tubes of standardagar, such as those used by Burke, do not supply "conditionsfavorable for growth," as evidenced by the fact that developmentis slow and the colonies extremely small. In deep tubes of thesoft agar medium, on the other hand, colonies developed to amaximum size of 6 mm. in diameter in the same or shorter periodsof time. While it was the writers' belief that deep agar culturein any form is not favorable for the germination of aerobic spores,the convenience of the method and the fact that B. subtilis grewwell in the soft agar led to its adoption for want of a bettermethod, particularly a surface cultivation method.
In the course of the work it was observed that cells developingfrom the inoculated spores were capable of motility in the 0.5per cent agar. It is obvious that such a condition would permitthe cells of the developing colonies to utilize the nutrient materialpresent in a considerable area surrounding the original loci ofdevelopment. Furthermore, it is conceivable that, althoughspores imprisoned in 1.5 per cent solid agar may germinate, thesubsequent growth may be checked or even ihhibited due to theaccumulation of metabolic products or to a mere mechanicalobstruction to the free movement of the cells. Mechanical andphysical factors such as density of the medium, surface tension.osmotic pressure and oxygen penetration, then, must be takeninto account as serious objections to the deep agar culture method
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of determining dormancy, particularly if solid agar of standardcomposition be used. In substantiation of this point it shouldbe said that spores of strain I of the milk organism, for example,develop promp'ly on the surface of plain agar while, as seen inthe results in table 7, the first sub-surface colonies to appear inthe deep 0.5 per cent plain agar did so only after 21 days.
b. Oxygen tension. An observation was made that suggests afurther explanation of the occurrence of dormancy under condi-tions of deep agar culture; namely, that with every organismtested the cells invariably moved through the soft agar from theoriginal loci of development toward the surface of the medium.In all cases, however, initial colonies developed at the points ofgermination of the spores. These were usually fluffy or of "puffball" appearance and on continued incubation tended to diffusethrough the medium. It was frequently observed that from amore or less dense initial locus the colony would spread outradially in the direction of the surface of the medium but nevertoward the bottom of the tube. It was also noted in a few in-stances in which there were several colonies distributed throughouta single tube that the colonies were increasingly larger as the sur-face of the medium was approached, those nearest the top beingmany times the size of those at the bottom of the tube. Thenatural conclusion from these observations is that environmentalconditions in the deep agar are unfavorable to aerobic bacteria,due to decreased oxygen accessibility. This factor alone maydetermine, or at least contribute to, dormancy of spores, althoughit was apparently not operative in the case of B. subtilis, whichshowed no delayed germination. It should be said, however,that the strain of B. subtilis used was capable of germination andgrowth under essentially anaerobic conditions.
c. Enzymic function. In a previous communication (1930) thewriters have shown that the milk organism produces an activesubstance that is capable of bringing about marked changes inmilk, probably a caseolytic enzyme, and the hypothesis wasadvanced that this enzyme may aid in, and perhaps account for,the prompt germination of spores of this organism in milk, evenafter severe heating. On the basis of the assumption that spore
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germination may be facilitated by enzyme secretion, anotherfactor contributing to the dormancy of aerobic spores in deepagar culture may be cited. Effront (1917) has shown that B.mesentericus, when cultivated on large surfaces, produces twentytimes as much protease as when grown below the surface. Thissuggests of course that the enzymic function of this organism isdependent upon oxygen accessibility. Since protein metabolismis probably of great importance during the process of germination,the function of the proteases alone may determine the dormancyof spores in some instances.
d. Single spore inoculum. There has been considerable contro-versy on the question of the mutual accelerative action of organ-isms in freshly seeded cultures. It is the view of numerousworkers that such an action is accountable for prompt growth ina new environment, and that the inevitable result of dilution andisolation of the cells is lag. Sherman and Albus (1924), however,in answer to the question "Is there an allelocatalytic effect in thegrowth of bacterial cultures?" take the stand that the rate ofgrowth of a culture is constant regardless of the size of theinoculum.Another view is that growth ensues more rapidly in cultures
started from large inoculums due to the fact that they containassortments of individuals, some of which are more vigorousor capable of adaptation to new environmental conditions thanothers. That this view does not necessarily apply to spore inocu-lums is shown in the present experiments with single spores ofB. subtilis, the individual spores of which developed as rapidly asdid larger spore seedings. The work of Barber (1920) adds weightto this contention; in using the single cell isolation technic, hefound that spores of B. subtilis gave 100 per cent positive cultures,and that the spores developed promptly even when transferred toglucose broth medium covered with oil or vaseline to provideanaerobic conditions. It may be concluded, then, that the isola-tion of the individual spores is unimportant as a factor contribut-ing to dormancy, at least with the organisms investigated here.
e. Nutrient value of the culture medium. The effect on germina-tion of the nutrient value of the culture medium has been already
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discussed to some extent. With washed unheated spores of anumber of organisms it has been shown that very definite retarda-tion or acceleration of germination may occur, depending uponthe kind of nutrient material that is present in the medium. Theexperiments on the dormancy of single washed unheated sporesfurther emphasize the importance of adequate nutritional stimuliin spore germin-ation. The fact that with some organisms theextent of dormancy is graduated in different mediums in whichthe spores of other organisms develop rapidly and equally wellindicates that there are definite nutritional requirements fordifferent organisms, and that the lack of these readily causes, orcontributes to, dormancy.
SUMMARY
Bacterial spore dormancy and germination have been investi-gated in relation to environmental factors.Because of the marked variability of germination, depending
upon the stimuli supplied in the environment, the deduction ismade that bacterial spores in the process of germination arevitally active bodies having requirements for metabolic functionwhich are the same as or more exacting and specific than those ofthe vegetative cells.Experimental evidence is presented to show that the dormancy
of aerobic bacterial sporesis largely, if not entirely, determinedby conditions in the environment of the spores, and that thesefactors must be taken into consideration, perhaps specifically foreach species, before so-called "inherent" or "normal" dormancy ofbacterial spores can be established;.
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