MANGANESE OF - Journal of Bacteriologyjb.asm.org/content/67/4/489.full.pdf · yield relatively few or no aerobic spores unless ... trivalent salts increased the percentage of ...

THE INFLUENCE OF IRON OR MANGANESE UPON THE FORMATION OFSPORES BY MESOPHILIC AEROBES IN FLUID ORGANIC MEDIA

HAROLD R. CURRAN AND FRED R. EVANSBureau of Dairy Industry, Agricultural Research Administration, United States Department of Agriculture,

Washington, D. C.

Received for publication October 22, 1953

There is growing evidence that many com-plex organic substrates, including most ofthe commonly used bacteriological fluid media,although supporting heavy vegetative growth,yield relatively few or no aerobic spores unlessfortified with certain minerals. The Salts which,when added to a particular substrate, promotethe formation of spores depend upon its com-position, upon the organism used, and otherfactors; thus, Fabian and Bryan (1933) notedthat the addition of monovalent but not bi- andtrivalent salts increased the percentage ofmesophilic spores formed in peptone solution.Knaysi (1945) observed that added magnesiumsulfate had a beneficial effect on the sporulationof Bacillwu mycoides in glucose broth but onlyif the oxygen supply was limited. Foster andHeiligman (1949) found that a deficiency ofpotassium in the usual organic media was limitingin the sporulation of Bacillus cereus. Morerecently Charney et al. (1951) have pointed to aspecific effect of manganese in trypticase andnutrient broths on the sporulation of Bacillussubtilis and certain other mesophiles.In the course of observations on the germina-

tion of spores in milk, we found that certainsalts, notably iron and manganese, althoughwithout effect upon the germnation of the spores,transformed the spore generating potential ofthe medium for certain species. Since normalmilk is a notoriously poor medium for theproduction of aerobic spores, the apparentspecific effect of the two salts led us to! study thisreaction in some detail. The results are presentedin this paper.

MATERIALS AND METHODS

The principal organisms used were: Bacillussubtilis (15u) American Can Company andB. brevis Temple University. Limited observa-tions were made on B. subtilis (9499), B. stearo-thermophilus (1518), and B. thermoacidurans

(B. coagulaus) (43P, 2353) from NationalCanners Association; B. subtilis (4149), B.stearothermophilus (CM2), and unidentifiedculture (3679) from American Can Company;B. subtilis (6051, 6634), B. brevis (8185), B.mycoides (B. cereus var. mycoides) (6462), andB. circulans (4513) from American Type CultureCollection; B. cereus (401), B. macerans (277),B. polymyxa (354), B. laterosporus (314), andB. megaterium (753) from the N. R. SmithCollection.The spores were produced, collected, and

prepared for use as described previously (Curranand Evans, 1945) except that dispersion of theclumps was accomplished by shaking with smallglass beads.The basal media were autoclaved skim milk,

usually unadjusted, and nutrient broths con-taining the following ingredients per liter, eachadjusted to a final pH 7.0:Nutrient broth-beef extract (Difco), 3 g;

peptone (Difco), 5 g.Yeast broth-yeast extract (Difoo), 10 g;

peptone (Difco), 10 g; K2HPO4, 5 g.Pork infusion-1 lb lean pork, 200 ml; peptone

(Difco), 5 g; tryptone (Difco), 1.6 g;K%HPO4, 1.25 g.

Peptone broth-peptone (Difco), 1 g.Chemically pure salts were used, solutions of

which were passed through fritted glass filtersand tested for sterility before use. Except asnoted the iron salt used was FeCl3.6H20, themanganese salt was MnCl2-4H20.

In general, the spores were seeded in the testmedia and the suspensions heated at 85 or 95 Cfor 10 minutes, cooled, and incubated at 30 or37 C. After incubation, the samples were heatedat 85 C for 15 minutes, cooled, and vigorouslyshaken with sterile sand or small glass beadssufficiently to disperse the clumps. Each samplewas plated in triplicate after heating, both beforeand after incubation. The plating medium was

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HAROLD R. CURRAN AND FRED R. EVANS

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HOURS AFTER INOCULATIONFigure 1. The effect of added iron or manganese (pg per ml) upon the sporulation of Bacillus subtils

(15u) in milk-iron as FeCls, manganese as MnC12. Temperature of incubation 37 C.

glucose nutrient agar containing 0.1 per centsoluble starch. The plates were counted after48 hours of incubation and occasionally recountedafter 48 hours of additional incubation; coloniesdeveloping on the plates were assumed to haveoriginated from spores.

EXPERIMENTAL RESULTS

Sporulion in milk. Sterile skim milk to whichhad been added different quantities of iron or

manganese was seeded uniformly with spores in

Erlenmeyer flasks, heated at 95 C for 10 minutes,cooled, and incubated as still cultures at 37 C.At intervals from 0 to 120 hours, spore countswere made by plating well mixed suspensionsafter heating at 85 C for 15 minutes. The resultsobtained with a strain each of B. subtilis andB. brevis are shown in figures 1 and 2. Sincevegetative growth was -abundant in both thetreated and untreated milk, it is clear that addediron or manganese provided something esentialfor rapid sporulation. In their absence, spore

synthesis, after rapid and almost completegermination, was negligible in 5 days althoughit occurred promptly and at high levels when themilk was fortified suitably with these minerals.It is noteworthy that manganese was effective inlow concentration while approximately 100 timesas much iron was required to produce a com-

parable result-a fact which probably explainswhy the spore promoting activity of this elementhas been previously overlooked. Increasing theconcentration of added iron in milk to 400 and600 &g per ml resulted usually in a furtherincrease of the spore crop when the pH was

adjusted to 6.4. Omission of the pre-incubationheating did not greatly affect the results. Iron ormanganese added directly to agar plates did notincrease the spore count.Examination of stained preparations made at

the plating periods after incubation revealedvisible spores in the presence of the addedminerals but no discernible spores in the controls;these preparations also indicated that the iron

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AEROBIC SPORES IN FLUID ORGANIC MEDIA 491

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Figure S. The effect of added iron or manganes (jg per ml) upon the sporulation of Bacillus brevs(Temple) in milk-iron as FeCl, manganese as MnCl1. Temperature of incubation 37 C.

(280 ,g per ml) increased the vegetative popula-tion an estimated 2 to 4-fold during comparableperiods of incubation; manganese (2.7 ,g perml) produced little or no increase in thevegetativepopulation.

Fssentially similar results were obtained withsix other strains of B. subtilis including (6051)(Marburg) and 6634 (Ford), B. brevie (8185),and B. laterosporus (314). Negative results orvery slight enhancement of sporulation wasnoted with B. megaterium (753), B. cereus (401),B. mycoidee (6462), B. macmrans (277), B.circlan (4513), B. polymyza (354), B. stearo-thrmnophilus (1518, CMg), B. Jirmacidurans(43P, 2353). The one anaerobe tested, 3679, wasnegative to iron and manganese.A large number of other metal salts used in the

same and lower concentrations either reducedthe spore crop or had no significant influence;these included the chlorides of potassium,sodium, magesium, calcium, cadmium, zinc,cobalt, nickel, strontium, tin (stannous), copper(cupric), and cerium (cerous).

Sporulation in broth media. In view of theresults obtained with milk, it was of interest toascertain if the spore promoting function ofiron and manganese could be demonstratedsimilarly in other complex organic media,particularly those used in the laboratory for thegrowth of sporogenic species. Figures 3 and 4show the development of spores in several brothmedia supplemented with iron or manganese.The methods were similar to those used in theprevious experiment except that after adding theiron the pH was readjusted to 7.0. Under thetest conditions the normal, untreated brothmedia like milk were not conducive to sporeformation,' a deficiency which was correctedby suitable additions of either iron or manganese.Each of the several unfortified media supportedheavy vegetative growth but produced few or nonew spores. Thus, manganese or iron was critical

1 Unfortified broths made from different batchlots of ingredients have shown differences intheir spore promoting capacity possibly due tovariations in their iron and manganese content.

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HOURS AFTER INOCULATIONFigure S. The effect of added iron or manganese (pg per ml) upon the sporulation of Bacilus subtilis

(15u) in broth media-iron as FeCls, manganese as MnCl,. Temperature of incubation 37 C.

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Figure 4. The effect of added iron or manganese (pg per ml) upon the sporulation of Bacillus brevis(Temple) in broth media- iron as FeCls, manganese as MnCl2. Temperature of incubation 37 C.

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AEROBIC SPORES IN FLUID ORGANIC MEDIA

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HOURS AFTER INOCULATIONFigure 5. The effect of different iron compounds (ug per ml of iron) upon the sporulation of Bacillus

subtilis (15u) in milk. Temperature of incubation 37 C.

for the formation of spores under the test condi-tions. It is apparent that the more complexinfusion and yeast media were less favorableto spore production than the simpler nutrientextract or peptone broth; the latter supple-mented with iron or manganese gave the bestspore yields. The observed response of B. subtilisto added manganese in nutrient broth is inagreement with the findings of Charney etal. (1951).

Sporulation as influenced by the form in whichiron i supplied. In this experiment sporulationof B. subtilis (15u) was measured in milk inresponse to a variety of iron containing com-pounds. The results are plotted semilogarith-mically in figure 5. It may be seen that iron waseffective in promoting the formation of sporesonly when it was present in a soluble form sinceneither the oxide nor the reduced iron sig-nificantly influenced the production of spores,whereas all soluble iron salts were sporogenicallyactive. The results obtained with iron thiocyanatesuggest that not only the ions but the iron in the

nonionic form contribute to the formation ofspores since this compound is feebly ionized.The rate at which spores became labile to heatwas unchanged by the addition of iron or manga-nese, indicating that their effect is primarilyupon a specific phase of cell metabolism-that is,sporulation.

Sporulation in milk with added rust or priorcntact with rusting container. The effects whichextraneous iron may induce in sporogenicorganims in milk suggest that contaminationwith iron may play a role in certain problemsin milk preservation; the wide distribution ofthis element in soil and the many routes bywhich it may enter milk lend substance to thisbelief. Of special interest is the effect uponsporulation of rust contamination in milkhandling equipment. To obtain some informationon this point, a sample of sterile skim milk wastreated with sterile rust scraped from a usedrusting dairy utensil while a second portion fromthe same source served as control; the twosamples were inoculated similarly with spores

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HAROLD R. CURRAN AND FRED R. EVANS

J 6 _ _1 o0'QlTRUING4 CA?Nl X

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HOURS AFTER INOCULATIONFigure 6. The effect of added rust (ug per ml) or prior contact with rusting cans upon the sporulation

of Bacillus subtilis (15u) in raw milk. Rust scraped from rusting cans was added directly to milk ormilk was held in rusting or rust-free cans at 0 C for 24 hours after which 10 ml samples were inoculatedwith spores, incubated at 37 C, and plate (spore) counts made as indicated. The spore count after re-frigeration before inoculation was: rusting can 0.5, rust-free can 2.5 per ml. Temperature of incubation37 C.

of B. subtiis, and plate (spore) counts weremade at sero hours and after the usual periods ofincubation as detailed. In a second experimentequal volumes of well mixed fresh raw milkwere stored at 0 C for 24 hours in each of twosimilar sterile milk cans, one of which was visiblyrust-free while the other contained on its innersurface large areas of rust. After preliminarystorage at 0 C, aliquots of each mple wereincubated at 37 C with and without previousinoculation with spores of B. subtilis. Sporecounts at the usual periods provided the desireddata. The results of each experiment are presentedin figure 6. Fint, it may be seen that the dataobtained in milk with added rust (figure 6)were comparable with those observed previouslywith purified soluble iron salts; also, it is evidentthat previous contact of milk with rustingsurfaces even at low temperature actively

promoted the formation of new spores whereasaliquots of the same bulk lot similarly refrigeratedin a rust-free container produced no significantnew spores at 37 C in 5 days. It may be notedthat the iron effects were produced in both thesample in which B. subtilis spores were addedand in the uninoculated sample, indicating thepresence of an iron responsive sporogenic meso-phile in the natural milk flora. The spore pro-moting activity of iron was pronounced in milkdiluted with water 1:10.

The effect of added iron or manganese i-n aeratedand in sealed cultures. The mechanism by whichiron or manganese promotes the formation ofspores in milk and broth solutions is not known.E6 measurements of milk showed the expectedshift to a more positive potential when chloridesof iron or manganese were added; the magnitudeof this change (0.02 to 0.06 v) in the used con-

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AEROBIC SPORES IN FLUID ORGANIC MEDIA

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Figure 7. The effect of added iron or manganese upon the sporulation of Bacillus subtili8 (15u) inmilk contained in open and sealed tubes-iron (200 sg per ml) as FeCis, manganese (2 pg per ml) as MnC12.Temperature of incubation 37 C.

centrations would seem to exclude a per se Eheffect. The possibility that soluble iron ormanganese salts might produce their effects byincreasing the available molecular oxygen was

considered. Two experiments were carried out toprovide information on this point.The effect of iron upon sporulation under

conditions of reduced oxygen concentration was

observed as follows: Eight ml volumes of sterilemilk in standard tubes with and without addediron were inoculated with spores and the samplespreheated and cooled; a portion of the tubes was

sealed quickly with agar containing thioglycolate,and the sealed and unsealed samples incubatedat 37 C, the plate (spore) counts made at theusual periods. In the samples under seal (figure 7),iron or manganese increased the total number ofspores, but the level so attained was muchless than in the open tubes similarly fortified.This result may be attributed to the very

limited oxygen supply of the sealed cultures,which by materially restricting growth militatedagainst the more effective utilization of the

salts. Of interest in figure 7, is the higher yield ofspores in the control under seal than in theabsence of the seal.The effect of added minerals upon sporulation

in aerated milk cultures was studied next.Forty ml volumes of well mixed milk weresterilized in 100 ml glass bottles. Individualsamples were treated then with iron or manganesesolution or an equivalent volume of distilledwater. After preliminary heating the sampleswere cooled to 30 C and shaken continuouslyon a reciprocating shaking machine; unshakensamples treated similarly in all other respectsserved as still controls. Plate (spore) counts weremade at the usual periods. The results (figure 8)show that vigorous and continued aerationincreased the spore level of the unfortifiedsamples, did not greatly affect the spore pro-moting activity of manganese, and measurablyreduced the spore level in the presence of addediron. The reason for the apparent fall in sporeconcentration in the shaken samples, evident in5 days, is not clear but may be due to the slow

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4HAROLD R. CURRAN AND FRED R. EVANS

Mn STILL

Mn SHAKEN -_ -0 Fe STILL .

/ -S- o-~ ~--_ _-- __ C- SHAKEN

Fe SHAKEN

I-/2 STILL

I I24 48 72 96

HOURS AFTER INOCULATION120

Figure 8. The effect of added iron upon the sporulation of Bacillus subtili8 (15u) in aerated (shaken)milk-iron (200 Ag per ml) as FeCls. Temperature of incubation 30 C.

germination of some spores whose developmentis influenced favorably by oxidation of growthinhibiting, metabolic by-products. The lowertemperature of incubation and the alteredvolume-surface ratio of the medium as comparedwith the previous tube cultures changed some-

what the development of spores in the stillcultures.

DISCUSSION

This study is concerned with the effect ofcertain mineral supplements upon the totalnumber of spores formed in complex fluid organicsubstrates. The effects produced by added ironor manganese in substrates which otherwiseproduced abundant vegetative celLs but fewspores invite speculation as to their mechanism ofaction.

Since the addition of 0.1 per cent solublestarch to the substrates did not appreciablyaffect the spore levels, antisporulation factorsare excluded from consideration.

A theory of sporogenesis has been advancedrecently by Hardwick and Foster (1952) basedupon the observed influence of various nutrientsupon the sporulation of washed vegetativecells in distilled water; they conclude that"sporulation results from the endogenousdegradation of pre-existing vegetative (enzyme)proteins which occurs in the absence of readilyutilizable exogenous sources of energy and ex- or

endogenous nitrogen". Since manganese seemsto be essential for substantial proteinase pro-duction by B. subtilis in complex organic media(Stockton and Wyss, 1946) and has an apparentspecific effect upon the sporulation of thisorganism over a wide range of oxygen con-

centration, this element may promote theformation of spores by contributing to thereserve of intracellular enzyme proteins believedby Hardwick and Foster to be the basic materialfor spore synthesis. It is of interest in thisconnection to note that the manganese contentof milk is extremely low (0.002 mg per 100 ml,

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AEROBIC SPORES IN FLUID ORGANIC MEDIA

Bulletin National Research Council, 1950), andthat Difco peptone and beef extract are re-portedly deficient in manganese for proteinaseproduction by B. subtilis (Stockton and Wyss,1946).Iron in contrast to manganese considerably

increased the vegetative population; however,the increment increase of spores was of a differentorder of magnitude. Since, for its maximalactivity, relatively large quantities of iron wererequired in a medium of limited oxygen con-centration, it might be supposed that ferric ironincreased significantly the amount of availableoxygen in the medium; however, this view seemsto be contradicted by the results obtained withferric and ferrous chloride and cyanate (figure 5).The action of iron in promoting the formation ofspores is apparently not direct and thereforenot specific to ferric or ferrous ions or to undis-sociated iron compounds but resides, perhaps, inthe common capacity of iron compounds tocreate for the responding organism a physico-chemical environment suited to the synthesis ofspores.The relationship between contamination of

milk with iron and the development of resistantspores is of manifest interest to the commercialfood canner. It may well explain the occasional,sudden appearance in condensery milk of largenumbers of spores which make sterilizationdifficult and uncertain; such outbreaks frequentlyare not explicable by external contaminationand yet are difficult to reproduce when inocula-tion is made into different milk. Emphasis thusis directed to the elimination of rust-proneequipment in food handling establishments,and since complete exclusion of iron from food isdifficult, new importance is attached to rapidheating and cooling and the maintenance of theproduct at subgrowth temperatures.

ACKNOWLEDGMENT

The authors are indebted to T. J. Mucha forEk determinations.

SUMMARY

Suitable additions of soluble iron or manganesesalts to fluid organic media were found toincrease greatly the total spore yields of Bacillus

subtilis, Bacillus brevis, and Bacillus laterosporus(one culture)-a variety of other salts waswithout significant effect or reduced the sporecrop. Manganese actively promoted sporesynthesis in relatively low concentration andwas effective through a wide range of oxygenconcentration. Comparable increases in sporeyield were obtained by iron supplements, butabout 100 times the concentration of manganesewas required and the stimulation did not occurwhen cultivation was carried out in an excess ofair (oxygen).Exposure of milk to rusting utensils was found

to stimulate the subsequent development ofspores comparable in degree to that obtainedwith purified iron salts. It is concluded that therelationship between iron-rust and spore develop-ment has practical significance in the commercialcanning of milk and other food products.

Possible mechanisms of the action of iron andmanganese are discussed.

REFERENCESBULLETIN NATiONAL RESEARCH COUNCIL 1950

The composition of milk. Published byNational Research Council, National Acad-emy Science, No. 119, Washington, D. C.

CHARNEY, J., FISHER, W. P., AND HEGARTY,C. P. 1951 Manganese as an essentialelement for sporulation in the genus Bacilus.J. Bact., 62, 145-148.

CURRAN, H. R., AND EVANS, F. R. 1945 Heatactivation inducing germination in thespores of thermotolerant and thermophilicaerobic bacteria. J. Bact., 49, 335-346.

FABIAN, F. W., AND BRYAN, C. S. 1933 Theinfluence of cations on aerobic sporogenesisin a liquid medium. J. Bact., 26, 543-558.

FoSTmR, J. W., AND HEILIGmAN, F. 1949 Mineraldeficiencies in complex organic media aslimiting factors in the sporulation of aerobicbacilli. J. Bact., 57, 613-615.

HARDwiCK, W. A., AND FOSTER, J. W. 1952On the nature of sporogenesis in some aerobicbacteria. J. Gen. Physiol., 35, 907-927.

KNAYSI, G. 1945 A study of some environ-mental factors which control endosporeformation by a strain of Bacillus mycoides.J. Bact., 49, 473-493.

STOCKTON, J. R., AND WYSS, 0. 1946 Proteinaseproduction by Bacillus subtilis. J. Bact.,52, 227-228.

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