T'H1E EFFECT OF METALLIC IONS ON THE GROWTH ANDMORPHOLOGY OF CLOSTRIDIUM PERFRINGENS

K. SHANKARI AND R. C. BARDDepartment of Bacteriology, Indiana University, Bloomington, Indiana

Received for publication August 6, 1951

The investigations of Pappenheimer and Shaskan (1944) and Bard andGunsalus (1950) indicate the important role played by iron in the growth andmetabolism of Clostridium perfringens. It is thus desirable to determine theadditional nutritional metallic ion requirements for this organism as a necessarypreliminary step to the investigation of the metabolic functions of other metallicions. Such a project requires at the onset a medium initially free of metallic ions,the latter ingredients being the sole nutritional factors limiting the rate andextent of growth. Use of a synthetic medium for the growth of this organism ispossible (Boyd, Logan, and Tytell, 1948) but such a medium still requirestreatment in some manner to remove the metallic ions present as normal con-taminants of medium ingredients. Moreover, very few reports exist describingtechnics for demetalling media composed of complex natural products, mediastill often employed for obligatory reasons in the study of the growth of numerousorgans.The methods used for the removal of metallic ions from bacteriological media

may be divided into three categories: use of chelating agents, removal of metalsby previous microbial growth in the medium, and by means of ion exchangesubstances. Waring and Werkman (1942) used 8-hydroxyquinoline (oxine) forthe removal of metallic ions from a synthetic medium. Albert et at. (1947) andGale and Mitchell (1949) reported that the oxine technic does not work satis-factorily with complex medium (beef broth). Hutner et al. (1950) recommendedthe use of ethylenediamine tetraacetic acid as a chelating agent to be addeddirectly to the medium; however, details of this procedure have not appeared.Pappenheimer and Shaskan (1944) and Bard and Gunsalus (1950) removed ironby adsorption on calcium phosphate. MacLeod and Snell (1947) removed K+and Mn from medium by growth of Lactobacillus arabinosus and eliminatedMg+ in the samne manner with Saccharomyces carlsbergensis. The medium soobtained was used for evaluating the requirements of these metals for severallactic acid bacteria. Certain cation exchange substances have been used for theremoval of cations from bacteriological media. Perlman, Dorrell, and Johnson(1946) employed the cation exchange resin, "zeo-carb H", for demetalling syn-thetic medium. Later, cation exchange resins were used by Webb (1948) andAbelson and Aldous (1950) for complex and synthetic media, respectively.

In this study "permutit H", a cation exchange resin (available from thePermutit Company), was used for the removal of metallic ions from complex

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medium. With this technic, the metallic ion growth requirements of C. per-fringens were determined both qualitatively and quantitatively, and data arealso presented concerning the inhibitory effects of certain other metallic ions.

MATERIALS AND METHODS

Clostridium perfringens, strain BP6K, from the departmental culture collec-tion, was used in this study and maintained by serial transfer in heart infusionbroth according to procedures described by Bard and Gunsalus (1950). To ob-tain cells for inocula in experiments measuring growth, 1 per cent inoculationfrom 1- to 4-day old heart infusion broth cultures was made into medium A.The latter medium consisted of tryptone, 10 g; yeast extract, 10 g; K2HPO4(anhydrous), 5 g; water, one liter; and was sterilized at 120 C for 20 minutes.Glucose, sterilized by filtration of a 20 per cent solution, was added asepticallyto a final concentration of 1 per cent. Medium A cultures (10 ml) were incubated10 to 12 hr in a 37 C water bath. The cells were separated by centrifugation,washed once in 10 ml sterile water, and suspended in the same volume of water.One drop (about 0.05 ml) of the cell suspension was used for inoculating tubescontaining the growth medium being tested.

All glassware, made of Pyrex glass, was treated for 24 hr with chromic acidsolution and rinsed six times with tap water and three times with distilled water.Final washing was done with high purity water obtained by passing distilledwater through a "deeminizer" (supplied by Crystal Research Laboratory, In-corporated, Hartford, Connecticut) which contains a mixed bed of anionic andcationic resins. This water was used for the preparation of solutions, suspensions,etc. Culture tubes were plugged with nonabsorbent cotton wrapped in cheese-cloth to avoid contamination of the medium with cotton fibers.To prepare the demetalled medium (termed basal medium), medium A was

made in concentrated form by dissolving 21.4 g tryptone and 21.4 g yeast ex-tract in 1,500 ml distilled water. Seven ml of this mixture are equivalent to 10ml of medium A. After heating for 15 minutes at 120 C, the medium was cooledand passed through a column of "permutit H", 20 inches long and 1.25 inchesin diameter, at the rate of 3 ml per minute at room temperature. The first 200ml were discarded and the subsequent 900 ml collected. The latter were heated15 minutes at 120 C, cooled, and then passed through a freshly regenerated col-umn. Resin regeneration was accomplished by passing 400 ml of 0.4 N HCl at arate of 5 to 7 ml per minute and then washing thoroughly with distilled water.The first 350 ml were allowed to run out, and the subsequent 250 ml were col-lected and used after adjustment to pH 7.4 with 1.2 N NH40H. The latter weredistributed in 7 ml quantities and after addition of various solutions (see here-after) the tubes were sterilized for 20 minutes at 120 C. After cooling, 0.5 ml of20 per cent glucose solution was added to each tube and the volume made to 10ml with sterile high purity water. The tubes were heated for 10 minutes in anArnold steamer, cooled, and inoculated with 1 drop of the cell suspension pre-pared from a medium A culture. All culture tubes were calibrated at 10.0 ml,and measurements of growth were made in an Evelyn photoelectric colorimeter

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with a 660 mu filter. The tubes were read after 2, 4, 6, 8, 10, and 24 hours' in-cubation in a 37 C water bath; as the positive control, the growth obtained inmedium A was taken as representing the optimum growth.

All solutions were made with chemically pure reagents. Ferrous sulfate and 20per cent glucose solutions were sterilized by passage through sterile ultrafinesintered glass filters. The buffer solution consisted of 10 g each of Na2HPO4* 7H20and K2HP04 (anhydrous) dissolved in high purity water to 100 ml with 17.1mg Na+ and 44.9 mg K+ present per ml of each solution. In all experimentsother than those concerning the determination of Na+ and K+ requirements,each ml of medium contained 684 and 449 ,ug of Na+ and K+, respectively.Amino acid and growth factor solutions were used at the concentrations de-scribed by Boyd, Logan, and Tytell (1948) for C. perfringens.

Iron was measured by a modification of the method described by Snell andSnell (1936). The procedure involved addition of 1 ml of one per cent hydro-quinone in acetate buffer (100 ml 2 per cent acetic acid and 100 ml 3 per centCH3COONa-3H20, pH 4.5) to the one ml sample. To this was added 1 ml of0.5 per cent a,a'-dipyridyl in the same acetate buffer. The mixture was allowedto stand for 1 hour, diluted to 5.0 ml with high purity water, and the red colorformed measured in an Evelyn colorimeter with a 515 mu filter. A blank wasalways included in which the colorless dipyridyl solution was replaced by water.The values obtained were converted to ,ug of ferrous iron per ml, compared to astandard curve prepared with ferrous iron using Fe(NH4)2(SO4)2.

RESULTS

The subsequent data represent results obtained and confirmed by numerousexperimental trials. Without the addition of certain metallic ions, the metal-deficient basal medium supports no growth. When the ashed constituents oftryptone and yeast extract are added to basal medium, almost optimum growthis obtained. In order to determine which metallic ions are essential for growth,various metallic salts (totaling about thirty) were added to the basal mediumin an attempt to simulate the ash. It soon became apparent, however, that rela-tively few metallic ions are required. By adding different amino acids and growthfactors in groups and then individually, it was observed that only L-lysine,L-arginine, and L-histidine are required as organic supplements to the basalmedium to yield optimum growth (see legend under figure 1). The succeedingdata refer to growth in terms of the optical density of the various cultures after24 hours' incubation at 37 C. After this period of incubation, no increase or de-crease in optical density was noted; most cultures had ceased growing after 8 to10 hours' incubation.

Effecd of Ca++, M++, and Fe++. The individual addition of Ca++ and Mg+to the basal medium yields suboptimal growth whereas addition of Fe+ alonesupports no growth at all (figure 1). Combination of Mg++ with Fe+ results ingrowth equivalent to about 85 per cent of the positive control, but the combina-tion of Ca+ plus Fe++ supports no more growth than Ca++ alone. These resultsclearly indicate the essential requirements for Mg++ and Fe+.

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The data presented in figures 2 and 3 show that addition of Ca++ and Mg++yields progressively greater growth but again indicate that for maxmum growth,Fe++ is necessary. As the Mg++ concentration is increased from 0.0 to 20.0 usgper ml, more growth is obtained at lower Ca++ levels indicating the partial re-placeability of the Ca++ requirement by Mg+. However, the fact that additionof Ca+ is still required at the highest Mg+ concentration tested points to theessential nature of Ca++. In the presence of Fe++ when maximum growth isattained, two combinations of Ca+ and Mg+ are adequate: 1.5 ,ug Ca" and10.0 ug Mg++ or 2.5 pg Ca++ and 7.5 ug Mg++, per ml.

0.8- -POSITIVE CONTROLII_MG+++ F£++

0.6

METALLIC ION COMGTP ONLY

OA ____

ol2 CA++ ONLYCA++ FE+

5 10 ~~15 20

METALLIC ION CONCENTRATION: jAG PER MLFgr .Effect on growthi of Ca++, Mg++, and Fe++ additions in the absence and presence

of Fe++ (2 p&g per ml). The test medium consists of basal (demetalled) medium, 7.0 Ml; L-arginine HCl, 3.0 Mg; L-lysine HCl, 6.2 mng; L-histidine HCl, 3.1 mg; glucose, 100 mg; sodiumand potassium phosphates (see text); water to total volume of 10.0 ml.

Attempts to replace Mg++ by Fe++ at a level of 2.5 pAg Ca++ per ml yieldednegative results. To medium containing 2.5 pg Ca++ and 2.0 pg Mg++ per ml (asuboptimal Mg++ concentration), increasing the Fe++ concentration progressivelyto 5.0 pug per ml failed to stimulate growth above that obtained at the Mg++ andCa++ levels indicated. These results demonstrate that Fe++ can not replace Mg++as a metallic ion growth requirement for C. perfringen2 and are of interest withregard to the role Mg++ plays in determining cellular morphology (see later). Ithas already been shown that Fe+ can not replace Ca++ (figure 3).The effect of various Fe++ concentrations was determined at the optimum

Mg++ and Ca++ concentrations of 7.5 and 2.5 pg per ml, respectively; the data

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z

w

a

0)

0L0

I MG4CNQ8 POSITIVE CONTROL Mra+CONC

0.6 1-0

0.0o l N N

I.l I I I

Z5 5.0 7.5 10.0 1.0 2.0 3.0

CA++ CONCENTRATION: AG PER MLFigure 2 (left). Effect on growth of Ca++ additions at various Mg+ levels, in the absence

of Fe++.Figure (right). Effect on growth of Ca+ additions at various Mg++ levels, in the pres-

ence of Fe+ (2,g per ml).

Q8

nCf)zZ Q6

-J

Ct)0L .1

02

FE++ CONCENTRATION:J.WG PER MLFigure 4. Effect on growth of Fe+ additions in the presence of optimum Ca+ and Mg+

levels.

are presented in figure 4. As may be seen, maximum growth is obtained at about2 ug added Fe++ per ml, and as the Fe++ concentration is increased, some growthinhibition is observed. Measurement of the total iron concentration of the sup-

plemented basal medium (but without added iron) revealed that 0.4 ,ug Fe+

WITHOUT FE++

I-POSITIVE CONTROL

~0- r lIN PRESENCE OMG++ 75 |

0 ~~~CA4+ 2.5 f.Ji.G PER MLS ~ ~~ ~~(A

__ I _ I II 2 3 4 5

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2R. SHANXAR AND R. C. BARD

per ml was present. This value was obtained by ashing 100 ml of this mediumand thus represents total iron (free plus bound). Moreover, the dipyridyl-ironassay method measures iron as Fe++, after reduction of any iron in the Fe+state, and the value obtained, therefore, also includes total iron in this respect.From these data the value of 2 ,ug Fe- per ml merely represents the amount ofadded iron required for maximum growth under the medium conditions obtainedin these experiments. Pappenheimer and Shaskan (1944) reported maximumgrowth at a level of 0.6 ,ug Fe++ per ml under different cultural conditions. Theextents of iron complexing and oxidation probably play significant roles inestablishing the actual concentrations of available Fe++ in different media. It

(n

Zi

4

0

'--.

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go-POSITIVE CONTROL

.6 III

u ~~I I ,C,++IN PRESENCE OFMG++ 7.5

02 CA++ 2.5 jLG PE R ML-FE ++ O.I

MO IIIO 0.1 0.2 0.3GLUTAMINE: MG PER ML

a I pI

0 5 lo Is 20Co++- CONCENTRATION:jj.G /ML

Figu,e 6. Effect on growth of glutamine and Co++ additions in the absence of Fe+ and inthe presence of optimum Ca" and Mg+ levels.

was observed, however, and in agreement with the authors mentioned, that as

the Fe+ concentration was increased, considerably more gas formation accom-

panied the increased growth.Noting the findings of Lerner and Mueller (1949) that the low glucose fer-

menting capacity of iron-deficient Clostridium tetani is restored by the additionof glutamine, the replaceability of iron by glutamine for growth of C. perfringenswas tested. It is observed from the data in figure 5 that glutamine does not re-

place Fe++ for growth of C. perfringens. Co++ also does not substitute for Fe++during growth of this organism and indeed exhibits a considerable growth in-hibitory function. The latter finding is interesting since Bard and Gunsalus(1950) found that Co++ plays a partial role in replacing Fe++ as the metallicion activator of aldolase in C. perfringens but that Co+ exerts a competitive

II GLUTAMINE_imhmE~4 o- O

u6oI_

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METALLIC IONS ON GROWTH AND MORPHOLOGY

inhibitory effect on the maximum activation obtainable with Fe+. The fore-going data with Co++ suggest that a specific site of Co++ action in this organismis the inhibition of the enzyme aldolase which in turn results in decreasedgrowth.

Effects of Na+ and K+. As described in Methods, the concentrations of Na+and K+ used were 684 ,ug and 449 ug of each per ml, respectively; the rationalefor the employment of these concentrations is indicated in table 1. In the absenceof added K+ only limited growth occurs whereas in the absence of added Na+maximum growth is obtained. However, in the latter case an increase in the lagphase of growth was noted, suggesting the requirement for Na+.

In determining the growth effects of Na+ and K+ under conditions of equimolartotal concentrations of these two ions, the data in figure 6 were obtained. It isagain clear that K+ is required for maximum growth whereas the absence ofadded Na+ does not affect total growth. To be noted also are the marked effectsupon cellular morphology as described hereafter.

TABLE 1Effects of Na+ and K+ on growth of Clostridium perfringens

NaHPOO KiHPO0 Na K+ GROWTH

ml per 10 ml medium pg per ml medium optical density

0.5 0.0 855 0 0.190.4 0.1 684 449 0.730.3 0.2 513 898 0.710.2 0.3 342 1347 0.690.1 0.4 171 1796 0.690.0 0.5 0 2245 0.71

Ten per cent solutions of Na2HP04-7H20 and K2HP04 (anhydrous).

Celular morphology. Although no special cytological studies were conductedin this investigation, several observations of cell morphology were made andappear to be related directly to the presence or absence of certain metallic ionsin the medium during growth. Pappenheimer and Shaskan (1944) reported thatiron-deficient C. perfringens is filamentous in shape whereas Webb (1948) ob-served a similar type morphology during growth in magnesium-deficient medium.Bard and Gunsalus (1950) noted no significant changes in the cell shape of thisorganism grown in iron-deficient medium and suggested that metallic ion de-ficiencies other than iron may be responsible for the altered cell morphology de-scribed by Pappenheimer and Shaskan (1944). In this study, it has been observedrepeatedly that magnesium deficiency, in the presence of optimum levels of othermetallic ions, does indeed lead to elongated or filamentous cells. Similarly, growthin the absence of added K+ but in the presence of optimum levels of othermetallic ions results in filamentous cells (figure 6). Such a potassium effect hasalso been noted with Moraxella lwoffi (Lwoff and Ionesco, 1950).As noted before, in the absence of added Na+ total growth is unaffected. How-

ever, the lag phase is prolonged under these conditions and chain formation-3 to

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K. SHANKAR AND R. C. BARD

8 cell-is noted (figure 6). IYom these findings, it is concluded that Na+ too isrequired for the optimum growth of C. perfringens.Study of the morphological changes noted in figure 6 suggests that an optimum

K+:Na+ ratio exists which, upon being changed significantly, leads to alteredcell morphology. It is possible that K+ is required specifically for cell divisionwhereas Na+ is involved in some process leading to the separation of individualcells. In the latter case, this postulated role of Na+ may be antagonized by excess

F-

zw0

0

F:a.0

- POSITIVE CONTROLI I o I

Q7 /l I

0.6 --NORMAL CHAIN

IN PRESENCE OFMG;++ 7.5

0.4 CA++ 2.5 ji#G PER MLFE~ 2.0)

0.3 1- X . 1 1 1

02 } / flILAMENTOUS

IUA %xI+U

5510 zo 30 445 35 25 1

JLM PER ML

Vo15 5 ONA

Figure 6. Effect on growth and cell morphology of Na+ and K+ additions in the presenceof optimum Ca++, Mg+, and Few levels.

K+, and the poor growth obtained at low K+ and high Na+ levels may be anothermanifestation of ionic competition.

Calcium deficiency also leads to morphological alterations. In the absence ofadded Ca+, and especially at suboptimal Mg++ levels, massive cellular ag-gregation occurs. A similar finding has been reported by Foster (1944) withRhodospirillum rubrum. If the Mg++ level is increased to the optimum concen-tration of 7.5 ,Ag per ml, macroscopic clumping is no longer apparent althoughmicroscopic aggregation still persists; the latter disappears only in the presenceof Ca++. As indicated previously (figures 2 and 3) Mg+ can substitute for Ca++to a large degree for an unknown number of processes involved in growth, and

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the partial cellular disaggregation phenomenon is probably another exampleof this substitution role.

Effects of Co++, Mn++, Zn++, and Cu++. Attempts were made to replace theMg++ requirement of C. perfringens with Co++ and Mn++. As may be seen infigures 7 and 8, both Co++ and Mn substitute for Mg++ to a slight degree whenno Mg++ is added to the basal medium. However, addition of Co++ to mediumcontaining a suboptimal Mg++ level (2.5 jig per ml) results in growth inhibitionwhich reaches complete inhibition at 110 ,g Co++ per ml (not shown in figure 7).In the absence of Mg++, only 65 ,ug Co++ per ml are required to inhibit growth

0.8 - POSITIVE'CONTROL POSITIVE 'CONTROL

Q6 H°(1)

zw

0 0

1=

0~

oL

O.C

I

jL,G MG*4PER ML.1: 7~52: 2.53: 0.

2 I... 4~~

)Li~._0~0 5 10 15 0 5 10 s

Co++:,j±G PER ML MN ++:.G PER MLFigure 7 (left). Effect on growth of Co++ additions in the presence of optimum Ca++ and

Fe+ levels at various Mg" concentrations.Figure 8 (right). Effect on growth of Mn " additions in the presence of optimum Cae

and Fe" levels at various Mg++ concentrations.

completely. At the optimal Mg++ level of 7.5 ,g per ml, Co++ inhibition is slight,and addition of 50 Mug Co++ per ml causes only slight inhibition (figure 9). At theCo+ concentration of 100 Ag per ml, 50 Mug Mg++ per ml are required to relievethe almost complete growth inhibition caused by Co++; neither 50 Mug Ca++ nor20 Mug Fe++ per ml relieve the inhibition at this Co+ level. Although Co++ ap-pears to play a partial role in fulfilling the Mg+ requirement for C. perfringensgrowth, it is not required for the growth of this organism, and its presence leadsto inhibition of growth when the Mg++ requirement is satisfied. The interactionapparently occurring between Mg++ and Co+ appears to represent another caseof ion antagonism and emphasizes the need for caution in interpreting the effectsof metallic ions upon growth.

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K. SHANKAR AND R. C. BARD

The effects of Mn++ addition are similar to those with Co ". In the absence ofMg, Mn " substitutes for Mg++ to a very limited extent (figure 8). At a sub-optimal Mg++ level (2.5 ug per ml), Mn " stimulates growth slightly, appearingto substitute partially for Mg-. But as the Mn " concentration is increased inthe presence of Mg++, Mn+ inhibits growth to approximately the same extentas Co++ (figure 9). Thus, r&,+ is not required for the growth of C. perfringensand at high levels is slightly gtowth inhibitory.The data in figure 9 indicate that Zn inhibits growth only slightly whereas

Cu § is strongly inhibitory. At a Cu++ level of 50 ,g per ml, increasing the Mg++

'I)z

LdJO

ct

F:

10 20 30 40 50METALLIC ION CONCENTRATION:).LG PER ML

Figure 9. Effect on growth of Zn++, Co++, Mn+, and Cut additions in the presence ofoptimum Ca++, Mg+, and Fe++ levels.

concentration to 100 ug per ml does not overcome the inhibition due to Cu++,nor do 50 ,ug Ca+ per ml or 20 pg Fe++ per, ml. The inhibition due to Cu " maybe due to an irreversible reaction, perhaps combination with sulfhydryl groups,and none of the metallic cations tested can compete with the undissociated Cu++complex formed. The interesting observation was made that in the presence ofCu"+ curved, vibrio-like cells are formed and the addition of Mg+, Ca++, orFe-++ does not alter this peculiar morphological effect.

DISCUSSION

The present state of knowledge concerning the specific roles played by metallicions in microbial growth has not attained sufficient volume nor breadth to permita significant discussion of the subject. This situation is quite analogous to the

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one concerned with growth factors little more than a decade ago. It is well toaccumulate data concerning the nutritional requirements of microorganisms formetallic ions, but these needs are best understood in terms of specific metabolicsites of ionic activity. The experimental approach described before permits suchstudy. It is now possible, at least in the case of C. perfringen8 and a few otherbacterial species, to cultivate metal-deficient cells whose metabolism may thenbe investigated. Any metabolic differences may be ascribed to the particular stateof metal deficiency, either directly or by one or many steps removed. Such anapproach has yielded interesting results in the case of iron-deficient C. per-fringens (Pappenheimer and Shaskan, 1944; Bard and Gunsalus, 1950), and thereis every indication that future efforts along these lines will prove equally fruitful.

ACKNOWLEDGMENTS

The authors take this opportunity to thank the Permutit Company, New York,New York, and Rohm and Haas, Philadelphia, Pennsylvania, for several gener-ous samples of ion exchange resins made available to them. One of us (K. S.)expresses his gratitude to Indiana University for making it possible to continuestudies in this country after the expiration of his India Government Scholarship.The work reported herein was supported in part by a grant from the GraduateSchool, Indiana University.

SUMbARY

Using a cationic exchange resin ("permutit H"), a metal-deficient complexmedium was obtained and employed to determine the major metallic ion growthrequirements of Clostridium perfringens. For optimum growth, Ca++, Mg++,Fe++, Na+, and K+ are required but not Zn++, Mn++, Co++, or Cu++. None ofthe latter metallic ions can replace those required for growth.

In the absence of Ca++, cells grow in an aggregated state whereas Mg++ or K+deficiency results in the growth of filamentous cells. In the presence of Cu++,which is growth-inhibitory, curved cells of normal size are obtained.

REFERENCESABELSON, P. H., AND ALDOUs, E. 1950 Ion antagonisms in microorganisms: interference

of normal magnesium metabolism by nickel, cobalt, cadmium, zinc, and manganese.J. Bact., 60, 401-413.

ALBERT, A., RUBBO, S. D., GOLDACRE, R. J., AND BALFOUR, B. G. 1947 The influence ofchemical constitution on anti-bacterial activity. Part III: A study of 8-hydroxyquino-line (oxine) and related compounds. Brit. J. Exptl. Path., 28, 69-87.

BARD, R. C., AND GUNSALUS, I. C. 1950 Glucose metabolism of Clostridium perfringens:existence of a metallo-aldolase. J. Bact., 59, 387-400.

BOYD, M. J., LOGAN, M. A., AND TYTELL, A. A. 1948 The growth requirements of Cloa-tridium perfringens (welchii) BP6K. J. Biol. Chem., 174, 1013-1025.

FOSTER, J. W. 1944 Oxidation of alcohols by nonsulfur photosynthetic bacteria. J.Bact., 47, 355-372.

GALE, E. F., AND MITCHELL, P. D. 1949 The assimilation of amino-acids by bacteria. 8.Trace metals in glutamic acid assimilation and their inactivation by 8-hydroxyquino-line. With a note on relative dissociation constants of some metal-oxine complexes.J. Gen. Microbiol., 3, 369-386.

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HUTNER, S. H., PROVASOLI, L., SCHATZ, A., AND HASKINS, C. P. 1950 Some approachesto the study of the role of metals in the metabolism of microorganisms. Proc. Am.Phil. Soc., 94, 152-170.

LERNER, E. M., AND MUELLER, J. H. 1949 The role of glutamine in the glucose metabo-lism of Clostridium tetani. J. Biol. Chem., 181, 43-45.

LWOFF, A., AND IONESCO, H. 1950 L'ion potassium et la croissancede labact6rie MoraxellaIwoffi. Ann. inst. Pasteur, 79, 14-19.

MACLEOD, R. A., AND SNELL, E. E. 1947 Some mineral requirements of the lactic acidbacteria. J. Biol. Chem., 170, 351-365.

PAPPENHEIMER, A. M., JR., AND SHASKAN, E. 1944 Effect of iron on carbohydrate metabo-lism of Clostridium welchii. J. Biol. Chem., 156, 265-275.

PERLMAN, D., DORRELL, W. W., AND JOHNSON, M. J. 1946 Effect of metallic ions on theproduction of citric acid by Aspergillus niger. Arch. Biochem., 11, 131-143.

SNELL, F. D., AND SNELL, C. T. 1936 Colorimetric methods of analysis. Vol. I. VanNostrand Company, New York, New York.

WARING, W. S., AND WERKMAN, C. H. 1942 Growth of bacteria in an iron-free medium.Arch. Biochem., 1, 303-310.

WEBB, M. 1948 The influence of magnesium on cell division. 1. The growth of Clos-tridium welchii in complex media deficient in magnesium. J. Gen. Microbiol.,2, 275-287.

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