NUTRITION AND METABOLISM OF MARINE BACTERIA

II. OBSERVATIONS ON THE RELATION OF SEA WATER TO THE GROWTH OF MARINE BACTERIA1

ROBERT A. MAcLEOD AND E. ONOFREYThe Fisherie8 Research Board of Canada, Pacific Fisheries Experimental Station, Vancouver, B.C.

Received for publication October 21, 1955

Marine bacteria have been arbitrarily definedas bacteria from the sea which on initial isolationrequire for growth a medium containing sea wateras the diluent (ZoBell and Upham, 1944). Thefunction of sea water in media for the growthof these organisms has never been clearly estab-lished. It has been stated, however, that neitherisotonic salt solution nor artificial sea water is asgood as natural sea water for the cultivation ofrecently isolated marine bacteria (ZoBell, 1946).The nutritional requirements of a number of

marine bacteria have been determined recently(MacLeod et al., 1954). In the course of thisstudy it was found that a mixture of inorganicsalts approximating the composition of sea watercould be used in place of natural sea water in achemically defined medium suitable for thegrowth of these organisms. A limited amount ofinformation regarding a requirement of the bac-teria for specific inorganic ions was also obtained.The present report presents the results of a

more detailed study of the relation of the con-stituents of sea water to the growth of marinebacteria.

MEHODS

Cultures. The sources of the organisms andthe general methods used in their study havebeen described (MacLeod et al., 1954). Six of theorganisms used in the previous study wereselected for this work. The organisms used havebeen tentatively identified as B9, a flavobac-terium; B10 and B20, corynebacteria; B16 andB26, mycoplana; and B30, a pseudomonad.Their organic growth requirements are knownand were reported in the previous communica-tion. The organisms all satisfy the criteria estab-lished for classifying them as marine bacteria,

1 A preliminary report of these findings wasmade at the Ninth Science Conference, B.C.Academy of Science, University of British Co-lumbia, April 28 and 29, 1955.

that is, on initial isolation they grew in a complexmedium prepared with sea water, but not withfresh water.

Inocula. The inoculum medium used has beendescribed previously (MacLeod et al., 1954). Inthis study cells were washed by centrifuging,removing the supernatant liquid and resus-pending them in 1.097 M glycerol solution. Thiswashing operation was repeated twice and onedrop of the final suspension used to inoculateeach flask. The concentration of glycerol in thewash solution was equal to the total molar ionconcentration of sea water.

Basal medium.' This medium was the simplestcapable of supporting the growth of all six or-ganisms. All solutions were prepared using waterdemineralized by passage through a bed of ionexchange resins (Bantam demineralizer, Barn-stead Still & Sterilizer Co., Boston, Mass.).The inorganic salts used were of reagent gradequality and were employed after recrystallizationfrom demineralized water. The three amino acidswere recrystallized from a solution adjusted topH 2 to 3 with HCI. Glucose was purified by con-secutive passage through "amberlite IRC-50"(H form) and "amberlite IR-4B" (OH form) ionexchange resin coluimns. Glycerol was distilledunder vacuum in an all-glass apparatus beforeuse. The NH40H and HCI solutions for neu-tralizing the medium were prepared by dissolvingNH8 and HCI gases, respectively, in de-mineralized water.Assay procedure. The procedures used in this

study differed from those employed previously(MacLeod et al., 1954) in only one importantrespect. In this investigation the growth responsein a particular assay was measured at frequent

'Composition of the basal medium (in mg per10 ml of final medium): Glucose, 30 mg; DL-a-alanine, 24 mg; DL-aspartic acid, 9.6 mg; L-glu-tamic acid, 12 mg; (NHi)2SO4, 8.7 mg;(NH4)2HPO4, 1.3 mg.

661

on June 23, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

MAcLEOD AND ONOFREY

TABLE 1Comparison of the ability of natural sea water, artificial sea water and fresh water, when each is acting as

the diluent in a chemically defined medium, to promote the growth of six marine bacteria

Organism Tested

|Fe++ | B9 B1O B16 B20 B26 | B30

Diluent Strength Added Incubation time (hr)

71 1 144 71 144 71 1441 71 144 71 1 1441 71 144

Per cent incident light transmittedt

psg/JO ml

Natural sea water .. . Full* 0 78 75 100 86 72 50 16 - 93 83 100 85Natural sea water ........ Full 50 19 21 90 21 28 30 2 - 87 29 98 80Natural sea water........ Half 0 66 53 58 36 73 59 36 - 68 51 58 35Natural sea water........ Half 50 19 20 5 24 30 50 27 - 19 20 29 49Artificial sea water....... Full 0 54 50 97 70 77 52 56 - 92 82 93 80Artificial sea water....... Full 50 25 23 100 85 56 31 11 - 89 71 92 67Artificial sea water....... Half 0 72 68 34 71 82 56 30 - 64 56 65 48Artificial sea water....... Half 50 19 21 12 50 32 62 28 - 16 22 21 49Distilled watert.e - 100 100 100 100 100 100 100 - 100 100 100 100

* Salinity: 35.4 g total salts per kg.t Evelyn colorimeter readings, 660 mlA filter, uninoculated blank = 100.

intervals during incubation instead of after a fixedperiod of incubation. The variation in pro-cedure was made possible by the use of 50 mlErlenmeyer flasks, to each of which a matched18 by 150 mm pyrex culture tube had beenattached. By tilting the contents of the flask intothe side tube at appropriate intervals, andinserting the side tube into a colorimeter, meas-urements of the change in turbidity of a culturewith time could be made without danger of con-tamination (Sherman and Grant, 1951). Unlessotherwise indicated, the results reported foreach organism in each experiment were obtainedafter a period of incubation which resulted in theachievement of maximum growth by theorganism. Certain of the organisms, however,autolyzed very rapidly after growth in the tubeshad ceased. If growth of the latter organisms wasslower in some tubes than in others, often somecultures would be autolyzing with a consequentlydecreasing turbidity while others were stillgrowing. In these cases, results in the particularexperiment were reported after a period ofincubation which represented the best possiblecompromise between those cultures which werestill growing and those which were autolyzing.

Salt solutions. The natural sea water used inthese experiments was collected in the vicinityof Departure Bay, Vancouver Island. We areindebted to the Pacific Biological Station for

making the sample available to us. Despite thefact that the sea water collection was made asfar from land as possible, the salinity was belowthat considered average for the open sea. The seawater used was therefore evaporated to thedesired salinity (35 to 40 g of total salts per kg)before use.For the composition of the artificial sea water

used in these experiments, see Lyman andFleming (1940).3

RESULTS

The comparative ability of natural sea water,artificial sea water and fresh water when each isacting as a diluent in a chemically defined mediumto promote the growth of the six marine bacteriainvestigated is shown in table 1. In this table, theresults are reported after two periods of incuba-tion except in the case of B20, which had reachedmaximum growth in all tubes in 71 hours. Incertain cases it will be noted that the turbidityof a culture was less after 144 hours than after71. This loss in turbidity on incubation was dueto autolysis. The results show that growth ofthe organisms occurred in media prepared with

' Composition of artificial sea water used: NaCl,23.476 g/kg; Na2SO4, 3.917 g/kg; NaHCO3, 0.192g/kg; KCI, 0.664 g/kg; KBr, 0.096 g/kg; MgCl2,4.981 g/kg; CaCl2, 1.102 g/kg; SrCl2, 0.024 g/kg;HaBO,, 0.026 g/kg.

662 [VOL. 71

on June 23, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

SEA WATER AND MARINE BACTERIAL GROWTH

both natural and artificial sea water, but notwith fresh water. Relatively poor growth of allorganisms except B20 was obtained in the mediasupporting growth unless a supplement of ironwas added. The table also shows that for all ofthe organisms except B20 both the rate andextent of their growth was increased by usinghalf rather than full strength sea water, ir-respectively of whether the sea water used was

natural or artificial. When both natural andartificial sea water were supplemented with ironand tested at half strength, there was no sig-nificant difference in the ability of the twodiluents to promote growth of any of the or-

ganisms. In separate experiments it was alsonoted that natural and artificial sea water couldbe used interchangeably for growth of the marinebacteria in the complex medium used in theirisolation. It would thus appear that the observeddependence of the marine bacteria investigatedhere on the presence of sea water in the mediumon initial isolation was due to the ability of sea

water to supply the inorganic environment re-

quired by the organisms for growth. There is noevidence that sea water was needed to supplyunknown organic factors necessary or stimulatoryfor the growth of these marine bacteria.When various groups of salts were omitted in

turn from artificial sea water, the growth re-

sponses obtained with the various organisms are

shown in table 2. It is evident that removingeither Nat or K+ salts completely prevented

growth in all cases. When Mg++, Ca++ and Sr++salts were omitted, four organisms failed to growand two grew only slightly. The removal of H3BOsdid not significantly affect the growth of any ofthe organisms. Since removing various salts,particularly the Na+ and Mg++ salts, wouldrender the solution hypotonic, the osmotic pres-

sure of the solution was restored by adding a

level of glycerol equal to the molar concentrationof the total number of ions removed. In no case

did the addition of glycerol permit growth of theorganisms in the absence of the salts. It was

established in separate experiments that none

of the organisms could utilize glycerol as a source

of carbon in the medium, and that cells harvestedfrom a medium containing glycerol were unableto oxidize glycerol in a Warburg respirometer.

Since each of the omissions shown in table 2involved more than one salt, it was of interestto know whether more than one salt in eachgroup was required for growth by the organisms.Three sodium salts, NaCl, Na2SO4, and NaHCO3,are used in the preparation of the artificial sea

water.3 NaCl and Na2SO4 were each added sepa-

rately to a medium deficient in Na+ salts at a

level which provided Na+ at the concentrationnormally made available by the mixture.NaHCOs could not be tested at this level becauseof the alkalinity of the resulting solution. Theresults (table 3) show that either salt was capableof satisfying the Nat salt requirements of all ofthe organisms if the period of incubation was

TABLE 2Growth response of marine bacteria in the presence of glycerol when various salts are removed from the

artificial sea water used as a diluent in the medium

Organism Tested

Composition of Artificial Sea Water* Glycerol B9 Blo I B16 I B20 B26 B30

Per cent incident light transmittedt

Complete.|- | 19 22 26 32 15 15No Na+salts. 100 100 100 100 100 100No Nae salts.+ 100 100 100 100 100 100No K salts................................. - 100 100 100 100 100 100No K+ salts.................................. + 100 100 100 100 100 100No Mg-', CaIIor Sr& salts.................. - 100 100 100 100 91 81No Mg++, Ca++ or Sr++ salts.................. + 100 100 100 100 91 86No HsBO. 19 13 24 28 15 17

* Supplemented with Fe+ 50 Ag per flask.t The molar concentration of glycerol added in each case was equal to the molar concentration of the

total number of ions removed.t See table 1. Incubation times: B10 and B20, 40 hr; B9, B16 and B30, 48 hr; B26, 72 hr.

1956 663

on June 23, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

MAcLEOD AND ONOFREY

TABLE 3The ability of single 8alt8 to replace groups of saltsfor the growth of marine bacteria in artifial sea

water medium

Organism Tested

Composition of Artificial Sea Water* Additions B9 B1O I B16 I B20 j B26 I B30

Per cent incident light transmittedt

Complete .......................... 19 6 24 26 17 17No Na+ salts 100 100 100 100 100 100No Na+ salts....................... NaCl (2.29 mM) 18 6 27 7 16 19No Na+ salts ....................... Na2S04 (1.145 mM) 20 16 28 100§ 16 16No Na+ salts....................... KCl (2.29 mM) 100 100 100 100 100 100No K+ salts 100 100 99 97 100 97No K+ salts........................ KCl (48.6 Amoles) 18 5 23 19 16 17No K+ salts ........................ KBr (48.6 pmoles) 20 4 23 26 16 17No K+ salts........................ NaCl (48.6 ;moles) 100 100 100 100 100 100No Mg++, Ca++, or Sr" 100 99 98 99 95 91NoMg+ 100 99 96 99 68 83NoCa+.......................... 96 95t 21 14 16 17No Sr r 19 16 23 11 16 17

* See table 2.t See table 1. Incubation times: B20, 40 hr; B10, 48 hr; B16 and B30, 66 hr; B9 and B26, 90 hr.$ Growth after 90 hr, 32.§ Growth after 120 hr, 28.

sufficiently long. Maximum growth was achievedmuch more rapidly in some cases, however, withNaCl than with Na2SO4. This was particularlynoticeable in the case of B20, where maximumgrowth in the presence of Na2SO4 was not reacheduntil after an incubation period of 120 hours. Alevel of KCl equal to that of the NaCl added didnot permit growth of the organi. Thesefindings indicate that lack of growth in theabsence of Na+ salts was due primarily to adeficiency of Na+ in the medium.Two K+ salts are present in artificial sea

water, KCl and KBr. Growth resulted when thetotal K+ provided by these two salts was madeavailable by either one, but not when the K+salts were replaced by equimolar concentrationsof NaCl (table 3). These results indicate that,in addition to a requirement for Nat, all of theorganisms tested need K+.When Mg+, Ca++, and SrK salts were omitted

from the medium, little or no growth of anyof the organisms was obtained. A deficiency ofMg++ alone prevented growth of four of theorganisms and greatly restricted the growth ofthe other two. Without added Ca++, B9 wouldnot grow, while B10 grew only after a relativelylong incubation period. A deficiency of Sr++ hadno significant effect on the growth of any of the

organisms. One can conclude, then, that at leastfour and probably all of the organisms testedhave an absolute requirement for Mg++ forgrowth, while one needs Ca++ as well.A salt solution was prepared4 which provided

those ions in sea water which this study hasshown to be required for the growth of the sixmarine bacteria. Na+, K+, Mg++, and Ca++ wereeach added to the medium at the level at whichthey are present in one-half concentrated artificialsea water. Growth of the bacteria in a mediumprepared with this salt solution was comparedwith growth when half strength artificial seawater (supplemented with Fe++) was used as thediluent. The results (table 4) reveal that growthin the medium prepared with the salt solution wasin every case equal to or better than growth inthe medium prepared with artificial sea water. Asupplement of yeast extract ash was also addedto tubes containing the salt solution to determinewhether further growth could be obtained bysupplying traces of other inorganic ions. In thecase of only one organism, B16, was any ap-

4Composition of salt solution containing ionsfound to be required for growth by the marinebacteria studied: NaCl, 12.705 g/L; KCI, 0.72g/L; FeSO4(NH4)2S04, 0.0254 g/L; MgCl1, 2.49g/L; CaC12, 0.551 g/L.

664 [VOL. 71

on June 23, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

SEA WATER AND MARINE BACTERIAL GROWTH

TABLE 4Comparison of the growth response of marine

bacteria in media prepared with artifial seawater and with salts providing ions known to berequired for growth

Organism Tested

Diluent B9 1B101B161B20 IB261B30Per cent incident light

transmitted*

Artificial sea H20 (halfstrength + Fe-+ (50 ogper 10 ml) ........... 19 12 32 32 43 18

Salt solution ........... 20 16 31 29 16 16Salt solution + 1 ml yeastextract ash ........... 17 23 23 40 15 15

* See table 1. Incubation times: B20, B26, andB30, 66 hr; B9, BlO, and B16, 71 hr.

preciable stimulation obtained when yeast extractash was added.

Since phosphorus is intimately involved in themetabolism of all living forms so far examined,and as sulfur amino acids are normal constituentsof proteins, one would expect that marine bac-teria would require sources of both phosphorusand sulfur in the medium for growth. To test theneed for a source of phosphate, a phosphatedeficient medium was prepared by replacing the(NH4)2HP04 used to introduce phosphate intothe medium with an equivalent amount of(NH4)2SO4. None of the organisms grew to more

than a very limited extent in this medium unless(NH4)2HP04 was added back (table 5). Theaddition of the same amount of NH4+ introducedas (NH4)2S04 did not permit growth, indicatingthat the response to (NH4)2HP04 was due to thephosphate it supplied. When the sulfate saltsin the medium were replaced by chloride, two ofthe organisms, B20 and B30, grew to an appreci-able extent. Even in the case of these organisms,

however, the addition of K2S04 to the S04--deficient medium greatly improved growth. Theresults indicate that four of the organisms defi-nitely require S04-- for growth, and two othersprobably require it also, although in amountscapable of being partially satisfied by contami-nating traces present in the medium.To determine if chloride was required for

growth by any of the organisms, a medium was

prepared using a salt mixture in which all of thechloride salts with the exception of Ca+, whichwas added as the nitrate, were replaced by theircorresponding sulfates. The yeast extract nutrientbroth medium used to grow the inoculum was

prepared, using this same chloride deficient saltmixture. The growth response of the organisin the chloride deficient basal medium is re-

corded in table 5. In the case of only one or-

ganism, B9, was no growth obtained in thismedium unless NaCl was added. For another,B20, the maximum growth achieved in theabsence of chloride was significantly less thanwas obtained in its presence. For the other or-

LBLE 5Some anion requirements of marine bacteria

Organisms Tested

Medium* Additions Per 10 MI B9 j B1O I B16 I B20 I B26 I B30

Per cent incident light transmittedt

P04-- deficient None 98 100 100 88 95 96(NH4)2HP04 11.3,moles 18 10 26 10 15 14(NH4)2504 11.3 jmoles 97 99 100 88 98 94

SO4 deficient None 100 100 100 57 97 66K2SO4 66.0 JAmoles 19 10 22 12 14 15KCl 132.0 pmoles 99 100 100 98 92 87

C1- deficient None 100 40 26 69 17 29NaCl replacing Na2SO4 16 34 23 30 15 222.89 mm NaCl 18 23 21 15 30 71

* The salt solution of footnote 4 was used in this medium with modifications as described in the text.t See table 1. Incubation times: For the P047- and SO4 experiments: B20, 24 hr; B9, B10, B16,

B30, 48 hr; B26, 72 hr. For the C1- experiment: B20 and B30, 48 hr; B10, B16, and B26, 72 hr; B9, 96 hr.

66519561

on June 23, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

MAcLEOD AND ONOFREY

ganisms studied, growth was either not affectedor only delayed by the absence of added chloride.It was necessary to establish whether or notgrowth of B9 and B20 on the addition of NaClto the C1- deficient medium was due to theintroduction of Cl- or to an increase in eitherthe Na+ concentration or the total ionic strengthof the solution. Since adding further Na2SO4to the medium at a level equivalent to the addedNaCl would have introduced a toxic level of SO4--,Na2SO4 present in the basal medium was replacedby a level of NaCl providing the same amount ofNa+. Although this did not provide two solutionsof exactly the same ionic strength, it did provideanother medium containing C1- ion in whichneither an increased ionic strength nor addi-tional Na+ could be the factor determininggrowth. Again, maxmum growth of B9 and B20occurred in the medium containing the chlorideion. It is thus evident that B9 requires Cl- forgrowth. It would appear also that B20 has anabsolute requirement for C1- but that theamounts needed are less than are required by B9,and can be partially provided for by contaminat-ing traces of chloride present in the medium.

ZoBell has reported that, after laboratory cul-tivation on sea-water media for periods of 2 to 12years, 56 of 60 species of marine bacteria de-veloped the ability to grow on fresh water media(ZoBell, 1946). It would appear from this ob-servation that marine bacteria lose comparativelyreadily the one characteristic which has beenused to distinguish them from land forms. Sinceit would be of interest to know what changes inspecific ion requirements would be representedby such a marked change in salt requirements,an attempt was made to train two of the marinebacteria used in this study to grow in a mediumprepared with fresh water. ZoBell's observationswere made using a complex laboratory mediumwhich would be expected to supply trace elementsin amounts sufficient for the growth of mostterrestrial bacteria. In the training studiescarried out here a medium of similar complexitywas used, i.e., yeast extract, 0.5 per cent; nutrientbroth, 0.8 per cent. If full strength sea waterwas used as the diluent in this medium, the seawater concentration was considered to be 100per cent. Lower sea water concentrations wereobtained by diluting the diluent appropriatelywith fresh water. Initially, the minimum concen-trations of sea water in the medium supportingjust visible growth of the two organisms studied

were 9 and 12 per cent, respectively. After seriallysubculturing the organisms into media containingprogressively lower concentrations of sea water,cultures were eventually obtained which couldproduce just visible growth at sea water concen-trations of, in one case, 3.25 per cent, and inthe other, 3.2 per cent. The organisms, whengrown at these low concentrations, however,autolyzed very rapidly and failed to survive morethan 1 or 2 subcultures into media of the samesea water concentrations. Repeated attempts toobtain growth at still lower sea water concentra-tions were unsuccessful.

DISCUSSION

Marine bacteria have been differentiatedfrom land forms on the basis of their need forsea water rather than fresh water for growth ina complex laboratory medium on initial isolation.The results of this investigation reveal that, atleast for the marine bacteria studied here, therequirement for sea water in the medium is basedon the capacity of sea water to supply the kindsand amounts of inorganic ions required forgrowth by the organisms. In addition it has beenobserved that none of the organisms investigatedhere has gained the capacity to grow in a complexmedium prepared with fresh water, even after2% years of cultivation on laboratory media.An attempt to develop such a capacity in two ofthe organisms by the application of trainingtechniques was unsuccessful.There are two unusual features of the qualita-

tive mineral requirements of the marine bacteriarevealed by this study. One is the fact that all ofthe organisms investigated require Na+ forgrowth and two of them need Cl-. Only one or-ganism from a terrestrial source, the red halophilePseudomona salinaria, has been reported torequire Na+ for growth (Brown and Gibbons,1955), although the presence of Na+ in themedium improved the response of Lactobacillusarabinos to pantothenic acid (Simy et al.,1954) and shortened the lag phase in the growthof Clostridium perfringens (Shankar and Bard,1952). True marine bacteria may well prove to bedistinguishable from land forms present as con-taminants in sea water not by having a require-ment for sea water but rather by having a readilydetectable need for Nat in the medium forgrowth.The only other report of a Cl- requirement

for the growth of bacteria is that for the halophile

666 [VOL. 71

on June 23, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

SEA WATER AND MARINE BACTERIAL GROWTH

P. salinaria (Brown and Gibbons, 1955). ThatC1- can be involved in the metabolism of micro-organisms in capacities other than thoseconcerned with growth, however, is apparentfrom the fact that two antibiotics produced bymicroorganisms-chlortetracycline (Broschard etal., 1949) and "chloromycetin" (Ehrlich et al.,1947)-contain chlorine.Sea water does not contain sufficient iron to

permit the marine bacteria investigated to groweither at their maximum rate or to the fullestextent. This, of course, applies only under thecultural conditions used in the laboratory. Intheir natural environment, the majority ofmarine bacteria are believed to be attached tothe surfaces of larger organisms and to particlessuspended in the sea. These surfaces, by adsorp-tion, would be able to concentrate nutrientssufficiently to enable better growth of the marinebacteria to take place than would occur if thebacteria lived free in the sea (ZoBell, 1946).

SUMMARY

The need of six marine bacteria for sea waterin a chemically defined medium has been shownto be due to the ability of sea water to supplythe inorganic ions required for growth by theorganisms. No evidence was obtained that seawater was capable of supplying unknown organicfactors either required by or stimulatory for thegrowth of these organisms.A supplement of iron in the sea water medium

increased both the rate and the extent of growthof all of the organisms tested. None of the or-ganisms grew unless Na+ and K+ were added tothe medium. Additions of Mg++ were requiredby four of the organisms and were stimulatoryfor the growth of the other two. Ca++ was re-quired by one organism and stimulated the earlygrowth of another. None of the organisms grewsignificantly without P04--- in the mediumwhile the absence of SO4-- prevented the growthof four organisms and reduced the amount ofgrowth of the other two. One organism required

Cl-, the growth of another was limited by its ab-sence, while the remainder either were unaffectedby its absence or needed Cl- for optimum rate ofgrowth. Attempts to train two of the marine bac-teria studied to grow in a complex mediumprepared with fresh water instead of artificial seawater were unsuccessful.

REFERENCES

BROSCHARD, R. W., DORNBUSH, A. C., GORDON, S.,HUTCHINGS, B. L., KOHLER, A. R., KRUPKA, G.KUSHNER, S., LEFEMINE, D. V. AND PIDACKS,C. 1949 Aureomycin, a new antibiotic.Science, 109, 199-200.

BROWN, H. J. AND GIBBONS, N. E. 1955 Theeffect of magnesium, potassium and iron onthe growth and morphology of red halophilicbacteria. Can. J. Microbiol., 1, 486-493

EHRLICH, J., BARTZ, Q. R., SMITH, R. M. ANDJOSLYN, D. A. 1947 Chloromycetin, a newantibiotic from a soil actinomycete. Science,106, 417.

LYMAN, J. AND FLEMING, R. H. 1940 Composi-tion of sea water. J. Marine Research (SearsFoundation), 3, 134-146.

MACLEOD, R. A., ONOFREY, E. AND NORRIS, M. E.1954 Nutrition and metabolism of marinebacteria. I. Survey of nutritional require-ments. J. Bacteriol., 68, 680-686.

SHANKAR, K. AND BARD, R. C. 1952 The effectof metallic ions on the growth and morphologyof Clo8tridium perfringens. J. Bacteriol., 63,279-290.

SHERMAN, F. G. AND GRANT, B. M. 1951 Aculture flask for the estimation of the growthof micro-organisms by optical methods. J.Lab. and Clin. Med., 37, 325-326.

SIRNY, R. J., BRAEKKAN, 0. R., KLUNG50YR, M.AND ELVEHJEM, C. A. 1954 Effects of potas-sium and sodium in microbiological assaymedia. J. Bacteriol., 68, 103-109.

ZOBELL, C. E. AND UPHAM, H. C. 1944 A list ofmarine bacteria including descriptions ofsixty new species. Bull. Scripps Inst.Oceanog., Univ. Calif., 5, 239-292.

ZOBELL, C. E. 1946 Marine microbiology.Chronica Botanica Co., Waltham, Mass.

66719561

on June 23, 2020 by guesthttp://jb.asm

.org/D

ownloaded from