ISOLATION AND CHARACTERIZATION OF A LARGE CELLPOSSIBLY POLYPLOID STRAIN OF ESCHERICHIA COLI'

JAMES E. OGG2 AND M. R. ZELLELaboratory of Bacteriology, Cornell University, Ithaca, New York

Received for publication April 18, 1957

A constantly increasing spectrum of bacterialmutations is under active investigation and theutility of bacterial mutation systems in studiesof mutagenesis has been amply demonstrated.Once bacteria were considered to be asexuallyreproducing organisms, but today at least threedistinct mechanisms of gene recombination areknown. The last decade has seen the introductionof many new techniques and new ideas intobacterial cytology, and although substantialprogress may have resulted, there still is nogeneral agreement as to the detailed structureand composition of the bacterial nucleus (De-Lamater, 1954; Knaysi, 1956; Robinow, 1956).Indeed, an active controversy is in progressconcerning the presence or absence of a mitoticcycle similar to that in higher organisms and theorganization of the chromatinic material intodiscrete chromosomes which behave duringdivision as do chromosomes in higher plantsand animals.

Lederberg et al. (1951) made comparativecytological studies of strains of Escherichia coliK-12 known to be haploid and diploid on thebasis of genetic evidence. Although they observedcertain characteristic differences in the cyto-logical appearances of the two strains, theirpreparations did not permit any clear interpreta-tion in terms of doubled chromosomes. A corre-lation between the ratio of uninucleate andmultinucleate E. coli strain B and E. coli strainB/r cells in different portions of the growthcurve and the ratio of intact to sectored lactose-negative, ultraviolet induced mutant colonieswas observed by Witkin (1951) who also noted acorrespondence between the modal number ofnuclei per cell in her different samples and themodal size of mutant sector. Her observations

1 This work has been supported jointly byCornell University and the Atomic Energy Com-mission through research contract No. AT(30-1)-1244.

2 Present address: Fort Detrick, S.O. Division,Frederick, Maryland.

show clearly that sectored mutant coloniesresult from mutation in only one of severalnuclei present in the cell. It is interesting thatthese observations of Lederberg et al. and Witkinprovide almost all of the direct evidence thatthe nucleus which has been the source of somuch controversy amongst bacterial cytologistsmay actually have a genetic function.The mass of data, on bacterial mutation

frequencies and expressZon, and the evidence fromgenetic recombination have shown conclusivelythat E. coli is normally hai3loid. Despite the factthat the cytological studies of haploid and di-ploid E. coli strains of Lederberg et al. (1951)were indecisive, such -comparative cytologicalinvestigations would°dm to be one of the mostcritical and most objective approaches to thequestion of the basic organization of the bac-terial nucleus and to the possible identificationof bacterial chromosomes. In addition, stablepolyploid strains would be of value in analysis ofthe mechanisms of radiobiological effects incomparative studies with their haploid parents.In view of their potential usefulness, an attemptwas made to isolate stable polyploid strains ofE. coli following treatment with camphor andother possible polyploidizing agents.

MATERIALS AND METHODS

The strain chosen for these studies was E.coli strain 82/r, a purine-requiring mutant ofE. coli strain B/r, obtained from Dr. E. H.Anderson who had employed it in studies of theinfluence of oxygen on the efficacy of X-rays ininactivation and induction of mutations (Ander-son, 1951). Camphor was selected because it hadbeen successfully employed in the production ofgiant, possibly polyploid cells of Pasteurellapestis (Won, 1950), of yeasts (Bauch, 1941; andothers), and of molds (Sansome, 1946; Roper,1952; and others). Natural camphor was used inthese experiments, since it is more effective thanthe synthetic product.

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RESULTS

Isolation of stable large cell strains. Camphor ishighly inhibitory to growth and the concentrationof camphor in the medium is difficult to controlbecause of the rapid volatilization at 37 C. Thefinal, successful technique consisted of growingthe cells in the presence of camphor vapors. Toaccomplish this, blocks of solid camphor ofapproximately 1 cu cm were suspended from thetops of petri dishes by means of cellophane tape.Many of the colonies developing in camphorvapors showed a coarser internal granularity andon microscopic examination were found to be

Figure 1. Internal colony morphology of 82/rand the large cell strain P6. Finely granularcolony of 82/r on left; coarsely granular colony ofP6 on right.

composed quite largely of obviously larger cells.After subculture, most such colonies proved to bemixtures of large and small cells and singlecolony strains of the coarsely granular type werein general unstabJe and reverted to the normaltype. However, on further treatment withcamphor vapors, several completely stablelarge-cell strains were isolated. A number of thesehave remained stable through repeated transferson stock culture agar slants. The large cellstrain selected for comparative studies with theparental culture E. coli strain 82/r was designatedE. coli strain P6 and has retained its large cellcharacteristic in the absence of camphor throughnumerous generations in a continuous growthapparatus and after 2 years of repeated transfers.

Colonial and cellular morphology. When viewedunder the steroscopic binocular microscope withoblique elimination (Henry, 1933), 82/r, thehaploid parental strain, is characterized bysmooth, finely granular colonies (figure 1, left).The large cell derivative P6, however, is charac-terized by colonies with a coarsely granularinternal structure (figure 1, right). No consistentdifference in the size of the colonies of the twostrains has been noted. In these strains, 82/rand P6, the occurrence of the coarsely granularcolony structure has been perfectly correlatedwith a larger than normal size of cell while thefinely granular colonies indicate the small cell

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Figure 2. Cell size of 82/r and P6 after different periods of growth on agar surfaces. Impression smears;tannic acid-crystal violet staining.

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size characteristic of 82/r. Thus, the colonialmorphology has been a useful index of cell sizethroughout these investigations. However, sinceE. coli strain B which is also presumably ahaploid strain and which is characterized bysmall cells comparable in size to 82/r also exhibitsunder our conditions a somewhat coarselygranular internal colony structure, this cor-relation between granularity and cell size is notgeneral and must be established for the par-ticular strains with which one is working.

Presented in figure 2 are photomicrographs ofcells of 82/r and P6 which were obtained fromcoverglass impression smears from agar plates atvarying times after inoculation. The smears wereair dried and stained with the tannic acid-crystalviolet cell wall technique (Robinow, 1945). It isreadily apparent that P6 exhibits significantlylarger cell size at all stages of incubation. Noquantitative estimates of the differences in cellsize have been made from microscopic measure-ments.

Nucleic acid content and dry weight per celldetermination. Having shown that cells of thecamphor treated P6 culture were significantlylarger at all stages of growth than the parental82/r strain, it was of interest to make determi-nations of the amount of DNA and RNA and ofthe dry wt per cell of these 2 strains in the station-ary phase of growth. The amounts of DNA andRNA were determined by the technique describedby Morse and Carter (1949). The dry wt, DNAand RNA contents were then made relative tothe total cell count as determined by micro-scopic counts in the Petroff-Hausser chamber(table 1). The cells in the counting chamber weresuspended in 0.6 per cent clarified Noble's agarand the counts were made under the oil immer-sion, phase contrast objective in an effort to in-crease accuracy. Individual determinations were

TABLE 1Relative DNA content, RNA content, and dryweight per cell for P6 and 82/r stationary phase

cells in aerated, nutrient broth cultures

No. of RatioAssays

P6:82/r Mean

DNA per cell ....... 4 2.35, 3.08, 2.752.33, 3.23

RNA per cell ....... 1 2.85 2.85Dry wt per cell. . 2 3.09, 3.38 3.24

quite variable, probably due more largely toinaccuracies in the total cell count than in thenucleic acid and dry wt determinations. In anycase, the data of table 1 support the conclusionthat the cell size of P6 is about 3 times greaterthan that of 82/r. This conclusion is supportedby the observation of identical DNA contentwhen equal weights of dried cells of P6 and82/r were analyzed. It seems safe to conclude thatthe protoplasmic composition of the P6 camphortreated strain is not different from that of itsparental strain, the primary difference merelybeing in cell size.

Growth rate determinations and fermentationtests. In general, polyploidy has an influence onthe growth rate in many higher organisms withthe growth rate becoming slower as the degree ofploidy increases (Lindstrom, 1936). Bauch (1941)also found that his presumed polyploid yeaststrains exhibited slower rates of growth than theoriginal strains. It was of interest, therefore, todetermine the relative growth rates of P6 and82/r. Typical growth rate curves are shown infigure 3 in which the samples were withdrawnfrom 300 ml nutrient broth cultures incubated at37 C on a mechanical shaker after inoculation

Figure 3. Comparative growth curves for 82/rand P6.

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with 0.5 ml of a 16 hr culture. On the basis of anumber of such experiments, it was concludedthat P6 and 82/r exhibit almost identical growthrates during the logarithmic phase of growth andhave similar lag periods. Although the growthcurves in figure 3 were not initiated with equalinocula, the difference in the maximum viablecell concentration shown is quite typical, themaximum stationary phase concentration of P6in general being from /' to k% that of 82/r. Thisobservation is compatible with the 3-fold greatercell size of P6.A number of carbohydrate substrates were

utilized in fermentation tests in Smith tubes todetermine if the camphor treatment had in anyway altered the fermentation reactions and alsoto compare the rates of fermentation of P6 and82/r since Ogur (1954) has shown that respira-tion and aerobic fermentation in a polyploidseries of yeasts ranging from haploid to tetra-ploid is ploidy dependent on a cellular basis.Although P6 appeared to ferment a number ofthe carbohydrates at a somewhat slower ratethan 82/r, the amount of acid and gas producedat the end of 72 hr incubation was the same forboth strains. The only apparent difference inmetabolic properties noted between the 2 strainswas the apparent failure of P6 to produce gaswhen grown in nutrient broth containing eithersodium pyruvate or formate. No study was madeof this difference.

Radiation induced mutations. In an organismwhere classical mendelian segregation studies arenot possible and where cytological foundationsare lacking for a critical diagnosis of polyploidy,an analysis of mutation frequencies is one of thefew remaining techniques for objectively testingfor the existence of polyploidy. If one accepts theconclusion that E. coli is normally haploid, thenthe simplest change in ploidy is to the diploid.With a mutation from a recessive to a dominantallele, a diploid would be expected to display afrequency of mutation twice that of a haploidsince there are in each diploid organism 2 genessubject to mutation. In the case of a mutationfrom a dorninant to a recessive allele, the apparentmutation rate in a diploid would be the square ofthat in a haploid since in a diploid, coincidentmutations at both homologous loci must occur inorder for the mutant phenotype to be expressed.With mutation rates of the order of 10ff to 10-7,such a difference should be easily detectablesince in the diploid, the comparable rates would

be 10-12 to 10-14. These differences would be evenmore pronounced in the event of higher degreesof ploidy.Three mutation systems have been studied

comparatively in 82/r and P6. These are back-mutation to purine independence, mutation toTi bacteriophage resistance and to strepto-mycin resistance or dependence. From Leder-berg's (1949) studies with partial diploids inE. coli strain K-12, purine independence wouldbe expected to be dominant to purineless, Tiphage resistance would be expected to be reces-sive to sensitivity, and Lederberg (1951) hasreported that streptomycin resistance is alsorecessive in E. coli K-12. Thus, the data obtainedwith Ti phage resistance and streptomycinresistance are especially critical.

In studies of purineless back mutation, 2537AUV, 250 kvp X-rays, and cobalt 60 y-rays havebeen used as mutagens.3 No data are presentedsince in general, similar frequencies of inducedmutations to purine independence were obtainedin both 82/r and P6 for comparable exposures.In figure 4 are presented typical mutation dose

curves obtained in comparative studies of mu-tation to streptomycin resistance following treat-ment with X-rays. The streptomycin resistancemutation assays were made with Newcombe's(1952) method which insures phenotypic expres-sion of the induced mutations. Washed cells sus-pended in oxygen saturated M/15 phosphatebuffer, pH 6.8, were irradiated with a GeneralElectric Maxitron 250 kvp X-ray tube with 3mm added aluminum filtration. As will be seenfrom the figure, the maximum frequency of strep-tomycin resistant induced mutations in 82/r wasabout 3 X 10-6. On the hypothesis that P6 werediploid, the expected frequency of streptomycinresistant mutants would be of the order of 10-11which would mean that none or a very few wouldbe expected in the present experiment.A similar result even more emphatically in

disagreement with the hypothesis that P6 is adiploid or polyploid derivative of 82/r wasobtained in studies of ultraviolet induced mu-tations to Ti phage resistance. Assays of phageresistant mutations were made using the spraymethod (Demerec and Laterjet, 1946) in order topermit phenotypic expression of the induced3The X-ray and gamma ray experiments were

carried out in the Biology Division of the OakRidge National Laboratory through the courtesyof Dr. Alexander Hollaender.

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101o phage resistant mutations. In the present experi-ments, samples of plates were withdrawn fromthe incubator at varying times of incubationafter seeding with irradiated cells. A represent-ative plate was used for an assay of growth by

3- washing the cells off the surface in nutrientbroth, and the remainder of the plates were

M simultaneously sprayed with Ti phage and

O0 82/r reincubated for eventual assay of phage resist-/ 82/r

ant clones. A typical curve of the appearance of

Ti phage resistant clones at various times ofincubation after exposure to equal doses of

zo incident 2537A ultraviolet is shown in figure 5.4 / o P6 Although P6 apparently undergoes a somewhat

greater initial delay in phenotypic expression, theeventual frequency of phage resistant mutant

10 clones is nearly equal to that of 82/r and both are

of the order of 1O-4. If the basic argument out-z lined above concerning the expected frequencies

of Ti phage resistance and streptomycin resist-ance is correct, these results are clearly incom-

_________________________________ _/.patible with the hypothesis that P6 is a diploid010 20 30 40 50 60 f_ 70 or polyploid derivative of 82/r.

X-RAY DOSE IN Kr Cytological investigations. Several workers haveFigure 4. Frequency of X-ray induced strepto- published observations of presumably polyploid

mycin resistant mutations in 82/r and P6. bacterial forms in which the diagnosis of thepolyploid nature was based largely or entirely on

105- cytological evidence. Thus DeLamater et al.(1953) and Minsavage and DeLamater (1955)have reported the production of transientpolyploids by treatment of various species of

E.COLI 82/r bacteria with colchicine and other drugs. Their4- conclusions were based entirely on cytologicalSlo / evidence. Clark and Webb (1955) have supported

-j

/ / their cytological diagnosis of polyploidy for the/ / large cells of Staphylococcus aureus with studies

o00 / /of the X-ray inactivation kinetics. However,m / l since it is not certain that radiation kills bac-

03- terial cells by inducing nuclear damage (Zelle,0 / / 1955) such irradiation inactivation analyses cano only be interpreted as indicative or corroborative

and not as supplying proof of the polyploidnature of the cell. Since the large cells studied

w 2- ( /E.COLI P6 by Clark and Webb do not persist and since,,lo10 unless cell division coincides perfectly withz chromosomal division in the division cycle of

bacteria, there is a "transient" diploid stage inthe division cycle of every bacterial cell, theexistence of a possible polyploid state which is

1010 2 4 6 8 10 12 only temporary is seemingly of no great signif-HOURS OF INCUBATION icance. Furthermore, Whitfield and Murray

Figure 5. Frequency of ultraviolet induced Ti (1956) have shown that chromatin bodies mayphage resistant mutations in 82/r and P6. reversibly aggregate into compact masses under

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a variety of circumstances which suggests thatthe supposed induction of "transient polyploidy"by arresting metaphase with presumed mitoticpoisons may actually indicate no more than adisturbance in metabolism which temporarilydisrupts the intracellular ionic equilibrium.

In the present state of bacterial cytology thereis no general agreement as to the structuraldetails of the bacterial nucleus and of the iden-tity of bacterial chromosomes. One needs onlyto examine recent reviews (DeLamater, 1954;Knaysi, 1956; Robinow, 1956) by different schoolsof thought in bacterial cytology to be impressedwith the fact that a diagnosis of polyploidy inbacteria resting solely on cytological evidencecould not be convincing at this time. Hence, noexhaustive cytological analysis has as yet beenundertaken.The only cytological analysis attempted has

been directed toward determination of theaverage number of nuclei per cell in both the82/r and P6 strains. Since evidence has beenadduced that P6 has about 3 times the DNAcontent per cell, it is of interest to know if theaverage number of nuclei per cell is the same inP6 as in 82/r. If this were true, it would suggestthat P6 may be a triploid derivative of 82/r ifone makes the further assumption that allbacterial DNA is nuclear. This latter assumptionseems reasonable since in general, DNA is con-sidered to be nuclear.For the nuclear counts, nutrient agar plates

previously warmed to 37 C were inoculated with20-hr aerated broth cultures of P6 and 82/r.Impression smears were made on glass cover-slips at varying times of incubation, air dried,then hydrolyzed in HCl and stained with thethionin SO2 method outlined by DeLamater(1951). The preparations obtained after 2 hrincubation of the plates were selected for thenuclear counts. In an effort to achieve objectiv-ity, three different observers made independentcounts of the number of discrete nuclear struc-tures or groups of nuclear structures in onehundred randomly selected cells of each culture.The results are presented in table 2. In making

these counts, no attempt was made at analyzingthe complex figures seen more frequently inP6, and the only datum recorded was the num-ber of stained structures per cell which appearednot to be connected to other similarly stainedstructures. Whether these discrete stainedbodies or groups of stained bodies actually are

TABLE 2Number of discrete stained structures or discretegroups of structures in 100 randomly selected cellsof P6 and 82/r after 2 hr of incubation on agarplates. HC1, thionin-SO2 staining

Observer P6 82/r Ratio-P6:82/r

A 284 183 1.55B 273 166 1.64C 299 202 1.48

Total .. 856 551 1.55

discrete nuclei is a debate we wish to avoid herebut it is on this basis that the nuclear numberwas estimated.

Although there are minor variations in thecounts obtained by the three observers, a x2 testfor homogeneity yields a value of 0.616 which for2 degrees of freedom indicates a probability ofabout 0.75 of obtaining as large or larger devi-ations by chance. The greatest deviation in thecounts of the different observers is in the numberof stained bodies in 82/r cells. However, thisdifference is not statistically significant, thex2 value for 2 degrees of freedom being 3.532yielding P greater than 0.15. It appears, there-fore, that the average ratio of the number ofdiscrete nuclear bodies between P6 and 82/r of1.55 is reliable statistically. Considered withthe approximately 3 times greater DNA contentper cell of P6, the ratio of 1.55 times as manynuclei per cell gives the interesting suggestionthat P6 may be a diploid derivative of 82/r.However, it must be emphasized that this isonly a suggestion for it is not certain that thenuclear counts are actually very accurate.Secondly, the nuclear counts were made on cellsjust entering the logarithmic phase of growthwhereas the DNA determinations were made onstationary phase cells and it is possible that thenuclear ratio between the strains may varyduring different growth phases.No more accurate studies of this nature have

been attempted, since at best, the evidence ofpolyploidy is somewhat indirect and it is hopedthat direct genetic evidence may be obtainedwhich will illuminate the nature of the changesinduced by camphor in P6. The most hopefulapproaches are further comparative studies ofmutation and comparative investigations ofgenetic recombination in 82/r and P6. Shouldsuch studies, which are now in progress, reveal

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unequivocally the genetic nature of P6, dstailedcomparative cytological analyses would begreatly enhanced.

DISCUSSION

A number of characteristics of P6 in comparisonwith its parental culture 82/r are compatiblewith the polyploid hypothesis. The significantlygreater cell size as evidenced microscopically andalso by comparative dry wt, the roughly 3 timesgreater DNA and RNA contents, and the tenta-tive conclusion that P6 may have about 1.5times as many nuclei per cell combine to suggesta possible diploid structure. The radiationinactivation data (Zelle and Ogg, 1957) are alsocompatible with a diploid constitution for P6, andthere are as yet no data from the attemptedgenetic recombination analysis which conflict.

However, the observations concerned withTI phage resistant and streptomycin resistantmutations following ultraviolet treatment areclearly at odds with a diploid or polyploid struc-ture if one accepts the recessive nature of thesemutations and assumes that chromosome dis-tribution in division of diploid or polyploidbacterial cells would be similar to that in poly-ploid somatic cells in higher organisms. Thus, ourdata at the moment cannot provide a clearanswer as to the genetic constitution of thelarge cell strain P6.

If one accepts the idea that all DNA is nuclear,then P6 would appear either to be a polyploid(presumably a triploid), to have about 3 timesas many nuclei per cell, or to be a combination ofthese 2 types of changes. If, as indicated by themutation data, P6 does not have diploid orpolyploid nuclei, then the camphor treatmentwould appear to have induced a change in themechanism regulating the nuclear constitution ina fashion such that apparently at all stages ofgrowth, the average number of nuclei is about 3times that of 82/r. Should this actually be provento be the case, comparative studies of the 2strains conceivably could yield informationconcerning the regulation of nuclear number inE. coli which is known to vary over a fairly widerange at different stages of growth (Witkin, 1951).

W1;hile considering possible nuclear impli-cations of our findings, it is of interest to recallthat the best available information indicates thatthere is only one linkage group in E. coli strainK-12 (Cavalli-Sforza and Jinks, 1956). Since atleast 13 different loci are known in this linkage

group, it seems highly improbable that E. colistrain K-12 contains more than one chromosomeunless the existence of very small or large butgenetically inert chromosomes is postulated. Ifindeed there is only one chromosome in haploidE. coli, then, since chromatin bodies have nodemonstrable membrane and lie separately inthe cytoplasm (Robinow, 1956), chromosome,chromatinic body, and nucleus all merge into oneand the same cytologically demonstrable struc-ture. It is not clear to the writers just whatcytological appearance would be exhibited by agenetically established polyploid under thehypothesis of a haploid, one chromosome nucleuswith no demonstrable membrane as the normalnuclear constitution.The data on induced phage resistant and

streptomycin resistant mutations can perhaps bereconciled with the other observations compatiblewith a polyploid constitution if one assumes thatthe radiation which induces the mutations alsoinduces a high rate of somatic segregation. Suchhigh rates of somatic segregation have beenobserved after ultraviolet irradiation of diploidheterozygous yeast (James, 1956). The inducedsegregation may be due to somatic crossing overor to a disjunctional separation of chromosomes.The phenotypic expression of recessive inducedmutations after irradiation of a diploid may alsobe due to a "haploidization" similar to thatobserved by Lederberg et al. (1951). They ob-served a high proportion of haploids displayingrecessive genes in the progeny of irradiatedheterozygous partial diploids. Neither of thesepossibilities has as yet been adequately testedalthough some information obtained from irradi-ation of prototrophs derived from crossingexperiments with P6, some of which could havebeen diploid or polyploid and heterozygous, sug-gest that neither haploidization nor iiiducedsomatic crossing over occurs. The observationthat Ti phage resistant mutant colonies of P6induced by ultraviolet appear to be coarselygranular and composed of large cells like P6supplies further evidence that haploidizationapparently does not occur after ultraviolet.

SUMMARY

A stable, large cell strain, P6, was isolatedfrom Escherichia coli strain 82/r, a purine requir-ing mutant of Escherichia coli strain B/r, follow-ing repeated exposure to natural camphor vapors.No significant change in growth rate or fermen-

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tation characteristics was noted in P6 except thefailure to produce any detectable gas when grown

in nutrient broth containing pyruvate or formate.Compared to the parent strain 82/r, P6 has

about three times greater dry weight, DNAcontent, and RNA content per cell.

Comparative frequencies of radiation inducedTi phage resistance and streptomycin resistancemutations in 82/r and P6 are at variance withthe hypothesis that P6 is a diploid or polyploidderivative of 82/r.

Estimates of the average nuclear number percell for 82/r and P6 suggest a possible diploidconstitution for P6.The nature of the stable, camphor induced

modification in P6 remains obscure.

REFERENCES

ANDERSON, E. H. 1951 The effect of oxygen onmutation induction by X-rays. Proc. Natl.Acad. Sci. U. S., 37, 340-349.

BAUCH, R. 1941 Experimentelle Mutationsl6-sung bei Hefe und andern Pilzen durch Be-handlung mit Campher, Acenaphthene, undColchicine. Naturwissenschaften, 29, 503-504.

CAVALLI-SFORZA, L. L. AND JINKS, J. L. 1956Studies on the genetic system of Escherichiacoli K-12. J. Genet., 54, 87-112.

CLARK, J. B. AND WEBB, R. B. 1955 Ploidystudies on the large cells of Micrococcusaureus. J. Bacteriol., 70, 454-463.

DELAMATER, E. 1951 A staining and dehydrat-ing procedure for the handling of micro-organisms. Stain Technol., 26, 199-204.

DELAMATER, E. D. 1954 Cytology of bacteria.II. The bacterial nucleus. Ann. Rev. Micro-biol., 8, 23-46.

DELAMATER, E. D., HUNTER, M. E., SZYBALSKI,W., AND BRYSON, V. 1953 Chemically in-duced aberrations of mitosis in bacteria. J.Gen. Microbiol., 12, 203-212.

DEMEREC, M. AND LATARJET, R. 1946 Muta-tions in bacteria induced by radiation. ColdSpring Harbor Symposia Quant. Biol., 11,38-49.

HENRY, B. S. 1933 Dissociation in the genus

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KNAYSI, G. 1956 Cytology of bacteria. Ann.Rev. Microbiol., 10, 253-274.

LEDERBERG, J. 1949 Aberrant heterozygous

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LEDERBERG, J. 1951 Streptomycin resistance: agenetically recessive mutation. J. Bacteriol.,61, 549-550.

LEDERBERG, J., LEDERBERG, E. M., ZINDER, N. D.,AND LIVELY, E. R. 1951 Recombinationanalysis of bacterial heredity. Cold SpringHarbor Symposia Quant. Biol., 16, 413-443.

LINDSTROM, E. W. 1936 Genetics of polyploidy.Botan. Rev., 2, 197-215.

MINSAVAGE, E. J. AND DELAMATER, E. 1955Some observations of the effect of colchicineupon Salmonella typhosa. J. Bacteriol., 70,501-509.

MORSE, M. L. AND CARTER, C. E. 1949 Thesynthesis of nucleic acids in cultures ofEscherichia coli strains B and B/r. J. Bac-teriol., 58, 317-326.

NEWCOMBE, H. B. 1952 Spontaneous and in-duced mutations. Symposia on RadiationMicrobiol. Biochem. Suppl. 1. J. CellularComp. Physiol., 39, 13-26.

OGUR, M. 1954 Respiration in a polyploidseries in Saccharomyces. Arch. Biochem. andBiophys. 53, 484-490.

ROBINOW, C. F. 1945 Nuclear apparatus andcell structure of rod-shaped bacteria. Adden-dum to R. J. Dubos' The bacterial cell. Har-vard University Press, Cambridge, Mass.

ROBINOW, C. F. 1956 The chromatin bodies ofbacteria. Bacteriol. Revs., 20, 207-242.

ROPER, J. A. 1952 Production of heterozygousdiploids in filamentous fungi. Experientia,8, 14-15.

SANSOME, E. R. 1946 Induction of 'gigas'forms of Penicillium notatum by treatmentwith camphor vapour. Nature, 157, 843.

WHITFIELD, J. F. AND MURRAY, R. G. E. 1956The effects of the ionic environment on thechromatin structures of bacteria. CanadianJ. Microbiol., 2, 245-260.

WITKIN, E. M. 1951 Nuclear segregation andthe delayed appearance of induced mutantsin Escherichia coli. Cold Spring HarborSymposia Quant. Biol., 16, 357-372.

WON, W. D. 1950 The production of giant cellsof Pasteurella pestis by treatment withcamphor. J. Bacteriol., 60, 102-104.

ZELLE, M. R. 1955 Radiation induced muta-tions and their implications on the mecha-nisms of radiation effects on bacteria. Bac-teriol. Revs., 19, 32-44.

ZELLE, M. R. AND OGG, J. E. 1957 Radiationresistance and genetic segregation in a largecell, possibly polyploid strain of Escherichiacoli. J. Bacteriol., 74, 485-493.

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