J. clin. Path. (1958), 11, 483.

THE BACTERIAL CELLBY

ROBERT CRUICKSHANKFrom the Bacteriology Department, University of Edinburgh

Our approach to the bacterial cell depends onour particular interests. The geneticist finds it amost useful plastic tool for studying mutations andvariations, transductions and transformations; thechemist is interested in it as a source of enzymesof bewildering variety ; he also helps in identifyingthe various components of the cell by fractiona-tion and detailed chemical analysis of bacterial cellmasses; the cytologist is mainly concerned withthe anatomy of the cell, using modern staining andmicroscopic methods, including phase-contrast andelectron microscopy; the bacteriologist is in-terested in the cell's interaction with its immediateenvironment, whether that be living tissue or deadand decaying matter.For clinical pathologists gathered together to do

honour to Virchow, it would seem most importantto discuss, if only in outline, our modern conceptsof the anatomy and physiology of the bacterialcell.

DefinitionBacteria belong to the kingdom of Protista,

which also includes plant and animal forms andhas many resemblances to the blue-green algae.According to majority opinion at the present time,the bacterium may be unicellular or multicellular,with haploid nuclear or chromatin material occur-ring as a single body and dividing by transversefission. Cell division depends on constrictiveingrowth of the cell wall and cytoplasmic mem-brane.

Let us start with a simple diagrammatic repre-sentation (Fig. 1) of the bacterial cell. It is en-closed in a cell wall from which there may beextruded flagella and fimbriae and may be sur-rounded by a capsule. The main function of thecell wall is to give form and rigidity and someprotection to the functional cell or protoplast. Thecytoplasm has a lining or membrane which acts asa selectively permeable osmotic barrier and con-tains within it many granules varying in size from10 to 20 mI,u composed mainly of ribonucleic acid(R.N.A.). Various inclusion bodies, such asvolutin granules, lipid granules, etc., may be con-tained within the cytoplasm, and we shall refer

to these later. The nuclear or chromatin materialhas the chemical and staining reactions of desoxy-ribonucleic acid (D.N.A.), but cannot be regardedas a nucleus in the sense that we use the term foranimal cells, since there is no nuclear membrane,no centriole, and no unequivocal evidence of divi-sion by mitosis.

Cell WallAlthough a rigid lining membrane for the bac-

terial cell, similar to that for the fungus mycelium,was postulated by Cohn in 1875, it is only sincethe introduction of electron microscopy that cellwalls have been clearly demonstrable. From bac-terial suspensions which have been mechanicallyor sonically disrupted, the cell walls can be separ-ated from the electronically more opaque cyto-plasm by high-speed centrifugation at 8,000 to10,000 r.p.m., after first removing any intact cellsat 2,000 to 3,000 r.p.m. It is less easy to get purecell walls with Gram-negative than with Gram-positive cells, and mechanical disruption givesbetter results than sonic disintegration. The cellwall constitutes about 20% of the dry weight ofGram-positive cocci and as much as 45 % forC. diphtheriae. The thinner walls of Gram-negative bacteria probably account for consider-ably less than 20% of the total weight. The thick-ness of the staphylococcal cell wall has been esti-mated at 15 to 20 mr,, while that of Gram-negativebacilli, like Bact. coli and salmonellae, is around10 to 15 m,u and of Mycobacterium tuberculosis23 mju. The selective rigidity of the outer casingis demonstrated by the finding that the cell wallsof rod-shaped organisms when specially preparedfor electron microscopy retain their cylindricalform, whereas the protoplast assumes a sphericalform when the cell wall is dissolved by lysozyme.Cell wall suspensions have a milky white opales-cent appearance, and in the case of chromogenicbacteria there is no contained pigment which isassociated with the small particles in the cyto-plasm.There is considerable variation in the chemical

composition of the cell walls in different bacterialspecies, the main constituents being peptide-poly-

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ROBERT CRUICKSHANK

MEMBRANE

%1NCLUSION GRANULES

'CELL WALL:

EXTRA- PROTOPLASTIC PROTOPLAStSTRUCTURES

FIG. 1.-Diagrammatic representation of a bacterial cell (extracellular structures omitted at bottom of figure).

saccharide complexes. Certain major differencesoccur between the Gram-positive and Gram-negative organisms, the former having a limitedrange of amino-acids, whereas the Gram-negativebacteria have the same full range of amino-acidsas have most proteins. The lipid content of thecell wall in Gram-negative bacteria is usually muchgreater than that of Gram-positive organisms(around 20% compared with 2-4%), and this highcontent of lipid may be related to the lipo-poly-saccharide of the 0 antigen or endotoxin. Poly-saccharides and hexosamine are present in aboutequal amounts in both Gram-positive and Gram-negative bacteria.

In the past few years more detailed analyseshave been made of the chemical content of the cellwall and its possible precursors (see Park, 1958).Thus, analysis of staphylococcal cell walls hasshown that muramic acid, D-glutamic acid, lysineand DL-alanine are present in a ratio of aboutI: 1: 1: 3, while examination of penicillin-inhibitedstaphylococci revealed an accumulation of auridine-5'-pyrophosphate complex which could be

a cell wall precursor, since it contained muramicacid and the amino-acids in the same ratio (Parkand Strominger, 1957). These findings may be linkedto the early suggestion by Duguid (1946), follow-ing observed morphological changes in penicillin-inhibited bacteria, that penicillin interferes speci-fically with the formation of the cell wall whileallowing growth to proceed until the organismbursts its defective envelope and so undergoeslysis, and to the recent observations by Cooper(1954, 1955) that penicillin is specifically bound toa bacterial lipid fraction which could be the cyto-plasmic membrane. At last we seem to be learningsomething about the mode and site of action ofpenicillin and perhaps of other antibiotics.

Another application of our knowledge of thechemistry of the cell wall concerns the action oflysozyme, which acts on a mucopolysaccharidewith the release of N-acetyl hexosamine. Whenresistant variants of the susceptible M. lysodeik-ticus are obtained, chemical analysis shows nochange in their sugar or amino-acid content buta 100-fold increase in the 0-acetyl group. These

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THE BACTERIAL CELL

findings suggest that lysozyme resistance may bedue to an ability of the organism to acetylatecertain cell wall hydroxyl groups associated withthe substrate, which in the sensitive strains are freeand receptive (Brumfitt, Wardlaw, and Park, 1958).

Other lytic enzymes, such as that derived fromStreptomyces albus, act on cell walls and can beused for preparing bacterial solutions for precipita-tion reaction, such as Lancefield grouping.

CapsulesMuch controversy and confusion have arisen

over the definition of bacterial capsules, and vari-ous alternative or additional names have been used,such as envelope, slimy layer, ectoplasm, and outercoat. We may define a capsule as a microscopi-cally demonstrable slimy or gelatinous layer cover-

ing the cell wall and having a definite external sur-

face. The term "envelope" is used by some

bacteriologists if the external boundary is notsharp or the slime has a tendency to dissolution;others have described a " capsule membrane," butthere seems no sound support for either of thesetwo terms. A good deal of the confusion hasarisen from difficulties in the demonstration of thecapsule. The two most reliable methods are thewet-film India ink technique, used as a negativestain, and the specific antigen-antibody reactionerroneously called the Neufeld Quellung or cap-sular swelling reaction. Duguid (1951), who gives a

detailed description of the India ink method, foundthat of dry-film staining techniques the eosin serum

method of Howie and Kirkpatrick (1934) was thebest. Recently, Novelli (1953) and others haveused Alcian blue as a capsule stain, since it is theonly dye so far recorded which will stain the bac-terial capsule without previous mordanting. Withone or other of these reliable methods, there isunanimity about the capsules of organisms like thepneumococcus, the anthrax bacillus, or Fried-lInder's bacillus. With Pasteurella pestis, on theother hand, there has been much divided opinionabout capsule and envelope. Amies (1951) believedthat the " envelope," dissolving in saline at 600 C.,is no more than a particularly well developed cap-sule which can be shown to persist despite solvents.In addition to these demonstrable capsules, certainbacteria may carry surface antigens or "micro-capsules" (Wilkinson, 1958) separable and anti-genically distinct from the cell wall, for example,the M antigen of Streptococcus pyogenes, whichcan be dissolved off by trypsin, without affectingthe cell wall or viability.Tomcsik and his colleagues (see Tomcsik, 1956)

have used the specific capsular reaction (a more

correct term than capsular swelling reaction) tostudy the structural elements in the capsule andalso as an indicator of the localization of surfaceantigens. When specific antiserum is added tocapsulated bacteria, a precipitation reaction quicklyoccurs which renders the capsule visible withoutchanging its size or shape. This reaction has beenused in the identification of such bacterial speciesas pneumococcus, streptococcus, Klebsiella,Haemophilus, Neisseria, Pasteurella, and Bacillus,and has helped to show that the capsular substanceof the anthrax bacillus is not a polysaccharide buta glutamic acid polypeptide, while that ofPasteurella pestis is also a protein or a protein-polysaccharide complex.

Flagella

Flagella are unusual in that they lie outside thecell wall and may be removed without killing thecell, yet have a characteristic movement which in-dicates that they are part of the living cell. Despitethe ingenious artistry and arguments of Pijper, wemay accept the flagella as locomotor organelleswith their origin in a basal granule inside the cellwall. Structurally, they are non-tapering filamentsin the shape of a cylindrical helix, which may haveeither one of two different wavelengths. The fila-ments vary in length up to 12 ,u and are approxi-mately 12 mpt thick. Chemically they are com-posed predominantly of protein made up of elon-gated molecules (molecular weight 41,000) with acharacteristic amino-acid composition from whichhistamine, tryptophane, proline, and cysteine arelacking. The great variety of antigenic entitiesamong flagella with very similar protein contentsuggests that these antigenic differences depend onthe arrangement rather than the composition ofthe amino-acids; this plasticity may also accountfor the variation from specific to group phasewhich characterizes many salmonella flagella.

In stained preparations of flagellated bacteriathe actual number of flagella is much greater thanare seen in vivo under dark-ground or phase-contrast microscopy. This difference is probablydue to a tendency for flagella to aggregate withany motion; aggregation into quite thick filamentsis particularly well seen in the elongated forms ofB. proteus grown on agar in the presence of peni-cillin (see Fleming, Voureka, Kramer, and Hughes,1950). When flagella have been broken off froma young culture they regenerate within half anhour; on the other hand, there is no evidence thatthey wear themselves out or even become frayedby active movement.

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ROBERT CRUICKSHANK

Space does not allow any discussion of the in-teresting studies of Stocker and others on thegenetic transductions of flagella and motility.

FinbriaeBefore we pass inside the cell wall mention must

be made of another type of appendage, the short,slender and very numerous fibres, called fimbriaeby Duguid, Smith, Dempster, and Edmunds (1955)and Duguid and Gillies (1957) to denote a fringewhich surrounds many species of Gram-negativebacteria (Bact. coli, Bact. cloacae, Salmonella,Shigella flexneri, but not Sh. sonnei or shigae.They are about half the thickness of flagella, andfimbriation is enhanced by frequent passage inbroth culture but has no relationship to motility.These fimbriae have strong adhesive propertiesand are responsible for agglutination of the redcells of various animal species. The fimbriaefrom different serological types of Shigella flexneriare antigenically similar and may be responsiblefor some of the non-specific agglutinins present inthe blood in many individuals. It is still uncertainwhat function, if any, they serve.

The ProtoplastSeparation of the cytoplasm from the cell wall

was observed many years ago by Fischer (1900),who described as " plasmoptysis " the ejection ofthe cytoplasm from organisms like B. anthracisand Vibrio proteus. The empty casing either ad-hered to or was separated from the cytoplasm,which then assumed a spherical form. This pheno-menon was recently rediscovered by Stahelin(1953, 1954) in weak-walled anthrax bacilli. Theterm protoplast was, however, first used by Wei-bull, who dissolved the cell wall of B. megateriumby lysozyme and maintained the spherical nakedcell in a stabilizing solution of sucrose or poly-ethylene glycol for at least 24 hours. Attachedflagella can be demonstrated in the intact cell al-though motility does not occur, probably for phy-sical reasons. The protoplasts disintegrate rapidlyin non-stabilizing solutions or if the suspension isaerated and the lysate is seen to contain " ghosts "or empty vesicles and small granules. The ghosts,which probably represent the cytoplasmic lining,are composed of delicate membranes and containmost of the pigmented material of the cell. Themetabolism of protoplasts and whole bacterialcells is remarkably similar, so that, besides thecytochrome system, most of the cell's enzymicactivity is contained within the protoplast. Phageproduction and spore formation also occur, pro-vided these phenomena have been initiated beforedissolution of the cell wall. To quote McQuillen

(1956), who has carried out many interestingstudies with protoplasts:

" Intact bacteria and the protoplasts derived fromthem have closely parallel capabilities. Both formsrespire; both synthesize proteins and nucleic acidsand form adaptive enzymes; both can support themultiplication of virulent and temperate bacterio-phages; both can support the development of endo-spores; both grow in appropriate media; and bothcan divide. Moreover, many of these activities arecarried out at approximately similar rates and tosimilar extents by the two forms, the whole and thepart. And yet the part is not the whole ; there aredifferences in behaviour. To what extent some ofthese differences are due to the use of unsuitableconditions is not yet known."Protoplasts cannot build a cell wall, possibly

because a starter of cell wall material is neededbefore more can be laid down. Similarly, thefailure to sporulate unless the process has alreadybeen initiated probably means that cell wall isneeded to complete spore formation. Again, al-though phage will multiply inside the protoplast,this body cannot be infected with virulent phage,which seems to need the cell wall for initial pene-tration.

The Nuclear MaterialFierce arguments have been raging for some

years on the nature and interpretation of thechanges that are demonstrable in the chromatinbodies or nuclear material of the bacterial cellduring the process of growth and division. Theargument largely turns on whether the chromatinbodies which grow in number with the growth ofthe cell are composed of chromosomes whichdivide like the chromosomes of animal and plantcells, i.e., by mitosis, or whether they dividedirectly or amitotically, as happens with many ofthe higher bacteria and Protista. The opposingviews are set out by Robinow and DeLamater re-spectively in their chapters in the Symposium onBacterial Anatomy (1956). Robinow, using Feul-gen and acid Giemsa stains, has studied parti-cularly the development of the single chromatinbody in germinating spores, and his interpretationsof the observed phenomena carry convincing evi-dence for direct amitotic division. DeLamaterhas used the actively growing cell for his studies,and, having perhaps too readily assumed that bac-teria divide by mitosis, interprets his findings to fitthis hypothesis. He claims that both B. mega-terium and Bact. coli have three chromosomeswhich can be shown pictorially to divide simul-taneously by a complex mitotic mechanism insidethe nuclear membrane. He and his colleagues alsodescribe mitochondria and a centriole, whichRobinow, on the other hand, regards as an acces-

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THE BACTERIAL CELL

sory chromatin granule. It is impossible for thelayman to adjudicate on the merits of these diver-gent stories by highly specialized cytologists, butsupport can be given to DeLamater's viewpointthat the truth will best be elicited by the simul-taneous application of other techniques, such asthose of biochemistry and bacterial physiologyalong with the cytochemical methods of the cyto-logist.

If we turn for a moment to the evidence of themicrobial geneticist, it is obvious that genetic ex-change of nuclear material takes place as a resultof sexual pairing in certain species; in addition,transfer of genes occurs to cause the phenomenaof transformation in pneumococcus and haemo-philus types and of transduction of toxigenicity inC. diphtheriae or motility in salmonella. The cyto-logists have so far been unable to contribute to abetter understanding of these strange happenings,although there has been some correlation betweencytological changes and genetic exchange in higherorganisms like Drosophila and maize. There islittle doubt that D.N.A. is the active principle inthe genetic mutations of bacteria, mediated some-times through bacteriophage. It has also beenshown that genetic differentiation of the D.N.A.material is linear, i.e., the "bacterial chromo-some" may be regarded as a long, thread-likeD.N.A. molecule, carrying the genes in an orderlysequence like the inscription on a tape. TheD.N.A. molecule, pictured by Watson and Crick(1953) as a cylindrical double helix with a diameterof 2.5 m,u, would correspond to a thread about1,000 times the length of the parent bacterial cell.Attempts at cytological study of the D.N.A. mole-cule are now being made by electron microscopicexamination of ultra-thin sections of bacteria. Itis suggested that the thread is coiled up to form atube, and in the photomicrographs of Maal0e andBirch-Andersen (1956) tube-like structures cut inall different directions can be demonstrated. Itmay be possible in the future to learn more aboutthe nucleus and its mode of division by detailedstudies of this kind.

Cytoplasm and Inclusion BodiesIn electron photomicrographs of sectioned bac-

teria (staphylococci, streptococci, paracolon bacilli,aerobacter, mycobacteria) the cytoplasm seems toconsist of fine granules of 10 to 20 m,u with some-times larger granules, the total number in a singlecell being of the order of 104 to 105. Chemicallythe cytoplasm is composed mainly of ribonucleicacid and protein and contains most of the enzymicactivity of the cell, partly in its membrane. The

small size of the granule and the multiplicity ofenzymes in many bacteria suggest that there willbe numerous enzymically different types ofgranules. They are probably derived from thenucleus, growing by the formation or accumula-tion of enzyme proteins. In discussing the rela-tionship of bacterial cytoplasm to that of otherlowly creatures, Bradfield states that:

" the most striking structural difference betweenbacterial cytoplasm and the cytoplasm of animalsand plants is the absence from bacteria of all kindsof intra-cytoplasmic membranes; the oxidativegranules are not wrapped in membranes to formmitochondria; the RNA-rich granules are notarrayed on membranes to form endoplasmic reticu-lum; and finally the cytoplasm as a whole is notseparated from the nucleus by a nuclear mem-brane."Among inclusion bodies, the most commonly

seen are the Babes-Ernst metachromatic bodies,often called volutin granules. These meta-chromatic granules, seen best in C. diphtheriaeand ranging in size up to 0.6 /u, are very electronopaque and chemically contain polymetaphosphateand probably other phosphorus compounds.Smith, Wilkinson, and Duguid (1954) found that,although volutin granules might account for 20%of the bacterial volume, only 1% of the dry weightwas polymetaphosphate.

Wilkinson and his colleagues (Williamson andWilkinson, 1958; Macrae and Wilkinson, 1958)have also studied the lipid inclusions found parti-cularly in the larger varieties of the Bacillusspecies. These lipid granules, which are composedmainly of a polymer of f8 hydroxybutyric acid, aremost numerous in cells about to sporulate or whengrown in semi-synthetic culture media containingan excess of glucose, pyruvate, or ,B hydroxy-butyrate as the main carbon sources for metabol-ism. They are regarded by the Edinburgh workersas stores of carbon and energy and are probablyused up in the process of sporulation. Whetherinclusion bodies like volutin and the lipidglobules serve a specific function in cellularmetabolism, or whether they represent an ab-normal or pathological state of the bacterial cell,is a debatable point. As already mentioned, thepigments of chromogenic bacteria are found pre-dominantly within the cytoplasm, and Schachman,Pardee, and Stanier (1952) have observed manywell-defined chromatophores of approximately40 m,u diameter in the cytoplasm of Rhodo-spirillum rubrum. They were absent from organ-isms grown in the dark. Probably many otherphotosynthetic bacteria possess similar chromato-phores.

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ROBERT CRUICKSHANK

Osmotic Activity of the Bacterial Cell

Having discussed some aspects of bacterialanatomy, we may conclude with a reference to thebacterial cell's mode of living. As with other cells,the active life and growth of bacteria depend on

an interchange of nutrient and waste productsbetween the interior of the cell and the externalenvironment. This involves a selective intake ofnutrient molecules and the expulsion of wastemolecules, while the vital enzymic and nuclearsubstances of the cytoplasm are retained. Thereis good evidence that the cytoplasmic lining mem-brane functions as a selective osmotic barrier,while the outer wall maintains the structuralrigidity of the cell. The bacterial cell is remark-ably resistant to violent changes in its environ-ment and will remain intact in media ranging fromdistilled water to strong salt solutions, althoughthis tolerance is considerably reduced in the young,

actively growing phase. Bacteria react by swellingor shrinking in media of high or low osmotic pres-

sures, but the reaction is selective in that, as

Fischer showed more than 50 years ago, plasmo-lysis of Gram-negative bacteria occurs in high con-

centrations of salt or sucrose but not in similarconcentrations of glycerol or urea. Duringplasmolysis the cell wall does not shrink with theprotoplast, the contents of which stream out ina series of fine droplets. Gram-positive bacteriaare much more resistant to plasmolysis, but they,too, respond by contraction selectively to hyper-tonic solutes of sucrose but not of glycerol. Thisindicates that they resemble Gram-negative bac-teria except that the plasma membrane does noteasily separate from the cell wall, although thereis good evidence that it is a distinct structure.The osmotic pressure inside the cell may be as

high as 20 atmospheres, and the tensile strength ofthe cell wall, greater in spherical than in cylindricalorganisms, is responsible for withstanding the highpressures. Obviously if this tensile strength isweakened, as by the action of penicillin, the pheno-menon of plasmolysis or plasmoptosis will readilyoccur.

The functionally active plasma membrane iscomposed of lipid and protein and is estimatedto be 15 m,u thick if hydrated, or 5 mrnt if un-

hydrated. This corresponds to the classical semi-permeable membrane 2-4 molecules thick. The

cell wall must also be permeable, and Mitchelland Moyle (1956) suggested that it consists ofa network of chains or fibres, with an effectivepore diameter of about 1 m,u. Indeed, althoughthe plasma membrane is the important osmoticbarrier to small molecules, the cell wall probablyacts as the barrier to solutes of 10,000 molecularweight or over. However, the cytoplasmic mem-brane is, according to the experiments by Mitchelland Moyle and others, more than a semi-permeablebarrier, for it contains the bulk of the cytochromesystem and other enzymes, and it may act as acarrier of substances into and out of the cell, aprocess called exchange diffusion. This may ex-plain the selectivity of the osmotic barrier, whichwould on this hypothesis be dependent on thepresence of carrier enzymes which act as ferryboats for the transport of selected passengers intoand out of the cell.

REFERENCESAmies, C. R. (1951). Brit. J. exp. Path., 32, 259.Brumfitt, W., Wardlaw, A. C., and Park, J. T. (1958). Nature (Lond.),

181, 1783.Cohn, F. (1875). Beitr. Biol. Pfl., 1, Heft 2, p. 127.Cooper, P. D. (1954). J. gen. Microbiol., 10, 236.-(1955). Ibid., 12, 100.DeLamater, E. D. (1956). Bacterial Anatomy (6th Symposium of the

Society for General Microbiology), p. 215. University Press,Cambridge.

Duguid, J. P. (1946). Edinb. med. J., 53, 401.-(1951). J. Path. Bact., 63, 673.- and Gillies, R. R. (1957). Ibid., 74, 397.- Smith, I. W., Dempster, G., and Edmunds, P. N. (1955). Ibid.,

70, 335.Fischer, A. (1900). Z. Hyg. Infect.-Kr., 35, 1.Fleming, A., Voureka, A., Kramer, I. R. H., and Hughes, W. H.

(1950). J. gen. Microbiol., 4, 257.Howie, J. W., and Kirkpatrick, J. (1934). J. Path. Bact., 39, 165.Maaloe, O., and Birch-Andersen, A. (1956). Bacterial Anatomy

(6th Symposium of the Society for General Microbiology), p. 261.University Press, Cambridge.

McQuillen, K. (1956). Ibid., p. 127.Macrae, R. M., and Wilkinson, J. F. (1958). J. gen. Microbiol., 19,

210.Mitchell, P., and Moyle, J. (1956). Bacterial Anatomy (6th Sym-

posium of the Society for General Microbiology), p. 150.University Press, Cambridge.

Novelli, A. (1953). Experientia (Basel), 9, 34.Park, J. T. (1958). The Strategy of Chemotherapy (8th Symposium of

the Society for General Microbiology), p. 49. University Press,Cambridge.

-- and Strominger, J. L. (1957). Science, 125, 99.Robinow, C. F. (1956). Bacterial Anatomy (6th Symposium of the

Society for General Microbiology), p. 181. University Press,Cambridge.

Schachman, H. K., Pardee, A. B., and Stanier, R. Y. (1952). Arch.Biochem., 38, 245.

Smith, I. W., Wilkinson, J. F., and Duguid, J. P. (1954). J. Bact.,68, 450.

Stahelin, H. (1953). Schweiz. Z. allg. Path., 16, 892.- (1954). Ibid., 17, 296.Tomcsik, J. (1956). Bacterial Anatomy (6th Symposium of the

Society for General Microbiology), p. 41. University Press,Cambridge.

Watson, J. D., and Crick, F. H. C. (1953). Nature (Lond.), 171, 737.Wilkinson, J. F. (1958). Bact. Rev., 22, 46.Williamson, D. H., and Wilkinson, J. F. (1958). J. gen. Microbiol.,

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