- Email: [email protected]
Bacterial Cytology
Bacterial Cytology ALFRED MARSHAK Marine Biological Laboratory, Woods Hole, Massachusetts
I. 11. 111. IV. V. VI. VII. VIII. IX. X. XI. XII. XIII.
Introduction ........................................................ Nuclei ............................................................. Centrioles .......................................................... Chromosomes ...................................................... Mitochondria ....................................................... Vacuoles ........................................................... Membranes ........................................................ Transverse Wall: Flagella .......................................... Nucleus of Bacterial Spores ........................................ Formation of Giant and Dwarf Farms ............................... Comparison with Cells of Higher Forms ............................. Conclusions ........................................................ References .........................................................
Page 103 104 105 106 107 107 108 108 109 110 110 112 113
I. INTRODUCTION In the study of the fine details of the structure of bacteria, as in other human activities, the turning of the wheels of progress appears in retrospect to have been amazingly slow while in the turmoil and excitement of present activity much seems to be happening, although it is not easy to see how much of this is pure spin and how much represents traction onto new ground. The whims of fashion have again brought the bacterial nucleus into prominence, not only as judged by the volume of words about it appearing in the literature, but apparently also in the thinking of bacteriologists not formerly accustomed to giving it much consideration. The magic of the word chromosome, reintroduced into bacterial cytology in 1945, may have had a good deal to do with the increase in interest in the bacterial nucleus, but more likely the development of microbial genetics, with its concentration on genetic factors involved in the control of familiar biochemical systems, has prepared more minds for thinking in terms of nuclear phenomena in bacteria. Certainly the development of fundamentally new techniques has had little to do with it. We find the same words (chromosomes, centrioles, nucleolus, mitotic process) being applied now, as in the past, to images obtained by staining procedures not very different from those used 20-30 years ago. There are in the current literature indications of a very natural tendency to seek in bacteria morphological counterparts of intracellular structures seen in higher forms. It Seems apropos to quote some words of caution given some 20 years ago ..... if taking the point of view of the uniformity of the living world, we 103
104
ALFRED M A R S H A K
have all reason to expect the presence of a nucleus in bacteria as well as in any other form, we have not any right to demand that in the morphological respect their nuclear apparatus should entirely coincide with nuclei of different organisms. I t is the duty of the biologist to apprehend its actual structure and not lay it forcibly on the Procrustes bed of preconceived notions, and, by the way, many shrink from it only because they cannot seem to make it fit this bed.” (Epstein et al., 1936.)
11. NUCLEI Although Feulgen and Rossenbeck (1924) applied their new procedure to bacteria they obtained negative results, but in the following year Voit (1925) presented evidence for a positive nucleal reaction in bacteria, although localization of the thymonucleic acid in nuclei was not proven. However, by 1936 the technique was sufficiently well advanced for a clear demonstration of nuclei in particularly difficult material, M . tuberculosis, to be presented by Epstein et al. (1936). They found that, after lipid extraction with carbon &sulfide as well as alcohol, Feulgen-positive nuclei could be seen and readily distinguished from volutin granules. Piekarski (1937), with a fortunate choice of bacteria which did not require such extraction. demonstrated nuclei by the Feulgen procedure and also with the Giemsa stain, as modified by Badian, using aqueous eosin for differentiation and counterstaining. The latter procedure in various modified forms has become popular in recent years although it lacks the specificity of the Feulgen method, and the necessity for thorough lipid extraction prior to staining by either the Feulgen or modified Giemsa methods is still usually overlooked. I have applied the Feulgen procedure after thorough lipid extraction to a large variety of bacteria and with a single exception found very obviously identifiable nuclei ( Marshak, unpublished data ) , The exception was an unidentified mycobacterium isolated from soil in which no nucleus could be detected by either the Feulgen or acidGiemsa method, but which on chemical analysis was found to contain large quantities of thymine, the nitrogenous base considered to be peculiar to deoxyribonucleic acid (Marshak and Vogel, 1951) . The significance of this observation remains undetermined, but it is mentioned here to emphasize the desirability of exploring not only phenomena related to the bacterial nucleus which conform to an expected pattern, but also those apparent exceptions which may ultimately throw some light on the general problem of nuclear organization.
BACTERIAL CYTOLOGY
10s
111. CENTRIOLES Having become aware of the existence of bacterial nuclei, it was to be expected that the appurtenances of the nuclear apparatus would be sought and, as in other instances in cytology, where observations are made on structures near the limit of resolution of the light microscope, the inevitable controversy over interpretation has again arisen. DeLamater (1953) has described granules which he considers to be centrioles. They are stained purple by his double staining procedure, which is a combination of the thionine-SO2 procedure he describGd previously, and a modification of the Cassel procedure ( 1951) in which the cells are mordanted with tannic acid before staining with basic fuchsin. The spindle and chromosomes are also purple, the cell wall red and “mitochondria” a “redviolet”. Bisset (1952, 1953a, b), using the combination stain, Giemsa and tannic acid-crystal violet, but avoiding dehydration by making examinations in water mounts, concludes that the structures called centrioles and mitochondria by DeLamater, Mudd et aE. (19Sl), and Mudd (1953) are nothing more than areas of greater basophilia at the growing tips and points of division of the cells. The picture is complicated by the designation of some granules (which also appear in association with the spindle but not at its poles) as “mitochondria,” i.e. those granules that stain redviolet rather than purple, which seems to be a rather fine distinction (DeLamater, 1953). By treatment with drugs (colchicine, formaldehyde, and antibiotics) DeLamater finds arrests or deviations from the normal behavior which support his contention that the granules associated with the spindle are centrioles. According to his interpretation the centriole is at first associated with the nuclear membrane, then migrates away from it as the spindle develops and elongates. In prometaphase there is a single centriole which then divides, each half going to the spindle poles as the cell proceeds into metaphase and anaphase. It is claimed that the drug action enlarges the centriole. Arrests at various phases of the centriole division cycle may be produced by the actinomycete-derived antibiotics which also inhibit mitosis in B. megatherium. Para-aminosalicylic acid differs from the above in inhibiting centriolar division resulting in unipolar spindles and increase in size of the nuclear mass suggestive of polyploidy. In the recovery from antibiotic inhibition there is segmentalization of the elongated nuclear mass into portions, each of which eventually contain the three chromosomes of the haploid complement. Colchicine acts differently in these organisms in producing directly a doubling of the chromosomes. Work by Szybalski and Hunter is cited by DeLamater and illustrations are given of stages purported to be zygotene and pachytene.
106
ALFRED MARSHAK
Hunter (cited by D e b a t e r ) has described a complete meiotic cycle for Micrococcus cryophilus from zygotene through the two meiotic divisions, but this reviewer has not yet seen the published descriptions. IV.
CHROMOSOMES
A fine series of parallel observations made with the electron microscope and with the light microscope after acid-Giemsa staining on strains of E . coli has been given by Kellenberger (1953). During the exponential phase of growth all of the nucleoid bodies have the form of chromosomes, as described by Robinow (1944, 1945). At the end of the growth phase the chromosome-like appearance is no longer found but instead there are larger masses of ellipsoidal, dumbbell-shaped, and rod-like bodies. Strain C of E . coli shows a characteristic peripheral disposition of the “chromosomes’’ instead of the paracentral one found in strains S and K12. In the presence of low concentrations of aureomycin or chloromycetin, the nucleoid body assumes a globular shape with a denser portion usually more or less centrally located in the less dense mass of the nucleoid. In strain C the formation of a similar centrally located body is induced by action of the antibiotic. Upon returning the bacteria to medium free of antibiotic, the “chromosomes” again appear at first in numbers much greater than normal (polychromosome phase), after which they regroup to form the normal nucleoids following which cell division takes place. The polychromosomal condition is also induced by exposure to ultraviolet light or to methyl-bis-chloroethylamine, but the polychromosome stage in these cases is followed by one suggesting fragmentation of the chromosomes to such an extent that they appear to fill the cell. Subsequently in those cells which do not lyse there is a regrouping to form the usual nucleoids. The amount of fragmentation appears to be dependent upon dose. Kellenberger makes the interesting observation that the agents which are effective in the conversion of prophage to phage, all induce the “fragmented chromosome” stage in E. coli. H e suggests that the mechanical rupturing of the chromosomes liberates the prophage from inhibiting influences so that it may then proceed to develop at the expense of the bacterial cell. Using staining procedures on B . megatherium, Bergersen (1953a) reports apparent fusion of paired nuclear bodies, when the culture proceeds beyond the logarithmic phase, to result in the formation of elongated chromatinic bars which then become converted to “zigzags” of short filaments. Subsequently on division of the cells typical bacterial rods are formed with a single, central, elliptical nuclear body. H e finds that chloroamphenicol appears to inhibit cell division while cell growth and increase in the chromatinic material goes on ( Bergersen, 1953b).
BACTERIAL CYTOLOGY
107
V. MITOCHONDRIA Mudd (1953) and his co-workers (Mudd et al. 1951; Winterschied and Mudd, 1953) have described localization of Janus green, the indophenol blue of the Nadi reagent, and the formazan of the tetrazoliums in definite regions or granules. This localization has been taken as an index of oxidation-reduction activity which is assumed to be characteristic of mitochondria, and hence that the regions defined by accumulation of these materials are mitochondria. Bisset (1952, 1953a, b) has pointed out that the granules designated as mitochondria are in regions of active growth, and hence of synthesis and intense oxidation-reduction activity, so that the agents used as indicators would be expected to accumulate there even if they contained no specialized organelles analogous to the mitochondria of higher forms. Chapman and Hillier (1953) have published some very fine electron micrographs of thin sections of B. cereus which show “peripheral bodies” or vacuoles which appear empty except for small dense granules. These are located near the site of formation of new transverse septa corresponding to the regions of the mitochondria1 bodies of Mudd et al. However, there is no indication of any of the typical mitochondrial structure (Palade, 1952). It should be mentioned that granules of formazan from tetrazolium do not necessarily accumulate in mitochondria. The writer has observed formazan granules in psoas muscle of the rat, which has no mitochondria (unpublished data). In B. megatherium fixed in Os04 vapor and stained with acid-Giemsa, Bergersen (1953a) has found stained granules similar to those described by Bisset at the terminal growing points and at the lateral septa. These regions also stained with Janus green and the Nadi reagent. Weibull (1953a) has found by continuous observation in phase contrast of B. megatherium in triphenyltetrazolium solutions that the formazan first appears as minute granules which then coalesce to form the large secondary granules described as mitochondria by Mudd (1953). He pointed out that the primary granules may indicate sites of oxidation-reduction activity but the larger secondary ones certainly do not. I n mammalian tissue where succinic dehydrogenase is associated exclusively with mitochondria it has been impossible to obtain a clear indication of staining within the mitochondria, although formazan is abundantly produced at their surfaces. It has also been found that intracellular fat droplets will be colored by the tetrazolium salts (Shelton and Schneider, 1952). VI. VACUOLES
Schuster ( 1952) has described vacuoles, which are probably liquidfilled, in Malleomyces mallei; these were observed using both light and electron microscopes. On further growth in high phosphate media,
10s
ALFRED MARSHAK
granules appear at the vacuolar sites which consist primarily of metaphosphate as determined by toluidine blue staining and extraction techniques. VII. MEMBRANES M‘eibull (1953b) has been able to remove the cell walls of B. megnthe-
riUnt with lysozyme and preserve the protoplast by keeping the cells in
sucrose solution. When they are transferred to buffer or saline solutions they lyse. By centrifugation of the lysed cells, ghosts were isolated which were considered to be the cytoplasmic membranes. However, since the ghosts are pigmented and contain enzymes it seems to the writer more likely that the ghosts are cytoplasmic residua rather than membranes. The absence of any true cytoplasmic membrane in electron micrographs of thin sections of bacteria (Chapman and Hillier, 1953) also indicates the necessity for caution in the use of the term membrane in such cases as these. Weibull was unable to recover nuclei from his protoplasts but did obtain a rapidly sedimenting gel which appeared to be made up primarily of deoxyribonucleic acid. Robinow and Murray ( 1953) have described specific staining of the cytoplasmic surface of some bacteria. They find a thin cytoplasmic membrane in electron micrographs of cells in which retraction of the protoplast from the cell wall is induced by fixation in formalin. However, judging from the micrographs published, the “menibrane” may be the residual cytoplasm not clumped with the main mass. This interpretation seems all the more plausible since inspection of the micrograph reveals at least two lines of density demarcation instead of the one expected on the membrane hypothesis. The protoplast surface does appear to provide a limiting surface which precipitates or accumulates the dye, but these observations raise again rather than settle the old question of whether there is a membrane in the structural sense, or a surface with differentiated physiological properties. Chapman and Hillier ( 1953) deduce from their studies of thin sections of E. coli that if a structural cytoplasmic membrane does exist, it must be less than 40 d wide.
VIII. TRANSVERSE WALL:FLAGELLA Analyzing the behavior of lysozynie-treated B. megatheritim towards stains, Welshimer (1953) finds evidence for different actions of the enzyme on the wall and on the capsular substances, an observation which is correlated with previous reports of polypeptides found in the capsule but not in the wall (see also Tomcsik and Guex-Holtzer, 1951j . The formation of the transverse wall by growth through invagination of the peripheral wall is clearly shown in the electron micrographs of Chapman and Hillier. They find the wall to be 200 d thick, with a n inner
BACTERIAL CYTOLOGY
109
portion which is denser and distinct from the outer portion. The transverse wall material is laid down as an annulus growing inward until a solid disc is formed, after which it thickens and divides into two layers. The cytoplasmic surface is always in contact with the forming wall. Van Iterson ( 1953) has described flagella in Spirillum, Proteus, and Vibrio, with definite basal granules which appear to be located in the cytoplasm beneath the cell wall. Piper et aE. (1953) believe that the flagellum of Spirillurn serpens is a continuation of the cell wall to which it is attached by a narrow stem. They find marked differences in the structure of flagella of Vibrio, Salmonella, and Spirillum. Van Iterson (1953) has shown that the flagellum of Vibrio has a definite sheath of material differing from that of the core. She concludes that motion of the flagellum cannot be as simple as the monomolecular mechanism proposed by Astbury ( 1949).
IX. NUCLEUS OF BACTERIAL SPORES Robinow (1953) now agrees with Bisset’s interpretation of the nuclear apparatus of bacterial spores. The spore contains a more or less central, vesicular, Feulgen-positive nucleus. Any of the procedures which involve acid hydrolysis result in the ejection of the nuclear material through a weak point in the spore wall to produce a diffusely Feulgen-positive mass on the surface of the spore. Chance (1953a, b) has described observations on stained preparations which suggest that in some species the transverse wall is laid down as a plate beginning at, or in the dividing nucleus in a manner resembling that found in higher plants. Clark and Webb (1953) have described instances where nuclei of the vesicular type, apparently in a state of division, are associated with a developing cell plate, suggesting to them that this type of nucleus is not limited to the resting stages. However, in Nocardia corallina, they found vesicular nuclei only in the coccal forms of old cultures and not in the rapidly growing filamentous forms which had only the chomosomal type of nucleus. Lack and Tanner (1953) have examined the changes in morphology of M . tuberculosis under conditions of nitrogen starvation which lead to lysis (Schaefer et al. 1949, Marshak & Schaefer 1952). They find that the nuclei swell to such an extent that the isolated cells take on a fusiform appearance. In those cells which are in cords or rafts, nuclear enlargement is accompanied by fusion of the cytoplasm of adjacent cells after which globules are formed which have the appearance of clusters of cocci. Similar coccoid forms are found in tissues of tuberculous organisms where the bacteria are growing under adverse conditions. Living forms may be distinguished from the dead ones by staining with malachite green and safranin, the viable cells staining green.
110
ALFRED M A R S H A K
X. FORMATION OF GIANTAND DWARF FORMS In a series of investigations, employing continuous observation with phase contrast, staining reactions, and electron microscopy, Tulasne (1953) has elucidated the sequence of events in the L cycle in Proteus and Salmonella. Under the appropriate conditions, the normal bacterium is converted to a giant form which may then become divided to reproduce the normal type. Alternatively, the giant cell may by rupture or other means give rise to minute bodies which are designated as dwarfs that have the same morphology as the pleuro-pneumonia-like organisms with dimensions in the range 0.1-0.3 p . The dwarfs may reproduce themselves, or in some circumstances give rise to giants. The formation of giants from the normal type begins with repeated chromosome division resulting in a filament containing as many as a hundred or more chromosomes. By swelling, the filament is then converted to a spherical body about 25 p in diameter which has no detectable cytoplasmic membrane. The dwarf has a Feulgen-positive nucleus and a very small amount of peripheral material whose composition is not defined but which appears to contain appreciable amounts of lipid and very little ribonucleic acid. That this peripheral substance plays the role of a kind of cytoplasm is deduced from the fact that the dwarf is metabolically active. Electron micrographs of tungstenshadowed dwarfs show crenulation of the surfaces parallel to the long axis of the particles, which Tulasne believes indicates a kind of granulation of the nuclear substance.
XI. COMPARISON WITH CELLSOF HIGHER FORMS One type of evidence needed to establish the homology of the chromatinic bodies of bacteria with nuclei and chromosomes of higher forms has, until recently, been lacking. Electron micrographs of whole bacteria have failed to reveal any internal chromosomal structure. Indeed, the bacterial nucleus rather surprisingly proved to be electron-optically less dense than the surrounding cytoplasm (Hillier et al., 1949). Even the electron micrographs of thin sections of bacteria, which show better resolution than those previously published, have not revealed any structures that might obviously be taken to be chromonemata or chromomeres. These sections showed the same less dense areas at the nuclear sites, seen in the whole bacteria, which appeared to have a fibrous structure and have contained within them bodies of a greater density, comparable to that of the cytoplasm (Chapman and Hillier, 1953). Unfortunately, criteria for clearly identifying chromosomal structures in electron micrographs of osmic acid-fixed preparations such as these are lacking even for the cells of higher plants and animals. Osmic acid has become the fixative of choice for electron microscopy, but it is not
BACTERIAL CYTOLOGY
111
necessarily the fixative best suited for revealing the relationship of objects visualized by this means to the familiar chromosome components of the cells of higher organisms as seen with the light microscope. Evidence for a chromonema-like internal structure in bacterial chromosomes has been obtained by procedures utilizing fixatives other than osmic acid (Marshak, 1951a, b) . By differential centrifugation of fragments obtained by subjecting E. coli (in the early log phase of growth) to sonic vibration in either acetic or citric acid solutions, a heavy particle fraction was isolated in which the particles had dimensions similar to those of the Feulgen and Giemsa-positive bodies of these bacteria. Electron micrographs of acetic acid-treated bacteria showed depressed empty areas in many of the cells which suggested loss by ejection of the nuclear mass. This phenomenon and the comparable one described by Bisset and by Robinow in spores indicates that the proteins of the bacterial nucleus are quite different from those of the cytoplasm. The heavy particle fraction, after being subjected to freeze-drying and chromium shadowing, was examined with the electron microscope. Almost all of the particles of this fraction showed a regular crenulation of the surfaces along the long axis. I n those particles which were not too dense, it could be seen that the crenulation was due to the presence of a helical structure, in which the coils of each helix were made up of bipartite strands each helically coiled. Fragments of about one-fourth the width of the usual heavy particle were found to contain a single helix composed of bipartite strands. The usual particle had four such helices. The structure seemed to be entirely comparable to the primary and secondary helices (major and minor coils) of the chromosomes of higher forms (Marshak, 1936). Although many types of structures examined in the electron microscope had been found to be helically coiled, this kind of system of helices had not been found elsewhere than in chromosomes. However, chemical analyses did not show any greater concentration of thymine in the heavy fraction than in either the light fraction or the whole bacteria. The significance of the chemical findings was not at all clear since loss of deoxyribonucleic acid to the small particle fraction during the vibration could not be excluded as a possibility. Most of the large particles could not be stained because they were coated with a dense black pigment, although occasionally such particles were found embedded in an amorphous matrix and these gave a positive Feulgen reaction. The morphology of the chromosome structures of E. coli described by Marsh& suggests comparison with the electron micrographs of the dwarf forms described by Tulasne (1953). Assuming that the surface lobulations in the electron micrographs of the dwarfs represent incompletely
112
ALFRED MARSHAK
resolved helices, the folIowing rough approximations may be made from measurements taken from Tulasne’s illustration (Tulasne, 1953, Fig. 12). The width of the dwarf, i.e. the diameter of the helix, is 0.32p, the pitch is 0 . 0 8 ~and the thickncss of the chromonema, i.e. the diameter of the minor coil, is 0.42,~.In E. coli chromosomes, the comparable measurements are 0.60~for the width of the chromosome pair or 0.30,~for the chromosome or chromatid width, depending upon whether the system of four helices or two helices is taken as the basic chromosome. The pitch of the primary helix (major coil) is 0 . 1 7 ~or exactly twice that of the dwarf, while the diameter of the secondary helix is 0.05p and its pitch 0.08~. In the bacterial viruses also, particularly in the immature forms, one finds an apparent helical structure. The electron micrographs by Wyckoff of immature E . coli bacteriophage T4 (Wyckoff, 1949; Fig. VIII, 32) and of immature Staphyocorrzcs K bacteriophage (Fig. VIII, 30) as well as those by Levinthal and Fisher (1952, Figs. 4 and 6) of E . coli bacteriophage T2 show that the principal structure of the phage at this stage is a helix with about one and one quarter turns. The dimensions of the diameter and the pitch, respectively, taken from the E . coli phage illustrations mentioned are approximately 0.7 and OSp which may he compared with the values 0.5 and 0.8,~for the E . coli chromosome. Although it must be recognized that these measurements are rough and limited, the simiIarity in the dimensions as well as the general morphology suggests the possibility of there being a basic structural unit common to the chromosomrs of normal bacteria, the dwarf or pleuro-pneumonia-like bacteria, and the bacterial viruses. It is of course quite possible that the correspondence in the dimensions found is accidental, but it would seem well worthwhile to make an extended series of accurate measurements in these superficially diverse organisms, to determine whether these indications are more than just coincidence. XII. CONCLUSIONS Perhaps our measures are still Procrustean. We may still be a long way from understanding the structural organization of the nucleus of the living bacterium as the disparity in the images observed with the electron microscope after osmic acid as compared with other fixatives suggests. The coordinated flocks of agglomerated flagella, such as those described by \-an Tterson (1953), may serve to remind us again of the possibility of obtaining regularities in fixed materials that are imposed by the procedures used. Nevertheless, since a uniform pattern seems to be emerging from diverse materials treated by various methods, we appear to be making a closer approach to reality. We do find sufficient points of resemblance between the chromosomes of higher organisms, the chromatinic bodies of
BACTERIAL CYTOLOGY
113
ordinary bacteria] the principal components of the pleuro-pneumonia-like organisms, and the bacterial viruses, to conclude with reasonable assurance that they are analogous in structure and probably in function.
XIII. REFERENCES Astbury, W. T., and Weibull, C. (1949) Nature 163, 280. Bergersen, F. J. (1953a) J. Gen. MicrobioZ. 9, 26. Bergersen, F. J. (1953b) J. Gm. Aficrobiol. 9, 353. Bisset, K. A. (1952) Exptl. Cell Research 3, 681. Bisset, K. A. (1953a) J. G m . Microbiol. 8, 50. Bisset, K. A. (1953b) Symposia on Bacterial Cytology. Fondazione Paterno, Rome, p. 9. Cassel, W. A. (1951) J . Bacteriol. 63, 239. Chance, H. L. (1953a) J. Bacteriol. 66, 593. Chance, H. L. (1953b) I. Bacteriol. 66, 239. Chapman, G. B., and Hillier, J. (1953) J. Bacteriol. 66, 362. Clark, J. B., and Webb, R. B. (1953) J. Bacteriol. 66, 498. DeLamater, E. D. (1953) Symposia on Bacterial Cytology, Fondazione Paterno, Rome, p. 108. Epstein, G. W., Ravich-Birger, E. D., and Svinkina, A. A. (1936) Giorn. battsriol. immunol. 16, 1. Feulgen, R., and Rossenbeck, H. (1924) 2. physiol. Chem. 135, 203. Hillier, J., Mudd, S., and Smith, A. G. (1949) I. Bacterial. 69, 319. Kellenberger, E. (1953) Symposia on Bacterial Cytology. Fondazione Paterno, Rome, p. 45. Lack, C. H., and Tanner, F. (1953) J. Ges. Microbiol. 8, 18. Levinthal, C., and Fisher, H. (1952) Biochim. e6 BiophysE Acta 9, 419. Marshak, A. (1936) J . Heredity 27, 459. Marshak, A. (1951a) Proc. Natl. Acad. Sci. US. 57, 38. Marshak, A. (19Slb) Exptl. Cell Research 2, 243. Marshak, A., and Schaefer, W. B. (1952) Am. Rev. Tuberc. 66, 75. Marshak, A., and Vogel, H. J. (1951) J. B i d . Chem. 189, 605. Mudd, S. (1953) Symposia on Bacterial Cytology. Fonsdazione Paterno, Rome, p. 67. Mudd, S., Winterschied, L C., DeLamater, E. D., and Henderson, H. J. (1951) I . Bacteriol. 62, 459. Palade, G. E. (1952) Anat. Record 114, 427. Piekarski, G. (1937) Arch. Mikrobiol. 8, 428. Piper, A., Crocker, C. G., Van der Walt, J. P., and Savage, N. (1953) J . Bacteriol. 66, 628. Robinow, C. F. (1944) J . H y g . 43, 413. Robinow, C. F. (1945) i s “The Bacterial Cell” (Dubs, ed.). Harvard Univ. Press, Cambridge. Robinow, C. F. (1953) J. Bacteriol. 66, 300. Robinow, C. F., and Murray, R. G. E. (1953) Exptl. Cell Research 4, 390. Shaefer, W. B., Marshak, A., and Burkhardt, B. (1949) I. Bacteriol. 68, 549. Schuster, G. P. K. (1952) 2. wis. Mikroskop. 61, 101. Shelton, E., and Schneider, W. C. (1952) Anat. Record 112, 61. Tomcsik, J., and Guex-Holtzer, S. (1951) Schweiz 2. allgem. Pathol. u. Baktrniol. 14, 515.
114
ALFRED M A R S H A K
Tulasne, R. (1953) Symposia on Bacterial Cytology. Fondazione Paterno, Rome, p. 144. van Iterson, W. (1953) Symposia on Bacterial Cytology. Fondazione Paterno, Rome, p. 24. Voit, K. (1925) Z . ges. exptl. Med. 47, 183. Weibull, C. (1953a) J . Bacteriol. 66, 137. Weibull, C. (1953b) J . Bacteriol. 66, 6%. Welshimer, H. J. (1953) I . Bacteriol. 66, 112. Winterseheid, L. C., and Mudd, S. (1953) Am. Rev. Tuberc. 67, 59. Wyckoff, R. W. G. (1949) “Electron Microscopy.” Interscience, N. Y .
I. 11. 111. IV. V. VI. VII. VIII. IX. X. XI. XII. XIII.
Introduction ........................................................ Nuclei ............................................................. Centrioles .......................................................... Chromosomes ...................................................... Mitochondria ....................................................... Vacuoles ........................................................... Membranes ........................................................ Transverse Wall: Flagella .......................................... Nucleus of Bacterial Spores ........................................ Formation of Giant and Dwarf Farms ............................... Comparison with Cells of Higher Forms ............................. Conclusions ........................................................ References .........................................................
Page 103 104 105 106 107 107 108 108 109 110 110 112 113
I. INTRODUCTION In the study of the fine details of the structure of bacteria, as in other human activities, the turning of the wheels of progress appears in retrospect to have been amazingly slow while in the turmoil and excitement of present activity much seems to be happening, although it is not easy to see how much of this is pure spin and how much represents traction onto new ground. The whims of fashion have again brought the bacterial nucleus into prominence, not only as judged by the volume of words about it appearing in the literature, but apparently also in the thinking of bacteriologists not formerly accustomed to giving it much consideration. The magic of the word chromosome, reintroduced into bacterial cytology in 1945, may have had a good deal to do with the increase in interest in the bacterial nucleus, but more likely the development of microbial genetics, with its concentration on genetic factors involved in the control of familiar biochemical systems, has prepared more minds for thinking in terms of nuclear phenomena in bacteria. Certainly the development of fundamentally new techniques has had little to do with it. We find the same words (chromosomes, centrioles, nucleolus, mitotic process) being applied now, as in the past, to images obtained by staining procedures not very different from those used 20-30 years ago. There are in the current literature indications of a very natural tendency to seek in bacteria morphological counterparts of intracellular structures seen in higher forms. It Seems apropos to quote some words of caution given some 20 years ago ..... if taking the point of view of the uniformity of the living world, we 103
104
ALFRED M A R S H A K
have all reason to expect the presence of a nucleus in bacteria as well as in any other form, we have not any right to demand that in the morphological respect their nuclear apparatus should entirely coincide with nuclei of different organisms. I t is the duty of the biologist to apprehend its actual structure and not lay it forcibly on the Procrustes bed of preconceived notions, and, by the way, many shrink from it only because they cannot seem to make it fit this bed.” (Epstein et al., 1936.)
11. NUCLEI Although Feulgen and Rossenbeck (1924) applied their new procedure to bacteria they obtained negative results, but in the following year Voit (1925) presented evidence for a positive nucleal reaction in bacteria, although localization of the thymonucleic acid in nuclei was not proven. However, by 1936 the technique was sufficiently well advanced for a clear demonstration of nuclei in particularly difficult material, M . tuberculosis, to be presented by Epstein et al. (1936). They found that, after lipid extraction with carbon &sulfide as well as alcohol, Feulgen-positive nuclei could be seen and readily distinguished from volutin granules. Piekarski (1937), with a fortunate choice of bacteria which did not require such extraction. demonstrated nuclei by the Feulgen procedure and also with the Giemsa stain, as modified by Badian, using aqueous eosin for differentiation and counterstaining. The latter procedure in various modified forms has become popular in recent years although it lacks the specificity of the Feulgen method, and the necessity for thorough lipid extraction prior to staining by either the Feulgen or modified Giemsa methods is still usually overlooked. I have applied the Feulgen procedure after thorough lipid extraction to a large variety of bacteria and with a single exception found very obviously identifiable nuclei ( Marshak, unpublished data ) , The exception was an unidentified mycobacterium isolated from soil in which no nucleus could be detected by either the Feulgen or acidGiemsa method, but which on chemical analysis was found to contain large quantities of thymine, the nitrogenous base considered to be peculiar to deoxyribonucleic acid (Marshak and Vogel, 1951) . The significance of this observation remains undetermined, but it is mentioned here to emphasize the desirability of exploring not only phenomena related to the bacterial nucleus which conform to an expected pattern, but also those apparent exceptions which may ultimately throw some light on the general problem of nuclear organization.
BACTERIAL CYTOLOGY
10s
111. CENTRIOLES Having become aware of the existence of bacterial nuclei, it was to be expected that the appurtenances of the nuclear apparatus would be sought and, as in other instances in cytology, where observations are made on structures near the limit of resolution of the light microscope, the inevitable controversy over interpretation has again arisen. DeLamater (1953) has described granules which he considers to be centrioles. They are stained purple by his double staining procedure, which is a combination of the thionine-SO2 procedure he describGd previously, and a modification of the Cassel procedure ( 1951) in which the cells are mordanted with tannic acid before staining with basic fuchsin. The spindle and chromosomes are also purple, the cell wall red and “mitochondria” a “redviolet”. Bisset (1952, 1953a, b), using the combination stain, Giemsa and tannic acid-crystal violet, but avoiding dehydration by making examinations in water mounts, concludes that the structures called centrioles and mitochondria by DeLamater, Mudd et aE. (19Sl), and Mudd (1953) are nothing more than areas of greater basophilia at the growing tips and points of division of the cells. The picture is complicated by the designation of some granules (which also appear in association with the spindle but not at its poles) as “mitochondria,” i.e. those granules that stain redviolet rather than purple, which seems to be a rather fine distinction (DeLamater, 1953). By treatment with drugs (colchicine, formaldehyde, and antibiotics) DeLamater finds arrests or deviations from the normal behavior which support his contention that the granules associated with the spindle are centrioles. According to his interpretation the centriole is at first associated with the nuclear membrane, then migrates away from it as the spindle develops and elongates. In prometaphase there is a single centriole which then divides, each half going to the spindle poles as the cell proceeds into metaphase and anaphase. It is claimed that the drug action enlarges the centriole. Arrests at various phases of the centriole division cycle may be produced by the actinomycete-derived antibiotics which also inhibit mitosis in B. megatherium. Para-aminosalicylic acid differs from the above in inhibiting centriolar division resulting in unipolar spindles and increase in size of the nuclear mass suggestive of polyploidy. In the recovery from antibiotic inhibition there is segmentalization of the elongated nuclear mass into portions, each of which eventually contain the three chromosomes of the haploid complement. Colchicine acts differently in these organisms in producing directly a doubling of the chromosomes. Work by Szybalski and Hunter is cited by DeLamater and illustrations are given of stages purported to be zygotene and pachytene.
106
ALFRED MARSHAK
Hunter (cited by D e b a t e r ) has described a complete meiotic cycle for Micrococcus cryophilus from zygotene through the two meiotic divisions, but this reviewer has not yet seen the published descriptions. IV.
CHROMOSOMES
A fine series of parallel observations made with the electron microscope and with the light microscope after acid-Giemsa staining on strains of E . coli has been given by Kellenberger (1953). During the exponential phase of growth all of the nucleoid bodies have the form of chromosomes, as described by Robinow (1944, 1945). At the end of the growth phase the chromosome-like appearance is no longer found but instead there are larger masses of ellipsoidal, dumbbell-shaped, and rod-like bodies. Strain C of E . coli shows a characteristic peripheral disposition of the “chromosomes’’ instead of the paracentral one found in strains S and K12. In the presence of low concentrations of aureomycin or chloromycetin, the nucleoid body assumes a globular shape with a denser portion usually more or less centrally located in the less dense mass of the nucleoid. In strain C the formation of a similar centrally located body is induced by action of the antibiotic. Upon returning the bacteria to medium free of antibiotic, the “chromosomes” again appear at first in numbers much greater than normal (polychromosome phase), after which they regroup to form the normal nucleoids following which cell division takes place. The polychromosomal condition is also induced by exposure to ultraviolet light or to methyl-bis-chloroethylamine, but the polychromosome stage in these cases is followed by one suggesting fragmentation of the chromosomes to such an extent that they appear to fill the cell. Subsequently in those cells which do not lyse there is a regrouping to form the usual nucleoids. The amount of fragmentation appears to be dependent upon dose. Kellenberger makes the interesting observation that the agents which are effective in the conversion of prophage to phage, all induce the “fragmented chromosome” stage in E. coli. H e suggests that the mechanical rupturing of the chromosomes liberates the prophage from inhibiting influences so that it may then proceed to develop at the expense of the bacterial cell. Using staining procedures on B . megatherium, Bergersen (1953a) reports apparent fusion of paired nuclear bodies, when the culture proceeds beyond the logarithmic phase, to result in the formation of elongated chromatinic bars which then become converted to “zigzags” of short filaments. Subsequently on division of the cells typical bacterial rods are formed with a single, central, elliptical nuclear body. H e finds that chloroamphenicol appears to inhibit cell division while cell growth and increase in the chromatinic material goes on ( Bergersen, 1953b).
BACTERIAL CYTOLOGY
107
V. MITOCHONDRIA Mudd (1953) and his co-workers (Mudd et al. 1951; Winterschied and Mudd, 1953) have described localization of Janus green, the indophenol blue of the Nadi reagent, and the formazan of the tetrazoliums in definite regions or granules. This localization has been taken as an index of oxidation-reduction activity which is assumed to be characteristic of mitochondria, and hence that the regions defined by accumulation of these materials are mitochondria. Bisset (1952, 1953a, b) has pointed out that the granules designated as mitochondria are in regions of active growth, and hence of synthesis and intense oxidation-reduction activity, so that the agents used as indicators would be expected to accumulate there even if they contained no specialized organelles analogous to the mitochondria of higher forms. Chapman and Hillier (1953) have published some very fine electron micrographs of thin sections of B. cereus which show “peripheral bodies” or vacuoles which appear empty except for small dense granules. These are located near the site of formation of new transverse septa corresponding to the regions of the mitochondria1 bodies of Mudd et al. However, there is no indication of any of the typical mitochondrial structure (Palade, 1952). It should be mentioned that granules of formazan from tetrazolium do not necessarily accumulate in mitochondria. The writer has observed formazan granules in psoas muscle of the rat, which has no mitochondria (unpublished data). In B. megatherium fixed in Os04 vapor and stained with acid-Giemsa, Bergersen (1953a) has found stained granules similar to those described by Bisset at the terminal growing points and at the lateral septa. These regions also stained with Janus green and the Nadi reagent. Weibull (1953a) has found by continuous observation in phase contrast of B. megatherium in triphenyltetrazolium solutions that the formazan first appears as minute granules which then coalesce to form the large secondary granules described as mitochondria by Mudd (1953). He pointed out that the primary granules may indicate sites of oxidation-reduction activity but the larger secondary ones certainly do not. I n mammalian tissue where succinic dehydrogenase is associated exclusively with mitochondria it has been impossible to obtain a clear indication of staining within the mitochondria, although formazan is abundantly produced at their surfaces. It has also been found that intracellular fat droplets will be colored by the tetrazolium salts (Shelton and Schneider, 1952). VI. VACUOLES
Schuster ( 1952) has described vacuoles, which are probably liquidfilled, in Malleomyces mallei; these were observed using both light and electron microscopes. On further growth in high phosphate media,
10s
ALFRED MARSHAK
granules appear at the vacuolar sites which consist primarily of metaphosphate as determined by toluidine blue staining and extraction techniques. VII. MEMBRANES M‘eibull (1953b) has been able to remove the cell walls of B. megnthe-
riUnt with lysozyme and preserve the protoplast by keeping the cells in
sucrose solution. When they are transferred to buffer or saline solutions they lyse. By centrifugation of the lysed cells, ghosts were isolated which were considered to be the cytoplasmic membranes. However, since the ghosts are pigmented and contain enzymes it seems to the writer more likely that the ghosts are cytoplasmic residua rather than membranes. The absence of any true cytoplasmic membrane in electron micrographs of thin sections of bacteria (Chapman and Hillier, 1953) also indicates the necessity for caution in the use of the term membrane in such cases as these. Weibull was unable to recover nuclei from his protoplasts but did obtain a rapidly sedimenting gel which appeared to be made up primarily of deoxyribonucleic acid. Robinow and Murray ( 1953) have described specific staining of the cytoplasmic surface of some bacteria. They find a thin cytoplasmic membrane in electron micrographs of cells in which retraction of the protoplast from the cell wall is induced by fixation in formalin. However, judging from the micrographs published, the “menibrane” may be the residual cytoplasm not clumped with the main mass. This interpretation seems all the more plausible since inspection of the micrograph reveals at least two lines of density demarcation instead of the one expected on the membrane hypothesis. The protoplast surface does appear to provide a limiting surface which precipitates or accumulates the dye, but these observations raise again rather than settle the old question of whether there is a membrane in the structural sense, or a surface with differentiated physiological properties. Chapman and Hillier ( 1953) deduce from their studies of thin sections of E. coli that if a structural cytoplasmic membrane does exist, it must be less than 40 d wide.
VIII. TRANSVERSE WALL:FLAGELLA Analyzing the behavior of lysozynie-treated B. megatheritim towards stains, Welshimer (1953) finds evidence for different actions of the enzyme on the wall and on the capsular substances, an observation which is correlated with previous reports of polypeptides found in the capsule but not in the wall (see also Tomcsik and Guex-Holtzer, 1951j . The formation of the transverse wall by growth through invagination of the peripheral wall is clearly shown in the electron micrographs of Chapman and Hillier. They find the wall to be 200 d thick, with a n inner
BACTERIAL CYTOLOGY
109
portion which is denser and distinct from the outer portion. The transverse wall material is laid down as an annulus growing inward until a solid disc is formed, after which it thickens and divides into two layers. The cytoplasmic surface is always in contact with the forming wall. Van Iterson ( 1953) has described flagella in Spirillum, Proteus, and Vibrio, with definite basal granules which appear to be located in the cytoplasm beneath the cell wall. Piper et aE. (1953) believe that the flagellum of Spirillurn serpens is a continuation of the cell wall to which it is attached by a narrow stem. They find marked differences in the structure of flagella of Vibrio, Salmonella, and Spirillum. Van Iterson (1953) has shown that the flagellum of Vibrio has a definite sheath of material differing from that of the core. She concludes that motion of the flagellum cannot be as simple as the monomolecular mechanism proposed by Astbury ( 1949).
IX. NUCLEUS OF BACTERIAL SPORES Robinow (1953) now agrees with Bisset’s interpretation of the nuclear apparatus of bacterial spores. The spore contains a more or less central, vesicular, Feulgen-positive nucleus. Any of the procedures which involve acid hydrolysis result in the ejection of the nuclear material through a weak point in the spore wall to produce a diffusely Feulgen-positive mass on the surface of the spore. Chance (1953a, b) has described observations on stained preparations which suggest that in some species the transverse wall is laid down as a plate beginning at, or in the dividing nucleus in a manner resembling that found in higher plants. Clark and Webb (1953) have described instances where nuclei of the vesicular type, apparently in a state of division, are associated with a developing cell plate, suggesting to them that this type of nucleus is not limited to the resting stages. However, in Nocardia corallina, they found vesicular nuclei only in the coccal forms of old cultures and not in the rapidly growing filamentous forms which had only the chomosomal type of nucleus. Lack and Tanner (1953) have examined the changes in morphology of M . tuberculosis under conditions of nitrogen starvation which lead to lysis (Schaefer et al. 1949, Marshak & Schaefer 1952). They find that the nuclei swell to such an extent that the isolated cells take on a fusiform appearance. In those cells which are in cords or rafts, nuclear enlargement is accompanied by fusion of the cytoplasm of adjacent cells after which globules are formed which have the appearance of clusters of cocci. Similar coccoid forms are found in tissues of tuberculous organisms where the bacteria are growing under adverse conditions. Living forms may be distinguished from the dead ones by staining with malachite green and safranin, the viable cells staining green.
110
ALFRED M A R S H A K
X. FORMATION OF GIANTAND DWARF FORMS In a series of investigations, employing continuous observation with phase contrast, staining reactions, and electron microscopy, Tulasne (1953) has elucidated the sequence of events in the L cycle in Proteus and Salmonella. Under the appropriate conditions, the normal bacterium is converted to a giant form which may then become divided to reproduce the normal type. Alternatively, the giant cell may by rupture or other means give rise to minute bodies which are designated as dwarfs that have the same morphology as the pleuro-pneumonia-like organisms with dimensions in the range 0.1-0.3 p . The dwarfs may reproduce themselves, or in some circumstances give rise to giants. The formation of giants from the normal type begins with repeated chromosome division resulting in a filament containing as many as a hundred or more chromosomes. By swelling, the filament is then converted to a spherical body about 25 p in diameter which has no detectable cytoplasmic membrane. The dwarf has a Feulgen-positive nucleus and a very small amount of peripheral material whose composition is not defined but which appears to contain appreciable amounts of lipid and very little ribonucleic acid. That this peripheral substance plays the role of a kind of cytoplasm is deduced from the fact that the dwarf is metabolically active. Electron micrographs of tungstenshadowed dwarfs show crenulation of the surfaces parallel to the long axis of the particles, which Tulasne believes indicates a kind of granulation of the nuclear substance.
XI. COMPARISON WITH CELLSOF HIGHER FORMS One type of evidence needed to establish the homology of the chromatinic bodies of bacteria with nuclei and chromosomes of higher forms has, until recently, been lacking. Electron micrographs of whole bacteria have failed to reveal any internal chromosomal structure. Indeed, the bacterial nucleus rather surprisingly proved to be electron-optically less dense than the surrounding cytoplasm (Hillier et al., 1949). Even the electron micrographs of thin sections of bacteria, which show better resolution than those previously published, have not revealed any structures that might obviously be taken to be chromonemata or chromomeres. These sections showed the same less dense areas at the nuclear sites, seen in the whole bacteria, which appeared to have a fibrous structure and have contained within them bodies of a greater density, comparable to that of the cytoplasm (Chapman and Hillier, 1953). Unfortunately, criteria for clearly identifying chromosomal structures in electron micrographs of osmic acid-fixed preparations such as these are lacking even for the cells of higher plants and animals. Osmic acid has become the fixative of choice for electron microscopy, but it is not
BACTERIAL CYTOLOGY
111
necessarily the fixative best suited for revealing the relationship of objects visualized by this means to the familiar chromosome components of the cells of higher organisms as seen with the light microscope. Evidence for a chromonema-like internal structure in bacterial chromosomes has been obtained by procedures utilizing fixatives other than osmic acid (Marshak, 1951a, b) . By differential centrifugation of fragments obtained by subjecting E. coli (in the early log phase of growth) to sonic vibration in either acetic or citric acid solutions, a heavy particle fraction was isolated in which the particles had dimensions similar to those of the Feulgen and Giemsa-positive bodies of these bacteria. Electron micrographs of acetic acid-treated bacteria showed depressed empty areas in many of the cells which suggested loss by ejection of the nuclear mass. This phenomenon and the comparable one described by Bisset and by Robinow in spores indicates that the proteins of the bacterial nucleus are quite different from those of the cytoplasm. The heavy particle fraction, after being subjected to freeze-drying and chromium shadowing, was examined with the electron microscope. Almost all of the particles of this fraction showed a regular crenulation of the surfaces along the long axis. I n those particles which were not too dense, it could be seen that the crenulation was due to the presence of a helical structure, in which the coils of each helix were made up of bipartite strands each helically coiled. Fragments of about one-fourth the width of the usual heavy particle were found to contain a single helix composed of bipartite strands. The usual particle had four such helices. The structure seemed to be entirely comparable to the primary and secondary helices (major and minor coils) of the chromosomes of higher forms (Marshak, 1936). Although many types of structures examined in the electron microscope had been found to be helically coiled, this kind of system of helices had not been found elsewhere than in chromosomes. However, chemical analyses did not show any greater concentration of thymine in the heavy fraction than in either the light fraction or the whole bacteria. The significance of the chemical findings was not at all clear since loss of deoxyribonucleic acid to the small particle fraction during the vibration could not be excluded as a possibility. Most of the large particles could not be stained because they were coated with a dense black pigment, although occasionally such particles were found embedded in an amorphous matrix and these gave a positive Feulgen reaction. The morphology of the chromosome structures of E. coli described by Marsh& suggests comparison with the electron micrographs of the dwarf forms described by Tulasne (1953). Assuming that the surface lobulations in the electron micrographs of the dwarfs represent incompletely
112
ALFRED MARSHAK
resolved helices, the folIowing rough approximations may be made from measurements taken from Tulasne’s illustration (Tulasne, 1953, Fig. 12). The width of the dwarf, i.e. the diameter of the helix, is 0.32p, the pitch is 0 . 0 8 ~and the thickncss of the chromonema, i.e. the diameter of the minor coil, is 0.42,~.In E. coli chromosomes, the comparable measurements are 0.60~for the width of the chromosome pair or 0.30,~for the chromosome or chromatid width, depending upon whether the system of four helices or two helices is taken as the basic chromosome. The pitch of the primary helix (major coil) is 0 . 1 7 ~or exactly twice that of the dwarf, while the diameter of the secondary helix is 0.05p and its pitch 0.08~. In the bacterial viruses also, particularly in the immature forms, one finds an apparent helical structure. The electron micrographs by Wyckoff of immature E . coli bacteriophage T4 (Wyckoff, 1949; Fig. VIII, 32) and of immature Staphyocorrzcs K bacteriophage (Fig. VIII, 30) as well as those by Levinthal and Fisher (1952, Figs. 4 and 6) of E . coli bacteriophage T2 show that the principal structure of the phage at this stage is a helix with about one and one quarter turns. The dimensions of the diameter and the pitch, respectively, taken from the E . coli phage illustrations mentioned are approximately 0.7 and OSp which may he compared with the values 0.5 and 0.8,~for the E . coli chromosome. Although it must be recognized that these measurements are rough and limited, the simiIarity in the dimensions as well as the general morphology suggests the possibility of there being a basic structural unit common to the chromosomrs of normal bacteria, the dwarf or pleuro-pneumonia-like bacteria, and the bacterial viruses. It is of course quite possible that the correspondence in the dimensions found is accidental, but it would seem well worthwhile to make an extended series of accurate measurements in these superficially diverse organisms, to determine whether these indications are more than just coincidence. XII. CONCLUSIONS Perhaps our measures are still Procrustean. We may still be a long way from understanding the structural organization of the nucleus of the living bacterium as the disparity in the images observed with the electron microscope after osmic acid as compared with other fixatives suggests. The coordinated flocks of agglomerated flagella, such as those described by \-an Tterson (1953), may serve to remind us again of the possibility of obtaining regularities in fixed materials that are imposed by the procedures used. Nevertheless, since a uniform pattern seems to be emerging from diverse materials treated by various methods, we appear to be making a closer approach to reality. We do find sufficient points of resemblance between the chromosomes of higher organisms, the chromatinic bodies of
BACTERIAL CYTOLOGY
113
ordinary bacteria] the principal components of the pleuro-pneumonia-like organisms, and the bacterial viruses, to conclude with reasonable assurance that they are analogous in structure and probably in function.
XIII. REFERENCES Astbury, W. T., and Weibull, C. (1949) Nature 163, 280. Bergersen, F. J. (1953a) J. Gen. MicrobioZ. 9, 26. Bergersen, F. J. (1953b) J. Gm. Aficrobiol. 9, 353. Bisset, K. A. (1952) Exptl. Cell Research 3, 681. Bisset, K. A. (1953a) J. G m . Microbiol. 8, 50. Bisset, K. A. (1953b) Symposia on Bacterial Cytology. Fondazione Paterno, Rome, p. 9. Cassel, W. A. (1951) J . Bacteriol. 63, 239. Chance, H. L. (1953a) J. Bacteriol. 66, 593. Chance, H. L. (1953b) I. Bacteriol. 66, 239. Chapman, G. B., and Hillier, J. (1953) J. Bacteriol. 66, 362. Clark, J. B., and Webb, R. B. (1953) J. Bacteriol. 66, 498. DeLamater, E. D. (1953) Symposia on Bacterial Cytology, Fondazione Paterno, Rome, p. 108. Epstein, G. W., Ravich-Birger, E. D., and Svinkina, A. A. (1936) Giorn. battsriol. immunol. 16, 1. Feulgen, R., and Rossenbeck, H. (1924) 2. physiol. Chem. 135, 203. Hillier, J., Mudd, S., and Smith, A. G. (1949) I. Bacterial. 69, 319. Kellenberger, E. (1953) Symposia on Bacterial Cytology. Fondazione Paterno, Rome, p. 45. Lack, C. H., and Tanner, F. (1953) J. Ges. Microbiol. 8, 18. Levinthal, C., and Fisher, H. (1952) Biochim. e6 BiophysE Acta 9, 419. Marshak, A. (1936) J . Heredity 27, 459. Marshak, A. (1951a) Proc. Natl. Acad. Sci. US. 57, 38. Marshak, A. (19Slb) Exptl. Cell Research 2, 243. Marshak, A., and Schaefer, W. B. (1952) Am. Rev. Tuberc. 66, 75. Marshak, A., and Vogel, H. J. (1951) J. B i d . Chem. 189, 605. Mudd, S. (1953) Symposia on Bacterial Cytology. Fonsdazione Paterno, Rome, p. 67. Mudd, S., Winterschied, L C., DeLamater, E. D., and Henderson, H. J. (1951) I . Bacteriol. 62, 459. Palade, G. E. (1952) Anat. Record 114, 427. Piekarski, G. (1937) Arch. Mikrobiol. 8, 428. Piper, A., Crocker, C. G., Van der Walt, J. P., and Savage, N. (1953) J . Bacteriol. 66, 628. Robinow, C. F. (1944) J . H y g . 43, 413. Robinow, C. F. (1945) i s “The Bacterial Cell” (Dubs, ed.). Harvard Univ. Press, Cambridge. Robinow, C. F. (1953) J. Bacteriol. 66, 300. Robinow, C. F., and Murray, R. G. E. (1953) Exptl. Cell Research 4, 390. Shaefer, W. B., Marshak, A., and Burkhardt, B. (1949) I. Bacteriol. 68, 549. Schuster, G. P. K. (1952) 2. wis. Mikroskop. 61, 101. Shelton, E., and Schneider, W. C. (1952) Anat. Record 112, 61. Tomcsik, J., and Guex-Holtzer, S. (1951) Schweiz 2. allgem. Pathol. u. Baktrniol. 14, 515.
114
ALFRED M A R S H A K
Tulasne, R. (1953) Symposia on Bacterial Cytology. Fondazione Paterno, Rome, p. 144. van Iterson, W. (1953) Symposia on Bacterial Cytology. Fondazione Paterno, Rome, p. 24. Voit, K. (1925) Z . ges. exptl. Med. 47, 183. Weibull, C. (1953a) J . Bacteriol. 66, 137. Weibull, C. (1953b) J . Bacteriol. 66, 6%. Welshimer, H. J. (1953) I . Bacteriol. 66, 112. Winterseheid, L. C., and Mudd, S. (1953) Am. Rev. Tuberc. 67, 59. Wyckoff, R. W. G. (1949) “Electron Microscopy.” Interscience, N. Y .