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Electron micrographs of sections of E. coli cells infected with the bacteriophage T4
12
BIOCHIMICA ET BIOPHYSICA ACTA
ELECTRON CELLS
MICROGRAPHS
INFECTED
WITH
OF SECTIONS THE
VOL. 15 (1954)
OF E. COLI
BACTERIOPHAGE
T4
by O. MAALOE AND A. BIRCH-ANDERSEN* Statens Serum Institut, Copenhagen (Denmark) AND
F. S. SJOSTRAND Department o] Anatomy, Karolinska Institute/., Stockholm (Sweden)
I n this j o u r n e l we r e c e n t l y described a m e t h o d for o b t a i n i n g t h i n sections of bacterial cells, suitable for h i g h - r e s o lu ti o n e l e c t r o n m i c r o s c o p y 1. I t was m e n t i o n e d t h a t m i erographs of coli cells infected w i t h t h e b a c t e r i o p h a g e T 4 suggest t h a t c e r t a i n s t r u c t u r e s seen inside t h e n o r m a l cell c o n t a i n desoxyribosenucleic acid (DNA) ; we shall n o w pres e n t some of these m i c r o g r a p h s a n d discuss t h e possibilities a n d l i m i t a t i o n s of t h e techniqu e from t h e p o in t of v i e w of s t u d y i n g cell m o r p h o l o g y d u r i n g p h a g e m u l t i p l i c a t i o n . METHODS AND MATERIAL The fixation, embedding and sectioning techniques as well as the equipment used for taking the micrographs were described in our first communication 1. Strain B, of E. coli, has again been used, this time after infection with phage T4r+. Vigorously aerated broth cultures with logarithmically growing bacteria at a density of about io 8 ceils per ml were multiply infected, and at various times phage synthesis was stopped by addition of cyanide and immediately thereafter OsO 4 was added to initiate fixation as described previouslyx. Techniques similar to those given by ADAMS~ were used to control multiplicity of infection and phage development. It was observed that OsO~ added z7 or more minutes after infection causes rapid lysis of part of the cellg; the later the addition the larger the fraction of cells that lyse. It was attempted, but without success, to counteract this effect of OsO 4 by reinfecting the cultures at i2 mill with IO T4 r+ particles per cell, thus establishing conditions known to inhibit normal lysis. Micrographs were also made of free phage particles which for technical reasons were agglutinated with anti-phage serum prior to fixation, embedding and sectioning.
MORPHOLOGICAL
OBSERVATIONS
T h e m i c r o g r a p h s of infected cells are best described b y c o m p a r i s o n w i t h those of norm al , growing cells. I n th e la tt e r , t h e b u l k of t h e m a t e r i a l seen in t h e m i c r o g r a p h s presents itself as a " s p o n g y " s t r u c t u r e w h i c h su r r o u n d s large " v a c u o l e s " c o n t a i n i n g t i g h t l y compressed, or wound, c e n t r a l s t r u c t u r e s of h i g h o p a c i t y ; t h e s p o n g y s t r u c t u r e is a c c e n t u a t e d a n d v e r y o p a q u e in a n a r r o w zone a r o u n d t h e vacuoles. I n our first p a p e r a l e n g t h w i s e section of such a cell, showing t w o vacuoles was p r e s e n t e d ((I), Fig. 2); * Working on this study in the Department of Anatomy, Karolinska Institutet, Stockholm; aided by a grant from the P. Carl Petersen Foundation. Re#rences p. z 9.
VOL. 15 (1954)
E. coli CELLS INFECTED WITH BACTERIOPHAGE T 4
13
Fig. I. Section of cell in l o g a r i t h m i c p h a s e of g r o w t h , s h o w i n g c o m p a c t a n d o p a q u e central s t r u c t u r e s in t h e " v a c u o l e " . Magnification 64,00o. T h e b l a c k a r e a s to t h e r i g h t a n d on t h e lower cell b o r d e r s h o w particles of c a r b o n b l a c k d e p o s i t e d on t h e m o u n t s to facilitate focussing.
the micrograph presented as Fig. I of the present paper originates from the same preparation, is technically better but shows only one vacuole. Infected samples of the culture producing the normal cells just described were used to make the preparations of which micrographs will now be presented. Thes.e samples were fixed 2 ~ , 8, 18 and 25 min after infection and they are described below in that order. To study changes that might occur very soon after infection, bacteria were mixed with about 15o T4r+ particles per cell in order to ensure complete infection of the whole culture within less than 15 seconds. After 2 ~ min cyanide was added and fixation initiated. Fig. 2 shows a typical central section in which all the elements of the normal cell can still be identified. The most obvious change caused by infection concerns the central bodies of the vacuoles, which, after infection, present themselves as a loose network of threads. In addition the vacuoles have become elongated and quite irregular; it is not possible to decide whether threads from neighbouring vacuoles mix at this stage although some micrographs suggest a fusion of vacuoles.--We have not been able to observe phage particles attached to the cell surface despite the high multiplicity of infection; the extensive damage done to the cell membranes during and after dehydration 1 m a y account for this failure. The morphological picture just described changes rapidly. Micrographs not to be presented here show that 3-4 min after infection m a n y cells already resemble those of Fig. 3, which were fixed 8 min afteri nfection. At that stage radical changes in the organisation of the intracellular structures are observed; the central regions are filled with an irregular structure of low opacity while in the periphery of the cells a large number of distinct loci have developed. These foci present themselves as light areas with a round or elongated dark body inside, and bordered b y regions with accentuated and very opaque Re/erences p. 19.
14
o . MA&LOE, A. BIRCH-ANDERSEN, F. S. SJOSTRAND
VOL. 1 5
(1954)
l"ig. 2. (;ell fixed 2 I/~ minutes after heavy infection with phage T4 r~, presenting very large vacuoles with loose n e t w o r k s of coarse threads. Magnification 7S,ooo.
voL. 15 (1954)
E. coli CELLS INFECTED WITH BACTERIOPHAGE T4
15
Fig. 3. Cells fixed 8 m i n u t e s after xo-fold infection with T4r+. Marginal foci surrounded by irregular dark structures and containing rounded opaque bodies seen m all ceils. Magnification 65,000.
16
O. MAALOE, A. BIRCH-ANDERSEN, F. S. SJ6STRAND
VOL. 1 5 (1954)
Figs. 4, 5, a n d 6. Portions of sections of ceils fixed i8 m i n u t e s after lo-fold infection with T4r+. T h e central regions h a v e a g a i n become clear a n d f r e q u e n t l y s h o w webs of fine t h r e a d s (Fig. 4); occasionally o p a q u e bodies, v e r y similar to t h e sections of free p h a g e particles, are seen (Figs. 5 a n d 6). Magnifications 5i,ooo, 61,ooo a n d 74,ooo, respectively.
VOL. 15 (1954)
E. coli CELLS INFECTED WITH BACTERIOPHAGE T 4
17
spongy structure of the type found in the vicinity of the central vacuoles of the normal growing cell. The bodies inside the marginal foci have the same high opacity as the threads of Figs. I and 2, and usually show circular or elliptical cross-sections with diameters of about 400 A, although in some cases long threads with the same diameter have been found in elongated peripheral foci. Assuming that the loci are independent units, it m a y be estimated that cells of varying size contain 5o--2oo such units; it cannot be excluded, however, that the loci represent sections through peripheral ducts contairing long threads of material of the same type as that which constitutes the compact central bodies of the vacuoles of the uninfected cells and which forms a looser network just after infection. It will be recalled that the infected cells gradually become susceptible to Os04, partial lysis occurring if fixation is not initiated until 17 or more min after infection. Thus in preparations fixed late only the cells resistant to OsO 4 can be studied, and it may be assumed that these cells constitute the fraction of the population which progresses most slowly towards spontaneous lysis. These cells show a clear central region extending throughout the length of the cell and bordered by a distinct zone of granular and very opaque structure within, or outside of, which are seen the few bodies which we tentatively identify with newly formed phage particles (see below). There is thus a striking difference between the micrographs of cells fixed 8 and, say, 18 min after infection repectively; the appearance of a clear central region and the disappearance of the marginal foci being characteristic of the change in morphology as the time for normal lysis approaches. It cannot at the moment be said whether the marginal foci correspond in number and localisation to the newly formed phage particles. In micrographs such as Figs 4, 5 and 6 newly formed particles may be looked for. Before attempting to identify any intracellular structures with phage, micrographs were made of free particles fixed, embedded and sectioned in the same way as the bacterial cells. Phage particles treated in this way show very opaque, non-differentiated crosssections with hexagonal outlines and diameters of about 500 A; in some cases a faint halo of much lighter material can be seen surrounding the dark core.-- Inside some sectioned cells bodies are seen that look like the sectioned phage particles; they are usually observed within the diffuse structures in the periphery of the cells and therefore more or less masked. In this connection it must be remembered that the volume of a longitudinal section, about 200 A units in thickness, is about 3-4% of the volume of the bacterial cell, and that 18 min after infection the average number of finished phage particles per ceil is only about 20. For these reasons, and because the cells that lyse on addition of OsO 4 probably are those containing the greatest number of new phage particles, only relatively few sections can be expected to contain complete new particles. We are thus not in a position, at the present time, to say anything definite about the distribution of the new phage particles inside the cells; a better technique permitting fixation of cells containing large numbers of new particles must be developed. DISCUSSION
In our first paper I reference was made to the present study and to the results obtained by LURIA AND HUMAN3 and by MURRAY, GILLEN AND HEAGY4 with staining techniques. It was noted that the redistribution of the material which the staining method suggests is DNA, corresponds to the redistribution observed after infection of the opaque Re/erences p. 19.
I8
O. MAALOE, A. BIRCH-ANDERSEN, F. S. SJOSTRAND
VOL. 15 (~954)
material constituting the central bodies inside the vacuoles of the uninfected cells (Figs. 2 and 3); we concluded t h a t the opaque material probably contains DNA. This interpretation is strongly supported b y the finding that the cores of free phage particles, known to be composed largely of DNA, show the same high opacity as the central bacterial bodies. This observation is difficult to reconcile with the view of ROBINOWs, that the light, or "empty-looking" regions, seen in OsO,-fixed, embedded and sectioned bacterial spores, are the sites of the nuclear DNA. MURRAY, who has employed several fixing and staining methods in a study of the nuclei of bacillus cereus 8, t e n d s to believe t h a t the basophilic materi31, left in the nuclear regions after hydrolysis or treatment with ribonuclease, is located in relatively light areas in the periphery of these regions. Such an arrangement is not suggested by our micrographs; as mentioned above, the pictures of sectioned bacteriophage particles indicate that the opaque central threads or bodies seen in the vacuoles of the sectioned coli cells m a y well represent tightly coiled DNA-structures. I t is not possible, at this moment, to say whether fixation or dehydration cause the DNA-structures of the living cell to contract to some extent. However this m a y be, our a s s u m p t i o n - - t h a t DNA-structures are present as highly opaque bodies in the central regions of the "nuclear vacuoles" seen in sectioned coli cells--is strikingly different from the arrangements proposed by ROBINOW5 and MURRAYs for bacterial spores and the nuclei of bacillus cereus. In particular we believe that accumulations of DNA (or RNA) must reveal themselves as opaque areas in electron micrographs, and that " e m p t y areas" cannot correspond to regions with appreciable concentrations of nucleic acid. The above paragraphs essentially concern the interpretation of the central structures seen in uninfected cells. The present material should be examined from the point of view of studying phage multiplication also. In this respect, however, the amount of new information so far obtained is slight. It has been pointed out that technical difficulties have prevented us from making sections showing phage particles absorbed on the cell membranes and sections with m a n y newly formed particles. These difficulties m a y be overcome by developing better fixation techniques; it is more doubtful whether it will be possible ever to study the initiation of phage multiplication by means of the E.M. FRASER AND WILLIAMS7 have obtained micrographs of phage particles in the process of liberating their DNA contents; the extruded material appears as an irregular bundle of very long threads, 20-25 A in diameter, and if this is the state in which the DNA of the infecting phage particle enters the bacterial cell 8, it will be impossible to identify this material after it has become mixed with the structures of the normal bacterium, the elements of which are irregularly arranged and of about the same dimensions. W h a t we can obtain, on the other hand, is evidence of the changes in the microstructures of the host cell, brought about b y infection with phage: at this level we have seen how the "nuclear" apparatus of the cell is almost instantaneously affected (Fig. 2) in a way suggesting loosening of and fusion throughout the cell of the material normally tightly wound in the centers of distinct vacuoles. It must be recalled, however, that the micrographs showing this change are produced after infection with about Ioo phage particles per cell ; it is thus not certain that such profound changes necessarily follow after infection with one or a few particles. Unfortunately the cytological method does not lend itself readily to a study of cells infected singly or with very few particles since, in this case, a considerable fraction of the cells remain uninfected, without being distinguishable as such. In our exRelerences p. 19.
VOL. 15
(1954)
E. coli CELLS INFECTED WITH BACTERIOPHAGE
T4
19
p e r i m e n t s n o v i s i b l e lysis w a s i n d u c e d b y t h e h e a v y i n f e c t i o n u s e d in s o m e cases (~'ig.2) a n d t h e ' a d d i t i o n of OsO~ d i d n o t affect t h e cells; it m a y t h e r e f o r e b e a s s u m e d t h a t t h e c h a n g e s o b s e r v e d a r e c a u s e d b y n o r m a l i n f e c t i o n a n d n o t b y t h e p h e n o m e n o n k n o w n as " l y s i s f r o m w i t h o u t ''9. T h e n e x t s t a g e in t h e c o u r s e of n o r m a l i n f e c t i o n is c h a r a c t e r i z e d b y m a r g i n a l foci in w h i c h s t r a n d s of t h e o p a q u e " n u c l e a r " m a t e r i a l are f o u n d (Fig. 3). W e b e l i e v e t h a t t h e s e foci r e p r e s e n t s p o t s in w h i c h p h a g e s y n t h e s i s , or m a t u r a t i o n , t a k e s place. T h e e s t i m a t e d n u m b e r of m a r g i n a l foci is s u c h t h a t one or a f e w p h a g e p a r t i c l e s p r o d u c e d p e r focus w o u l d a c c o u n t for t h e y i e l d of n e w p a r t i c l e s f r o m "a b a c t e r i u m . As m e n t i o n e d before, a t t e m p t s a t m a k i n g s e c t i o n s of b a c t e r i a c o n t a i n i n g m a n y n e w p a r t i c l e s h a v e failed, a n d w e a r e t h e r e f o r e n o t in a p o s i t i o n to s a y w h e t h e r t h e l o c a l i s a t i o n of n e w p a r t i c l e s inside t h e cell c o r r e s p o n d s w i t h t h e p o s i t i o n of t h e m a r g i n a l foci. T h e e v i d e n c e we h a v e p o i n t s in t h a t d i r e c t i o n b u t is n o t c o n c l u s i v e (Figs. 4, 5, a n d 6). SUMMARY Micrographs are presented of sections of coli cells fixed at various times after infection with the bacteriophage T4r+. The changes in the distribution of "nuclear material" within the ceils, noted in stained preparations by other investigators, have been studied in greater detail. Based upon sections of free bacteriophage particles the opaque threads and bodies, characteristic of the "nuclear sites" of these bacteria, are tentatively identified as DNA-structures. R~SUMt~ Les auteurs donnent des microphotographies de sections de cellules de Cell fixees ~. des temps variables suivant l'infection par le bacteriophage T4r +. Les changements dans la distribution du "'materiel nucleaire" ~ l'interieur des cellules, notes dans des preparations colorees par des observateurs anterieurs, ont ~t6 6tudi~s avec plus de d~tail. Par comparaison avec des sections de phages libres, les auteurs proposent de considerer les filaments et les corpuscules opaques, caracteristiques des "localisations nucMaires" de ces bacteries, comme constitu~s par du DNA. ZUSAMMENFASSUNG Es werden Mikrophotographien von Schnitten yon ColizeUen gezeigt, die verschiedene Zeiten nach der Infektion mit dem Bakteriophagenstamm T4r+ fixiert wurden. Die Ver~inderungen in der Verteilung des Kernmaterials innerhalb der Zellen, die in gef/trbten Pr/iparaten von anderen Autoren beobachtet wurden, ist genau untersucht worden. Auf Grund der Beobachtung an Schnitten yon bakteriophagenfreien Teilchen wurden die opaken F/iden und K6rper, die fiir die Kernanaloge dieser Bakterien charakteristisch sind, als DNA-Strukturen identifiziert. REFERENCES x A. BIRCH-ANDERSEN, O. MAALOE AND F. S. SJOSTRAND, Biochim. Biophys. Acta, I2 (1953) 395. 2 M. H. ADAMS,in Methods o/Medical Research, Vol. 2, J. H. COMROE, Ed., The Year Book Publisher, Chicago, 195 ° . a S. E. LURIA AND M. L. HUMAN, J. Bact., 59 (195 o) 551. * R. G. E. MURRAY, D. H. GILLEN AND F. C. HEAGY, J. Bact., 59 (195o) 603. C. F. ROBINOW, J. Bact., 66 (I953) 3 °o. 4 R. G. E. MURRAY, Symposium on Bacterial Cytology, VI Internat. Microbiol. Congress, Rome, 1953. D. FRASER AND R. C. WILLIAMS, Prec. Nat. Acad. Sci., 39 (1953) 75 °. s A. D. HERSHEY.ANU M. CHASE, J. Gen. Physiol., 3~ (I9.j2) 39. M. DELBROCK,J. Gen. Physiol., 23 (I94o) 643. R e c e i v e d A p r i l I 7 t h , I95 4
BIOCHIMICA ET BIOPHYSICA ACTA
ELECTRON CELLS
MICROGRAPHS
INFECTED
WITH
OF SECTIONS THE
VOL. 15 (1954)
OF E. COLI
BACTERIOPHAGE
T4
by O. MAALOE AND A. BIRCH-ANDERSEN* Statens Serum Institut, Copenhagen (Denmark) AND
F. S. SJOSTRAND Department o] Anatomy, Karolinska Institute/., Stockholm (Sweden)
I n this j o u r n e l we r e c e n t l y described a m e t h o d for o b t a i n i n g t h i n sections of bacterial cells, suitable for h i g h - r e s o lu ti o n e l e c t r o n m i c r o s c o p y 1. I t was m e n t i o n e d t h a t m i erographs of coli cells infected w i t h t h e b a c t e r i o p h a g e T 4 suggest t h a t c e r t a i n s t r u c t u r e s seen inside t h e n o r m a l cell c o n t a i n desoxyribosenucleic acid (DNA) ; we shall n o w pres e n t some of these m i c r o g r a p h s a n d discuss t h e possibilities a n d l i m i t a t i o n s of t h e techniqu e from t h e p o in t of v i e w of s t u d y i n g cell m o r p h o l o g y d u r i n g p h a g e m u l t i p l i c a t i o n . METHODS AND MATERIAL The fixation, embedding and sectioning techniques as well as the equipment used for taking the micrographs were described in our first communication 1. Strain B, of E. coli, has again been used, this time after infection with phage T4r+. Vigorously aerated broth cultures with logarithmically growing bacteria at a density of about io 8 ceils per ml were multiply infected, and at various times phage synthesis was stopped by addition of cyanide and immediately thereafter OsO 4 was added to initiate fixation as described previouslyx. Techniques similar to those given by ADAMS~ were used to control multiplicity of infection and phage development. It was observed that OsO~ added z7 or more minutes after infection causes rapid lysis of part of the cellg; the later the addition the larger the fraction of cells that lyse. It was attempted, but without success, to counteract this effect of OsO 4 by reinfecting the cultures at i2 mill with IO T4 r+ particles per cell, thus establishing conditions known to inhibit normal lysis. Micrographs were also made of free phage particles which for technical reasons were agglutinated with anti-phage serum prior to fixation, embedding and sectioning.
MORPHOLOGICAL
OBSERVATIONS
T h e m i c r o g r a p h s of infected cells are best described b y c o m p a r i s o n w i t h those of norm al , growing cells. I n th e la tt e r , t h e b u l k of t h e m a t e r i a l seen in t h e m i c r o g r a p h s presents itself as a " s p o n g y " s t r u c t u r e w h i c h su r r o u n d s large " v a c u o l e s " c o n t a i n i n g t i g h t l y compressed, or wound, c e n t r a l s t r u c t u r e s of h i g h o p a c i t y ; t h e s p o n g y s t r u c t u r e is a c c e n t u a t e d a n d v e r y o p a q u e in a n a r r o w zone a r o u n d t h e vacuoles. I n our first p a p e r a l e n g t h w i s e section of such a cell, showing t w o vacuoles was p r e s e n t e d ((I), Fig. 2); * Working on this study in the Department of Anatomy, Karolinska Institutet, Stockholm; aided by a grant from the P. Carl Petersen Foundation. Re#rences p. z 9.
VOL. 15 (1954)
E. coli CELLS INFECTED WITH BACTERIOPHAGE T 4
13
Fig. I. Section of cell in l o g a r i t h m i c p h a s e of g r o w t h , s h o w i n g c o m p a c t a n d o p a q u e central s t r u c t u r e s in t h e " v a c u o l e " . Magnification 64,00o. T h e b l a c k a r e a s to t h e r i g h t a n d on t h e lower cell b o r d e r s h o w particles of c a r b o n b l a c k d e p o s i t e d on t h e m o u n t s to facilitate focussing.
the micrograph presented as Fig. I of the present paper originates from the same preparation, is technically better but shows only one vacuole. Infected samples of the culture producing the normal cells just described were used to make the preparations of which micrographs will now be presented. Thes.e samples were fixed 2 ~ , 8, 18 and 25 min after infection and they are described below in that order. To study changes that might occur very soon after infection, bacteria were mixed with about 15o T4r+ particles per cell in order to ensure complete infection of the whole culture within less than 15 seconds. After 2 ~ min cyanide was added and fixation initiated. Fig. 2 shows a typical central section in which all the elements of the normal cell can still be identified. The most obvious change caused by infection concerns the central bodies of the vacuoles, which, after infection, present themselves as a loose network of threads. In addition the vacuoles have become elongated and quite irregular; it is not possible to decide whether threads from neighbouring vacuoles mix at this stage although some micrographs suggest a fusion of vacuoles.--We have not been able to observe phage particles attached to the cell surface despite the high multiplicity of infection; the extensive damage done to the cell membranes during and after dehydration 1 m a y account for this failure. The morphological picture just described changes rapidly. Micrographs not to be presented here show that 3-4 min after infection m a n y cells already resemble those of Fig. 3, which were fixed 8 min afteri nfection. At that stage radical changes in the organisation of the intracellular structures are observed; the central regions are filled with an irregular structure of low opacity while in the periphery of the cells a large number of distinct loci have developed. These foci present themselves as light areas with a round or elongated dark body inside, and bordered b y regions with accentuated and very opaque Re/erences p. 19.
14
o . MA&LOE, A. BIRCH-ANDERSEN, F. S. SJOSTRAND
VOL. 1 5
(1954)
l"ig. 2. (;ell fixed 2 I/~ minutes after heavy infection with phage T4 r~, presenting very large vacuoles with loose n e t w o r k s of coarse threads. Magnification 7S,ooo.
voL. 15 (1954)
E. coli CELLS INFECTED WITH BACTERIOPHAGE T4
15
Fig. 3. Cells fixed 8 m i n u t e s after xo-fold infection with T4r+. Marginal foci surrounded by irregular dark structures and containing rounded opaque bodies seen m all ceils. Magnification 65,000.
16
O. MAALOE, A. BIRCH-ANDERSEN, F. S. SJ6STRAND
VOL. 1 5 (1954)
Figs. 4, 5, a n d 6. Portions of sections of ceils fixed i8 m i n u t e s after lo-fold infection with T4r+. T h e central regions h a v e a g a i n become clear a n d f r e q u e n t l y s h o w webs of fine t h r e a d s (Fig. 4); occasionally o p a q u e bodies, v e r y similar to t h e sections of free p h a g e particles, are seen (Figs. 5 a n d 6). Magnifications 5i,ooo, 61,ooo a n d 74,ooo, respectively.
VOL. 15 (1954)
E. coli CELLS INFECTED WITH BACTERIOPHAGE T 4
17
spongy structure of the type found in the vicinity of the central vacuoles of the normal growing cell. The bodies inside the marginal foci have the same high opacity as the threads of Figs. I and 2, and usually show circular or elliptical cross-sections with diameters of about 400 A, although in some cases long threads with the same diameter have been found in elongated peripheral foci. Assuming that the loci are independent units, it m a y be estimated that cells of varying size contain 5o--2oo such units; it cannot be excluded, however, that the loci represent sections through peripheral ducts contairing long threads of material of the same type as that which constitutes the compact central bodies of the vacuoles of the uninfected cells and which forms a looser network just after infection. It will be recalled that the infected cells gradually become susceptible to Os04, partial lysis occurring if fixation is not initiated until 17 or more min after infection. Thus in preparations fixed late only the cells resistant to OsO 4 can be studied, and it may be assumed that these cells constitute the fraction of the population which progresses most slowly towards spontaneous lysis. These cells show a clear central region extending throughout the length of the cell and bordered by a distinct zone of granular and very opaque structure within, or outside of, which are seen the few bodies which we tentatively identify with newly formed phage particles (see below). There is thus a striking difference between the micrographs of cells fixed 8 and, say, 18 min after infection repectively; the appearance of a clear central region and the disappearance of the marginal foci being characteristic of the change in morphology as the time for normal lysis approaches. It cannot at the moment be said whether the marginal foci correspond in number and localisation to the newly formed phage particles. In micrographs such as Figs 4, 5 and 6 newly formed particles may be looked for. Before attempting to identify any intracellular structures with phage, micrographs were made of free particles fixed, embedded and sectioned in the same way as the bacterial cells. Phage particles treated in this way show very opaque, non-differentiated crosssections with hexagonal outlines and diameters of about 500 A; in some cases a faint halo of much lighter material can be seen surrounding the dark core.-- Inside some sectioned cells bodies are seen that look like the sectioned phage particles; they are usually observed within the diffuse structures in the periphery of the cells and therefore more or less masked. In this connection it must be remembered that the volume of a longitudinal section, about 200 A units in thickness, is about 3-4% of the volume of the bacterial cell, and that 18 min after infection the average number of finished phage particles per ceil is only about 20. For these reasons, and because the cells that lyse on addition of OsO 4 probably are those containing the greatest number of new phage particles, only relatively few sections can be expected to contain complete new particles. We are thus not in a position, at the present time, to say anything definite about the distribution of the new phage particles inside the cells; a better technique permitting fixation of cells containing large numbers of new particles must be developed. DISCUSSION
In our first paper I reference was made to the present study and to the results obtained by LURIA AND HUMAN3 and by MURRAY, GILLEN AND HEAGY4 with staining techniques. It was noted that the redistribution of the material which the staining method suggests is DNA, corresponds to the redistribution observed after infection of the opaque Re/erences p. 19.
I8
O. MAALOE, A. BIRCH-ANDERSEN, F. S. SJOSTRAND
VOL. 15 (~954)
material constituting the central bodies inside the vacuoles of the uninfected cells (Figs. 2 and 3); we concluded t h a t the opaque material probably contains DNA. This interpretation is strongly supported b y the finding that the cores of free phage particles, known to be composed largely of DNA, show the same high opacity as the central bacterial bodies. This observation is difficult to reconcile with the view of ROBINOWs, that the light, or "empty-looking" regions, seen in OsO,-fixed, embedded and sectioned bacterial spores, are the sites of the nuclear DNA. MURRAY, who has employed several fixing and staining methods in a study of the nuclei of bacillus cereus 8, t e n d s to believe t h a t the basophilic materi31, left in the nuclear regions after hydrolysis or treatment with ribonuclease, is located in relatively light areas in the periphery of these regions. Such an arrangement is not suggested by our micrographs; as mentioned above, the pictures of sectioned bacteriophage particles indicate that the opaque central threads or bodies seen in the vacuoles of the sectioned coli cells m a y well represent tightly coiled DNA-structures. I t is not possible, at this moment, to say whether fixation or dehydration cause the DNA-structures of the living cell to contract to some extent. However this m a y be, our a s s u m p t i o n - - t h a t DNA-structures are present as highly opaque bodies in the central regions of the "nuclear vacuoles" seen in sectioned coli cells--is strikingly different from the arrangements proposed by ROBINOW5 and MURRAYs for bacterial spores and the nuclei of bacillus cereus. In particular we believe that accumulations of DNA (or RNA) must reveal themselves as opaque areas in electron micrographs, and that " e m p t y areas" cannot correspond to regions with appreciable concentrations of nucleic acid. The above paragraphs essentially concern the interpretation of the central structures seen in uninfected cells. The present material should be examined from the point of view of studying phage multiplication also. In this respect, however, the amount of new information so far obtained is slight. It has been pointed out that technical difficulties have prevented us from making sections showing phage particles absorbed on the cell membranes and sections with m a n y newly formed particles. These difficulties m a y be overcome by developing better fixation techniques; it is more doubtful whether it will be possible ever to study the initiation of phage multiplication by means of the E.M. FRASER AND WILLIAMS7 have obtained micrographs of phage particles in the process of liberating their DNA contents; the extruded material appears as an irregular bundle of very long threads, 20-25 A in diameter, and if this is the state in which the DNA of the infecting phage particle enters the bacterial cell 8, it will be impossible to identify this material after it has become mixed with the structures of the normal bacterium, the elements of which are irregularly arranged and of about the same dimensions. W h a t we can obtain, on the other hand, is evidence of the changes in the microstructures of the host cell, brought about b y infection with phage: at this level we have seen how the "nuclear" apparatus of the cell is almost instantaneously affected (Fig. 2) in a way suggesting loosening of and fusion throughout the cell of the material normally tightly wound in the centers of distinct vacuoles. It must be recalled, however, that the micrographs showing this change are produced after infection with about Ioo phage particles per cell ; it is thus not certain that such profound changes necessarily follow after infection with one or a few particles. Unfortunately the cytological method does not lend itself readily to a study of cells infected singly or with very few particles since, in this case, a considerable fraction of the cells remain uninfected, without being distinguishable as such. In our exRelerences p. 19.
VOL. 15
(1954)
E. coli CELLS INFECTED WITH BACTERIOPHAGE
T4
19
p e r i m e n t s n o v i s i b l e lysis w a s i n d u c e d b y t h e h e a v y i n f e c t i o n u s e d in s o m e cases (~'ig.2) a n d t h e ' a d d i t i o n of OsO~ d i d n o t affect t h e cells; it m a y t h e r e f o r e b e a s s u m e d t h a t t h e c h a n g e s o b s e r v e d a r e c a u s e d b y n o r m a l i n f e c t i o n a n d n o t b y t h e p h e n o m e n o n k n o w n as " l y s i s f r o m w i t h o u t ''9. T h e n e x t s t a g e in t h e c o u r s e of n o r m a l i n f e c t i o n is c h a r a c t e r i z e d b y m a r g i n a l foci in w h i c h s t r a n d s of t h e o p a q u e " n u c l e a r " m a t e r i a l are f o u n d (Fig. 3). W e b e l i e v e t h a t t h e s e foci r e p r e s e n t s p o t s in w h i c h p h a g e s y n t h e s i s , or m a t u r a t i o n , t a k e s place. T h e e s t i m a t e d n u m b e r of m a r g i n a l foci is s u c h t h a t one or a f e w p h a g e p a r t i c l e s p r o d u c e d p e r focus w o u l d a c c o u n t for t h e y i e l d of n e w p a r t i c l e s f r o m "a b a c t e r i u m . As m e n t i o n e d before, a t t e m p t s a t m a k i n g s e c t i o n s of b a c t e r i a c o n t a i n i n g m a n y n e w p a r t i c l e s h a v e failed, a n d w e a r e t h e r e f o r e n o t in a p o s i t i o n to s a y w h e t h e r t h e l o c a l i s a t i o n of n e w p a r t i c l e s inside t h e cell c o r r e s p o n d s w i t h t h e p o s i t i o n of t h e m a r g i n a l foci. T h e e v i d e n c e we h a v e p o i n t s in t h a t d i r e c t i o n b u t is n o t c o n c l u s i v e (Figs. 4, 5, a n d 6). SUMMARY Micrographs are presented of sections of coli cells fixed at various times after infection with the bacteriophage T4r+. The changes in the distribution of "nuclear material" within the ceils, noted in stained preparations by other investigators, have been studied in greater detail. Based upon sections of free bacteriophage particles the opaque threads and bodies, characteristic of the "nuclear sites" of these bacteria, are tentatively identified as DNA-structures. R~SUMt~ Les auteurs donnent des microphotographies de sections de cellules de Cell fixees ~. des temps variables suivant l'infection par le bacteriophage T4r +. Les changements dans la distribution du "'materiel nucleaire" ~ l'interieur des cellules, notes dans des preparations colorees par des observateurs anterieurs, ont ~t6 6tudi~s avec plus de d~tail. Par comparaison avec des sections de phages libres, les auteurs proposent de considerer les filaments et les corpuscules opaques, caracteristiques des "localisations nucMaires" de ces bacteries, comme constitu~s par du DNA. ZUSAMMENFASSUNG Es werden Mikrophotographien von Schnitten yon ColizeUen gezeigt, die verschiedene Zeiten nach der Infektion mit dem Bakteriophagenstamm T4r+ fixiert wurden. Die Ver~inderungen in der Verteilung des Kernmaterials innerhalb der Zellen, die in gef/trbten Pr/iparaten von anderen Autoren beobachtet wurden, ist genau untersucht worden. Auf Grund der Beobachtung an Schnitten yon bakteriophagenfreien Teilchen wurden die opaken F/iden und K6rper, die fiir die Kernanaloge dieser Bakterien charakteristisch sind, als DNA-Strukturen identifiziert. REFERENCES x A. BIRCH-ANDERSEN, O. MAALOE AND F. S. SJOSTRAND, Biochim. Biophys. Acta, I2 (1953) 395. 2 M. H. ADAMS,in Methods o/Medical Research, Vol. 2, J. H. COMROE, Ed., The Year Book Publisher, Chicago, 195 ° . a S. E. LURIA AND M. L. HUMAN, J. Bact., 59 (195 o) 551. * R. G. E. MURRAY, D. H. GILLEN AND F. C. HEAGY, J. Bact., 59 (195o) 603. C. F. ROBINOW, J. Bact., 66 (I953) 3 °o. 4 R. G. E. MURRAY, Symposium on Bacterial Cytology, VI Internat. Microbiol. Congress, Rome, 1953. D. FRASER AND R. C. WILLIAMS, Prec. Nat. Acad. Sci., 39 (1953) 75 °. s A. D. HERSHEY.ANU M. CHASE, J. Gen. Physiol., 3~ (I9.j2) 39. M. DELBROCK,J. Gen. Physiol., 23 (I94o) 643. R e c e i v e d A p r i l I 7 t h , I95 4