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A cytological and chemical analysis of the bacterial nucleus
Experimental
636
A CYTOLOGICAL
I.
A
METHOD
MEGATERIUM
AND CHEMICAL ANALYSIS BACTERIAL NUCLEUS
FOR AND
Cell Research 16, 636-647 (1959)
THE THE
ISOLATION CYTOLOGY
OF OF THE
NUCLEI ISOLATED
OF THE
FROM
BACILLUS
STRUCTURES’
E. D. DELAMATER Section on Cytology and Genetics, Department of Physiology, School of Medicine, Philadelphia, Pa., U.S.A. University of Pennsylvania, Received July 31, 1958
structure and divisional mechanism of bacterial nuclei remains controversial [7, 9, 15, 161. Efforts to resolve the problems contingent to this controversy are at present following two general lines of endeavor. Thinsectioning and electron microscopy of bacteria are proceeding in many laboratories [ 1,2,3,4,13,14]. In these various studies both filamentous and tubular structures, thought to represent chromosomes and/or nuclei, have been observed. Improved methods appear to be required for further advance [lo]. Efforts to isolate bacterial nuclei have followed three avenues. Spiegelman ef al. [16] and Fitz-James [9] profess to have isolated nuclei from protoplasts of Bacillus megaferium. These studies constitute an excellent beginning, but as yet do not resolve the problems. These authors are able to account for all the DNA present in the intact cells in the isolated nuclei and washes obtained from them. Marshak [ll, 121 has ruptured Escherichia coli with sonication (Raytheon 10 KC) and has published electron photomicrographs and measurements of spiral structures which he believes to be bacterial chromosomes. DeLamater and Minsavage [s] have demonstrated and published in brief that bile salts act upon B. megaferium to produce a clearing of the cells under certain conditions, apparently leaving the nuclei visible and cytologically intact in situ. The cells are likewise made fragile and can be readily broken, either by sonic vibration or mechanical disruption with the Mickle disintegrator. The nuclear structures visible in the cleared cells are liberated and relatively easily concentrated in pure form. The purpose of the present paper is to present the methods in detail and to describe cytologically the isolated nuclear structures made available by these means. THE
1 This investigation was supported in part by research grants PHS#C-3010 and PHS#C-2189 from the National Institutes of Health, Public Health Service; by Grant #I29 from the National Multiple Sclerosis Society; and by a research grant from Eli Lilly and Company. Experimental
Cell Research 16
Cytological and chemical analysis 01 bacterial nucleus MATERIALS
AND
METRO
Organism The Pennsylvania strain of B. megaterium, the subject [I9 61, has been used exclusively in the present work. Methods
of numerous
prior
studies
of Cultivation
The organism is grown through two successive S-hour growth periods in (M-9) salt-glucose medium, and subsequently for 4 hours in casein hydrolysate medium. It has been found that the sensitivity to the bile salts is increased about 5- to fOfold by this brief growth period in nutrient medium. The cells are then centrifuged, washed once in M/15 phosphate buffer, and resuspended in M/15 phosphate buffer containing 1 per cent sodium glycocholate (Nutritional Biochemical Corporation Lot 86561). The reaction mixture is allowed to stand at room temperature. Periodically a drop of the cell suspension is removed, placed on a slide under a coverslip, and sealed with paraffin, “Lubriseal”, or nail polish, and observed under the phase cectrast microscope at 1212 x for progress in the clearing process presently to be described. Methods
of Isolation of Nuclei
Sonic vibration.When the cells have cleared sufficiently, they are centrifuged and washed with M/15 phosphate buffer or 0.25 per cent sucrose. They are then piaced in a 10 KC Raytheon sonic vibrator, cooled with circulating ice-water, and are sonicated for 14 minutes, at which time the degree of cellular disruption is checked by phase contrast microscopy, as indicated above. If further treatment in needed, an additional 30 to 60 seconds of exposure to sound is given, microscopic check being made every 30 seconds. The disrupted cells are removed from the machine, placed in polyethylene centrifuge tubes at 4°C. The disruptect cell walls may be removed either by differe~t~~~ centrifugation, which pever gives a clean separation because of the gelatinousness of the mixture, or by the following m&hod. If cells are treated for 1 to 2 hours or more in 1 per cent sodium glycocholate, they can no longer act as substrate to lysozyme, whereas cells grown in the manner described will readily form protoplasts in the presence of lysozyme. After disruption, however, the fragmented walls again readily act as substrate for lysozyme and may be eliminated from the mixture directly as follows. Following sonication, the cells are centrifuged at 250 g at 1°C for 20 minutes, which removes the remaining whole cells and a small percentage of cell fragments. The supernate, containing only cell fragments, is spun for IO minutes at 1000 g at 1°C. The pellet is washed twice in 0.0075 M sucrose (100 ml) and resuspended in 0.001 per cent lysozyme in 0.0075 M sucrose for 60 minutes at 4X, or for 15 to 20 minutes at R.T. The material is centrifuged at 4000 g for 30 minutes at 1”C, and the pellet washed with 0.0075 M sucrose. For electron microscopy, cytochemistry, or chemical analysis, the material is washed twice in distilled water at 1°C prior to further processing. Esperimerzfal
Cell Reseurch 16
E. D. DeLamater The methods described are straightforward and simple, and afford an opportunity to obtain large numbers of structures which appear to constitute both intact nuclei and chromosomes which have been freed from the nuclei. That these structures are believed to constitute both intact nuclei and free chromosomes, is based on the following observations. Mickle rlisintegrafion.-Rupture of cleared cells by the Mickle disintegrator was found to be accomplished better if cells were concentrated and shot into absolute alcohol, giving a final concentration of alcohol of 70 per cent, at - 30°C or below, and allowed to stand at this temperature for 5 to 6 days. This alcohol treatment appeared to make the already fragile cells more brittle so that they broke up more readily on the impact of the glass beads. Fragmentation of the cells here was carried out in the 70 per cent alcohol. This procedure also seems to have the advantage of holding the chromosomes and nuclei together more effectively, both chemically and physically, and of washing out any glycocholate. Cytologic
Procedures
Microscopy.-Phase contrast microscopic observations were made directly with a B & L dark phase 97 y: objective 1.0 NA, and a 1.0 NA long working distance phase contrast condenser. This optical system was completed with 12.5 x widefield oculars. The light source was a B & L 300 W zirconium arc lamp set at a sufficient distance to fill the back lens of the condenser, and focused in this plane. Neutral density filters were used to control light intensity. White light microscopy was accomplished with a B & L DDE research microscope equipped with a 97 x achromatic 1.25 NA objective, a 90 x apochromatic 1.4 NA objective, an achromatic 1.4 NA condenser, complemented with 12.5 x widefield oculars. The light source was a 100 W zirconium arc lamp (Mikrark) set, at a sufficient distance to fill the back lens of the condenser, and focused on this plane. Photograpi?y.-Photographs were taken with a B & L L-camera. Kodak microfile film was used for high contrast negatives; Kodak Panatomic X film was used for lower contrast negatives. Development was done at 20°C for 16 minutes in Kodak D-76 developer. Prints were made on the appropriate paper. Cytologic preparations.-Preparations for phase contrast microscopy were made by placing a small drop of material to be examined on a microscope slide and sealing a coverslip over it with “Lubriseal”, paraffin, or nail polish. Preparations of normal cells (for control) and cells cleared by sodium glycocholate were made either by spreading a film between two coverslips and fixing in osmium tetroxide vapor, or by transferring cells from 1 cc of suspension from a Millipore filter to a coverslip and fixing in like manner prior to staining. Drops of suspensions of isolated nuclei and chromosomes were placed between two coverslips and spread, and likewise fixed, or drops were spread with a needle and barely allowed to approach dryness before fixation. Preparations were stained by the azure A-SO, procedure and by the Feulgen procedure, as applied in previous studies [5]. Material stained with aceto-orcein was mixed in a test tube with several drops of stain. It was found that better preparations were produced if about 20 minutes were allowed before slides of this material were made. Slides were prepared by
Cyfological
and chemical analysis
of bacterial nucleus
63
a drop of the mixture of stain and cells and/or cell. fragments on a slide, gently pressing it out and sealing with nail polish. Gram-stained material was spread on a slide, dried and stained by routine prncedure.
placing
OBSERVATIONS
Clearing
Effect of Sodium Glycocholate
Cells grown as described and exposed to 1 per cent sodium g~y~ochol~te in either M/IS phosphate buffer or 0.0075 M sucrose at room temperature, without shaking, clear progressively. Areas corresponding to the sites of nuclei remain dense, while the cytoplasm loses its density. The whole process is easily and clearly followed by phase contrast microscopy. Fig. 1 shows two normal cells of B. megaterium for comparison with Figs. 2 and 3, in which the nuclear masses are visible. The discreteness of the individual nuclei is more clearly seen in Fig. 3. Figs. 18-26 and Fig. 33 show similar cleared cells stained with the azure A-SO, and the Feulgen stains, respectively. With these two met are chemically specific for DNA, the similarity between the stained material and that seen by phase contrast microscopy is readily observed. Fig. 39 shows a comparable cell stained with aceto-orcein, which is considered to be one of the better chromosomal stains, albeit a chemically non-specific one. Studies on Isolated Nuclei For detailed description the reader is referred to the escription of Plates. he isolate nuclei may appear as discrete structures within which the included chromosomes may appear either as threadlike structures (Figs. 4, 5, 6, 7, 8) by phase contrast, and similarly in stained preparations (Figs. 20, 22, 23, 24, Figs. 25-31, 36, 37, 41, 42, Figs. 44-46, 56, St). Occasionally, as in Fig. 9, the elongated c,hromosomes appear to be confined, whether by a membrane or other structure, is not clear; or they may appear as condensed structures in configurations compatible with divisional stages (Figs. 11-l 7) by phase contrast, and similarly in stained preparations (Fig. 21, Figs. 32, 35, Figs. 47, 48, 49). The figures in which the chromosomes are filamentous are interpreted as interphases; those in which the chromosomes are rod-like, as prophases; and those in which the chromosomes are contracted and condensed and apparently incorporated in what appears to be a mitotic apparatus, as divisional stages.
E. D. DeLamater Bacillus megaterium grown in salt-glucose medium (M-9) overnight at 30°C (2 transfers); and subsequently for 4 hours at 34°C in casein hydrolysate medium. Cultures then spun down; washed in phosphate buffer once at pH 7.3; then exposed to 1.0 per cent sodium glycocholate. They were observed or stained in this drug, or were spun down and resuspended in phosphate buffer at pH 7.3 or 0.25 per cent sucrose. Cells were exposed to sonic vibration in phosphate buffer at pH 7.3 or sucrose (0.25 per cent). Magnification x 4850, unless otherwise stated.
Figs. l-17
Phase Contrast
Fig. l.-Two cells of B. megaterium showing when grown in casein hydrolysate medium.
normal
phase density
and characteristic
rod form
Figs. 2, 3.-Cells grown as described in both media and exposed to 1.0 per cent sodium glycocholate, showing clearing of cytoplasm with the appearance of the nuclear structures. In Fig. 2 the chromosomes appear to be filamentous. In Fig. 3 the nuclei appear as separate structures. Figs. 4-l7.-Cells treated as described and exposed to 2 minutes sonicvibration in phosphate buffer (pH 7.3) or 0.25 per cent sucrose. Fig. 4.-Shows
clump
of isolated
chromosomes.
Many
chromosomes
(10 KC Raytheon)
appear threadlike.
Fig. 5.-Adjacent clusters of threadlike chromosomes which appear to be attached It is thought that these represent two joined nuclei from a single cell. Fig. 6.-Isolated
chromosomes
Fig. 7.-Chromosomes Fig. S.-Isolated
thought
to a granule.
lying free. to represent
those from two nuclei.
chromosomes.
Fig. 9.-Single
isolated
Fig. lO.-Clump
interphase
nucleus; chromosomes
of nuclei in divisional
Fig. Il.-Three
adherent
Fig. 12.--Isolated centriole.
divisional
prometaphase
threadlike.
stages, the chromosomes
(late prophase)
with
three
condensed
Figs. 13, 14.-Isolated metaphase stages showing condensed centrioles at the poles, and spindlelike structures visible. Fig. 15.-Isolated trioles at poles.
condensed.
nuclei from a single cell.
early anaphase showing
early separation
chromosomes
chromosomes
of condensed
and one
on the plate with
chromosomes
and cen-
Fig. 16.-Righthand figure: Isolated anaphase and prometaphase (late prophase). Lefthand figure shows centriole at one pole (other out of focus), and separating chromosomes as discrete granules. Centriole, spindle and three condensed chromosomes visible in prometaphase. Fig. 17.-Upper figure: Isolated telophase showing separated somes in each are visible. Lower figure: Isolated prometaphase and spindle and condensed chromosomes.
sister nuclei. The three chromo(late prophase) showing centriole
Figs. 1% 32 Azure A-SO2 Stain Figs. l&20.-Undisrupted glycocholate.
cells grown and cleared as described.
Fig. iS.-Early stage of clearing. Compare with Fig. 2. Figs. 19, 20.-Late Fig. 21.-Divisional Figs. 22-24.-Isolated Experimental
stages in clearing stages with
Nuclear
structures
of cytoplasm.
centrioles
becoming
Chromosomal
and condensed
clumps of nuclei and chromosomes.
Cell Research 16
Exposed
to 1.0 per cent sodium
visible structures
chromosomes.
as cytoplasm clearly
clears.
defined.
Cytological and chemical analysis of bacferial nucleus
641
b .
8
9
10
,”
71
Experimenful
12
Cell Research
13
16
E. D. DeLamafer
642
Fig. 12 and the lower configuration in Fig. 17, and the righthand configuration in Fig. 16 suggest the late prophase (prometaphase) configurations previously described in stained material in intact cells [5, 6, 71, in which the three condensed, beadlike chromosomes lie opposite and attached to a single granule thought to represent the centriole. Figs. 13 and 14 and Fig. 47 suggest metaphase configurations in isolated nuclei with the condensed chromosomes lying between the centrioles with evidence of their attachment to them. Fig. 15, is thought to represent an early anaphase, while the lefthand figure in Fig. 16, and upper figure in Fig. 17 and Fig. 32 (lower (figure) and Figs. 48 and 49 (lower left) depict late anaphase stages. Not infrequently the freed nuclei from a single cell remain attached or stuck together, as seen in Figs. 5, 11, 22 and 24 and Figs. 34 and 43. Fig. 25.-Isolated Fig. 26.-Shows mosomes.
clumps of nuclei and chromosomes. both an isolated
Fig. 27.--Isolated
metaphase
nucleus showing
with
threadlike
condensed
chromosomes
filamentous
Fig. 31.-Similar
larger
Fig. 32.-Anaphase daughter nuclei.
chromosome
spiral
with associated
chromosome
configuration
with
chro-
chromosomes.
Figs. 28, 29.-Tangle of threadlike chromosomes. Chromosomal lose “body” with prolonged exposure to phosphate buffer. Fig. 30.-Spiral
and threadlike
with
granule suggesting a nucleolus
associated
one centriole
threads appear to “unravel”
granule
and
( x 4500).
( x 4850).
and spindle showing,
and bridges between
Figs. 33-38 Feulgen Stain Fig. 33.-Undisrupted somes ( x 4500).
cell showing
Fig. 34.-Nuclei from disrupted structure appears to be divisional
many
condensed
cell. Structures ( x 4500).
divisional
nuclei with
are held together
condensed
by delicate
strand.
chromoUpper
Fig. 35.-Isolated anaphase showing separating daughter chromosomes which appear to be held together by a confining membrane. The centriolelike and spindlelike structures are visible ( x 4500). Fig. 36.-Clump
of isolated
Fig. 37.-Three
condensed
clumps of isolated
Fig. 38.-Isolated ( x 4500).
chromosomes
Figs. 39-51 Aceto-Orcein Fig. 39.-Undisrupted cytoplasm and partial
chromosomes
chromosomes and fragments
( x 4500).
and nuclei ( x 4850). in material
held in phosphate
buffer
too long
Stain cell in material subjected visualization of the nuclei.
to sonic vibration, showing early clearing of Fragments of walls and chromosomes in field.
Figs. 40P42.--Upper section: Disrupted cell showing emergence of nuclei from husk. Chromosomes mainly in filamentous form. Lower section: Clump of freed nuclei and chromosomes. Remnants of cell walls are present in field. Fig. 43.-Disrupted cell. Nuclei contained Nuclei appear to be in condensed phase. Experimental
Cell Research 16
in persistent
“protoplast”,
but shorn
of cell wall.
Cyfological and chemical analysis of bacferinl nucleus
25
26
1
127
.~28
; :
29.
643
E. D. DeLamater
644
Fig. 44.-Clumps of isolated filamentous chromosomes. Many chromosomes appear double. Fig. 45.~-Isolated clump of filamentous ribbonlike chromosomes. Fig. 46.-Large clump of nuclei in condensed divisional stages. Fig. 47.-Partially disrupted cell showing clear metaphase with polar centrioles and spindles in focus and chromosomes aligned on plate. Fig. 48.--Undisrupted cell showing anaphase with chromosomes separating toward centrioles. Fig. 49.-Isolated anaphase e@valcnt to Fig. 4X. Figs. 50, 51.--Clumps of isolated chromosomes. Many of the chromosomes are filament,ons, some ribbonlike.
If the isolated nuclear material is allowed to remain in M/l5 phosphate for any protracted period, it not only loses its stainability progressively, but also, if in the filamentous form, becomes more filamentous and delicate (Figs. 28, 29, 38). It may also become more obviously headed, as seen in Figs. 28 and 29 and Fig. 50. Occasionally configurations suggesting spiral structures, as seen in Figs. 30 and 31 are to be observed. buffer
Experimental
Cell Research 16
Cytological and chemical analysis of bacterial nucleus
DISCUSSJON
Methods have been evolved which have permitted the isolation of what appear to be bacterial nuclei and their constitutive chromosomes. These structures can be readily observed in both unstained and stained preparations. The configurations are strongly reminiscent of those described in intact cells by appropriate cytologic means by the author and his associates in the past [6, 71. The observations presented here appear to support the contention that the bacterial nucleus is a complex apparatus, including not only chromosomes but an organized divisional apparatus as well, and that the divisional mechanism is mitotic. The clearing eflects produced by sodium glycocholate on cells of B. rnqaferium, cytologically at least, simulate those produced by the action of when this enzyme acts to remove cytoplasmic (primarily) RS,4> leaving the nuclear areas relatively more clearly stainable. The same is true for the action of cold TCB and cold PCA. In each of these cases the stained nuclei, whether stained by Feulgen, azure A-SO,, or Giemsa, in a general way simulate those observed here. Likewise, visualization of the nuclei by the May-Griinwald method, in which the nuclei and cytoplasm are simultaneously stained but in different colors, produces a picture strongly suggestive of the gross picture seen here. That the bile salt may be producing coagulation artifacts is a possibility that cannot at present be excluded. However, the over-all cytologic similarities, disregarding for the moment the details of interpretation, support the view that we are dealing with nuclei and their intrinsic components. That the results are not at the present time in complete accord with those of Spiegelman ef ab. [16] and Fitz-James [9] who isolated their structures from protoplasts, may depend upon two things. In the first place, the metho are w-holly dissimilar, a fact which may largely account for the differences. Secondly, these authors have isolated their structures from protoplasts which are maintained of necessity in strongly hypertonic solutions. Tbe cells from which the protoplasts are derived are probably bi- or multinucleate, as can be seen in many of Fitz-James’s photographs, and because Qf the hypertonicity of the suspending medium, the nuclei may be aggregated or clumped, causing confusion. Fitz-James [9] recognizes these possibilities. The binuclear condition of Fitz-James’s protoplasts is obvious even in the disaggregated state. The electron photomicrographs they present [9, 161 of their isolated structures likewise are not interpretable in terms Qf the present Experimental
Cell Research 16
E. D. DeLamafer findings. The significance of the long streamers of material thought to be DNA is also not clear, nor do we find any evidence of the bipartite structure consisting of a core and chromatin, which they maintain exists. It is perhaps unfortunate that so much emphasis is placed upon this supposed bipartite structure of the nuclei of B. megaterium. This interpretation reflects other as yet unconfirmed studies on these and other organisms [15] currently derived from the same laboratory. It is felt that both these workers and ourselves have, from completely different approaches, evolved methods which will ultimately aid in the resolution of the controversy concerning the nature of the bacterial nucleus. To date the study of the bacterial nucleus by means of the electron microscope, whether on whole cells or thin sections, has not yet produced a sufficiently complete picture to afford opportunity for comprehensive interpretation. By isolation of these structures, opportunity is made for a possibly more constructive and enlightened approach with this instrument. Analysis of the isolated nuclei and chromosomes both by chemical means and by electron microscopy is proceeding, and the results will be presented at a later time. Likewise, a study of the effect of glycocholate on nuclear material is in progress and will be presented at a later time.
SUMMARY
The clearing action of sodium glycocholate on cells of B. megaterium is described. This bile salt also renders the organism more fragile and amenable to rupture by sonication or mechanical (Mickle) disruption, permitting the liberation and subsequent isolation of the nuclei and chromosomes. These have been studied by means of routine cytologic and cytochemical procedures and by phase contrast microscopy. The results indicate that the bacterial nucleus is a complex structure containing chromosomes, granules suggesting centrioles, and a spindle-like element. The stages observed suggest a mitotic cycle, and conform to those previously described by other means in intact cells. The significance of the findings is discussed.
The author would like to acknowledge the assistance of Dr. Edward J. Minsavage, who likewise contributed measurably in the discussion of results. He would also like to acknowledge the more recent participation of Mr. Patrick Echlin, who as part of his orientation training in the laboratory, undertook the task of repeating independently the procedures and observations presented here. Experimental
Cell Research 16
Cytological and chemical analysis of bacterial nucleus REFERENCES 1. BIRCII-AXDERSEN, A., S. Gen. Microbial. 13, 327 (1955). 2. BIRCFI-ANDEKSEN, A., MAALBE, 0. and SJ~STRAXD, F. S., Biochim. ei Riophys. AC& 42: 395 (1953). 3. BRADFIELD, J. FL G., Nature 173, 184 (1954). 4. CHAPMAN, G. B., and HILLIER, J., J. Bucteriol. 66, 362 (1953). 5. DELAMATER, E. D., Sfain Technol. 26, 199 (1951). 6. -Rend. Ist. Super. Snnitd 108 (1953). 7. -in Bacterial Anatomy. Cambridge Univ. Press, Cambridge. 1956. 8. DELAMATER. E. D., and MINSAVAGE, E. J.. Racferiol. Proc. 1, 4% (1957). 9. FITZ-JAM&P. C., J. Racteriol. 75. 369 (1958). 0. and 10. MAALBE, bridge, 1956.SMIRCH-ANDERSEN, A., in Bacterial Anatomv, Cambridge. Yniv. Press, Cam11. 12. 13. 14. 15. 16.
M~RSHAK, A., Expf!. Cell Research 2, 243 (1951). -Hiochim. ef Biophys. Acta 18, 140 (1955). REKARSKI, 6. and GIESBRECHT, P., Nafurwissensclzuffen 4, 1 (1955). PIEKAKSKI, G. and POXTIERI, G. M., Zentr. Bakteriol. Parasifenk. Ros~xow, C. I:., Hacteriol. Rev. 20, 207 (1957). SPIEGELMAK, S., ARONSON, A. I. and FITZ-JAXES, P. C., j. Bacferiol.
42 - Lw3’io.3
Qrig. 165, 212 (1956). 75 102 (1958).
Experimenful
Cell Research 16
636
A CYTOLOGICAL
I.
A
METHOD
MEGATERIUM
AND CHEMICAL ANALYSIS BACTERIAL NUCLEUS
FOR AND
Cell Research 16, 636-647 (1959)
THE THE
ISOLATION CYTOLOGY
OF OF THE
NUCLEI ISOLATED
OF THE
FROM
BACILLUS
STRUCTURES’
E. D. DELAMATER Section on Cytology and Genetics, Department of Physiology, School of Medicine, Philadelphia, Pa., U.S.A. University of Pennsylvania, Received July 31, 1958
structure and divisional mechanism of bacterial nuclei remains controversial [7, 9, 15, 161. Efforts to resolve the problems contingent to this controversy are at present following two general lines of endeavor. Thinsectioning and electron microscopy of bacteria are proceeding in many laboratories [ 1,2,3,4,13,14]. In these various studies both filamentous and tubular structures, thought to represent chromosomes and/or nuclei, have been observed. Improved methods appear to be required for further advance [lo]. Efforts to isolate bacterial nuclei have followed three avenues. Spiegelman ef al. [16] and Fitz-James [9] profess to have isolated nuclei from protoplasts of Bacillus megaferium. These studies constitute an excellent beginning, but as yet do not resolve the problems. These authors are able to account for all the DNA present in the intact cells in the isolated nuclei and washes obtained from them. Marshak [ll, 121 has ruptured Escherichia coli with sonication (Raytheon 10 KC) and has published electron photomicrographs and measurements of spiral structures which he believes to be bacterial chromosomes. DeLamater and Minsavage [s] have demonstrated and published in brief that bile salts act upon B. megaferium to produce a clearing of the cells under certain conditions, apparently leaving the nuclei visible and cytologically intact in situ. The cells are likewise made fragile and can be readily broken, either by sonic vibration or mechanical disruption with the Mickle disintegrator. The nuclear structures visible in the cleared cells are liberated and relatively easily concentrated in pure form. The purpose of the present paper is to present the methods in detail and to describe cytologically the isolated nuclear structures made available by these means. THE
1 This investigation was supported in part by research grants PHS#C-3010 and PHS#C-2189 from the National Institutes of Health, Public Health Service; by Grant #I29 from the National Multiple Sclerosis Society; and by a research grant from Eli Lilly and Company. Experimental
Cell Research 16
Cytological and chemical analysis 01 bacterial nucleus MATERIALS
AND
METRO
Organism The Pennsylvania strain of B. megaterium, the subject [I9 61, has been used exclusively in the present work. Methods
of numerous
prior
studies
of Cultivation
The organism is grown through two successive S-hour growth periods in (M-9) salt-glucose medium, and subsequently for 4 hours in casein hydrolysate medium. It has been found that the sensitivity to the bile salts is increased about 5- to fOfold by this brief growth period in nutrient medium. The cells are then centrifuged, washed once in M/15 phosphate buffer, and resuspended in M/15 phosphate buffer containing 1 per cent sodium glycocholate (Nutritional Biochemical Corporation Lot 86561). The reaction mixture is allowed to stand at room temperature. Periodically a drop of the cell suspension is removed, placed on a slide under a coverslip, and sealed with paraffin, “Lubriseal”, or nail polish, and observed under the phase cectrast microscope at 1212 x for progress in the clearing process presently to be described. Methods
of Isolation of Nuclei
Sonic vibration.When the cells have cleared sufficiently, they are centrifuged and washed with M/15 phosphate buffer or 0.25 per cent sucrose. They are then piaced in a 10 KC Raytheon sonic vibrator, cooled with circulating ice-water, and are sonicated for 14 minutes, at which time the degree of cellular disruption is checked by phase contrast microscopy, as indicated above. If further treatment in needed, an additional 30 to 60 seconds of exposure to sound is given, microscopic check being made every 30 seconds. The disrupted cells are removed from the machine, placed in polyethylene centrifuge tubes at 4°C. The disruptect cell walls may be removed either by differe~t~~~ centrifugation, which pever gives a clean separation because of the gelatinousness of the mixture, or by the following m&hod. If cells are treated for 1 to 2 hours or more in 1 per cent sodium glycocholate, they can no longer act as substrate to lysozyme, whereas cells grown in the manner described will readily form protoplasts in the presence of lysozyme. After disruption, however, the fragmented walls again readily act as substrate for lysozyme and may be eliminated from the mixture directly as follows. Following sonication, the cells are centrifuged at 250 g at 1°C for 20 minutes, which removes the remaining whole cells and a small percentage of cell fragments. The supernate, containing only cell fragments, is spun for IO minutes at 1000 g at 1°C. The pellet is washed twice in 0.0075 M sucrose (100 ml) and resuspended in 0.001 per cent lysozyme in 0.0075 M sucrose for 60 minutes at 4X, or for 15 to 20 minutes at R.T. The material is centrifuged at 4000 g for 30 minutes at 1”C, and the pellet washed with 0.0075 M sucrose. For electron microscopy, cytochemistry, or chemical analysis, the material is washed twice in distilled water at 1°C prior to further processing. Esperimerzfal
Cell Reseurch 16
E. D. DeLamater The methods described are straightforward and simple, and afford an opportunity to obtain large numbers of structures which appear to constitute both intact nuclei and chromosomes which have been freed from the nuclei. That these structures are believed to constitute both intact nuclei and free chromosomes, is based on the following observations. Mickle rlisintegrafion.-Rupture of cleared cells by the Mickle disintegrator was found to be accomplished better if cells were concentrated and shot into absolute alcohol, giving a final concentration of alcohol of 70 per cent, at - 30°C or below, and allowed to stand at this temperature for 5 to 6 days. This alcohol treatment appeared to make the already fragile cells more brittle so that they broke up more readily on the impact of the glass beads. Fragmentation of the cells here was carried out in the 70 per cent alcohol. This procedure also seems to have the advantage of holding the chromosomes and nuclei together more effectively, both chemically and physically, and of washing out any glycocholate. Cytologic
Procedures
Microscopy.-Phase contrast microscopic observations were made directly with a B & L dark phase 97 y: objective 1.0 NA, and a 1.0 NA long working distance phase contrast condenser. This optical system was completed with 12.5 x widefield oculars. The light source was a B & L 300 W zirconium arc lamp set at a sufficient distance to fill the back lens of the condenser, and focused in this plane. Neutral density filters were used to control light intensity. White light microscopy was accomplished with a B & L DDE research microscope equipped with a 97 x achromatic 1.25 NA objective, a 90 x apochromatic 1.4 NA objective, an achromatic 1.4 NA condenser, complemented with 12.5 x widefield oculars. The light source was a 100 W zirconium arc lamp (Mikrark) set, at a sufficient distance to fill the back lens of the condenser, and focused on this plane. Photograpi?y.-Photographs were taken with a B & L L-camera. Kodak microfile film was used for high contrast negatives; Kodak Panatomic X film was used for lower contrast negatives. Development was done at 20°C for 16 minutes in Kodak D-76 developer. Prints were made on the appropriate paper. Cytologic preparations.-Preparations for phase contrast microscopy were made by placing a small drop of material to be examined on a microscope slide and sealing a coverslip over it with “Lubriseal”, paraffin, or nail polish. Preparations of normal cells (for control) and cells cleared by sodium glycocholate were made either by spreading a film between two coverslips and fixing in osmium tetroxide vapor, or by transferring cells from 1 cc of suspension from a Millipore filter to a coverslip and fixing in like manner prior to staining. Drops of suspensions of isolated nuclei and chromosomes were placed between two coverslips and spread, and likewise fixed, or drops were spread with a needle and barely allowed to approach dryness before fixation. Preparations were stained by the azure A-SO, procedure and by the Feulgen procedure, as applied in previous studies [5]. Material stained with aceto-orcein was mixed in a test tube with several drops of stain. It was found that better preparations were produced if about 20 minutes were allowed before slides of this material were made. Slides were prepared by
Cyfological
and chemical analysis
of bacterial nucleus
63
a drop of the mixture of stain and cells and/or cell. fragments on a slide, gently pressing it out and sealing with nail polish. Gram-stained material was spread on a slide, dried and stained by routine prncedure.
placing
OBSERVATIONS
Clearing
Effect of Sodium Glycocholate
Cells grown as described and exposed to 1 per cent sodium g~y~ochol~te in either M/IS phosphate buffer or 0.0075 M sucrose at room temperature, without shaking, clear progressively. Areas corresponding to the sites of nuclei remain dense, while the cytoplasm loses its density. The whole process is easily and clearly followed by phase contrast microscopy. Fig. 1 shows two normal cells of B. megaterium for comparison with Figs. 2 and 3, in which the nuclear masses are visible. The discreteness of the individual nuclei is more clearly seen in Fig. 3. Figs. 18-26 and Fig. 33 show similar cleared cells stained with the azure A-SO, and the Feulgen stains, respectively. With these two met are chemically specific for DNA, the similarity between the stained material and that seen by phase contrast microscopy is readily observed. Fig. 39 shows a comparable cell stained with aceto-orcein, which is considered to be one of the better chromosomal stains, albeit a chemically non-specific one. Studies on Isolated Nuclei For detailed description the reader is referred to the escription of Plates. he isolate nuclei may appear as discrete structures within which the included chromosomes may appear either as threadlike structures (Figs. 4, 5, 6, 7, 8) by phase contrast, and similarly in stained preparations (Figs. 20, 22, 23, 24, Figs. 25-31, 36, 37, 41, 42, Figs. 44-46, 56, St). Occasionally, as in Fig. 9, the elongated c,hromosomes appear to be confined, whether by a membrane or other structure, is not clear; or they may appear as condensed structures in configurations compatible with divisional stages (Figs. 11-l 7) by phase contrast, and similarly in stained preparations (Fig. 21, Figs. 32, 35, Figs. 47, 48, 49). The figures in which the chromosomes are filamentous are interpreted as interphases; those in which the chromosomes are rod-like, as prophases; and those in which the chromosomes are contracted and condensed and apparently incorporated in what appears to be a mitotic apparatus, as divisional stages.
E. D. DeLamater Bacillus megaterium grown in salt-glucose medium (M-9) overnight at 30°C (2 transfers); and subsequently for 4 hours at 34°C in casein hydrolysate medium. Cultures then spun down; washed in phosphate buffer once at pH 7.3; then exposed to 1.0 per cent sodium glycocholate. They were observed or stained in this drug, or were spun down and resuspended in phosphate buffer at pH 7.3 or 0.25 per cent sucrose. Cells were exposed to sonic vibration in phosphate buffer at pH 7.3 or sucrose (0.25 per cent). Magnification x 4850, unless otherwise stated.
Figs. l-17
Phase Contrast
Fig. l.-Two cells of B. megaterium showing when grown in casein hydrolysate medium.
normal
phase density
and characteristic
rod form
Figs. 2, 3.-Cells grown as described in both media and exposed to 1.0 per cent sodium glycocholate, showing clearing of cytoplasm with the appearance of the nuclear structures. In Fig. 2 the chromosomes appear to be filamentous. In Fig. 3 the nuclei appear as separate structures. Figs. 4-l7.-Cells treated as described and exposed to 2 minutes sonicvibration in phosphate buffer (pH 7.3) or 0.25 per cent sucrose. Fig. 4.-Shows
clump
of isolated
chromosomes.
Many
chromosomes
(10 KC Raytheon)
appear threadlike.
Fig. 5.-Adjacent clusters of threadlike chromosomes which appear to be attached It is thought that these represent two joined nuclei from a single cell. Fig. 6.-Isolated
chromosomes
Fig. 7.-Chromosomes Fig. S.-Isolated
thought
to a granule.
lying free. to represent
those from two nuclei.
chromosomes.
Fig. 9.-Single
isolated
Fig. lO.-Clump
interphase
nucleus; chromosomes
of nuclei in divisional
Fig. Il.-Three
adherent
Fig. 12.--Isolated centriole.
divisional
prometaphase
threadlike.
stages, the chromosomes
(late prophase)
with
three
condensed
Figs. 13, 14.-Isolated metaphase stages showing condensed centrioles at the poles, and spindlelike structures visible. Fig. 15.-Isolated trioles at poles.
condensed.
nuclei from a single cell.
early anaphase showing
early separation
chromosomes
chromosomes
of condensed
and one
on the plate with
chromosomes
and cen-
Fig. 16.-Righthand figure: Isolated anaphase and prometaphase (late prophase). Lefthand figure shows centriole at one pole (other out of focus), and separating chromosomes as discrete granules. Centriole, spindle and three condensed chromosomes visible in prometaphase. Fig. 17.-Upper figure: Isolated telophase showing separated somes in each are visible. Lower figure: Isolated prometaphase and spindle and condensed chromosomes.
sister nuclei. The three chromo(late prophase) showing centriole
Figs. 1% 32 Azure A-SO2 Stain Figs. l&20.-Undisrupted glycocholate.
cells grown and cleared as described.
Fig. iS.-Early stage of clearing. Compare with Fig. 2. Figs. 19, 20.-Late Fig. 21.-Divisional Figs. 22-24.-Isolated Experimental
stages in clearing stages with
Nuclear
structures
of cytoplasm.
centrioles
becoming
Chromosomal
and condensed
clumps of nuclei and chromosomes.
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Exposed
to 1.0 per cent sodium
visible structures
chromosomes.
as cytoplasm clearly
clears.
defined.
Cytological and chemical analysis of bacferial nucleus
641
b .
8
9
10
,”
71
Experimenful
12
Cell Research
13
16
E. D. DeLamafer
642
Fig. 12 and the lower configuration in Fig. 17, and the righthand configuration in Fig. 16 suggest the late prophase (prometaphase) configurations previously described in stained material in intact cells [5, 6, 71, in which the three condensed, beadlike chromosomes lie opposite and attached to a single granule thought to represent the centriole. Figs. 13 and 14 and Fig. 47 suggest metaphase configurations in isolated nuclei with the condensed chromosomes lying between the centrioles with evidence of their attachment to them. Fig. 15, is thought to represent an early anaphase, while the lefthand figure in Fig. 16, and upper figure in Fig. 17 and Fig. 32 (lower (figure) and Figs. 48 and 49 (lower left) depict late anaphase stages. Not infrequently the freed nuclei from a single cell remain attached or stuck together, as seen in Figs. 5, 11, 22 and 24 and Figs. 34 and 43. Fig. 25.-Isolated Fig. 26.-Shows mosomes.
clumps of nuclei and chromosomes. both an isolated
Fig. 27.--Isolated
metaphase
nucleus showing
with
threadlike
condensed
chromosomes
filamentous
Fig. 31.-Similar
larger
Fig. 32.-Anaphase daughter nuclei.
chromosome
spiral
with associated
chromosome
configuration
with
chro-
chromosomes.
Figs. 28, 29.-Tangle of threadlike chromosomes. Chromosomal lose “body” with prolonged exposure to phosphate buffer. Fig. 30.-Spiral
and threadlike
with
granule suggesting a nucleolus
associated
one centriole
threads appear to “unravel”
granule
and
( x 4500).
( x 4850).
and spindle showing,
and bridges between
Figs. 33-38 Feulgen Stain Fig. 33.-Undisrupted somes ( x 4500).
cell showing
Fig. 34.-Nuclei from disrupted structure appears to be divisional
many
condensed
cell. Structures ( x 4500).
divisional
nuclei with
are held together
condensed
by delicate
strand.
chromoUpper
Fig. 35.-Isolated anaphase showing separating daughter chromosomes which appear to be held together by a confining membrane. The centriolelike and spindlelike structures are visible ( x 4500). Fig. 36.-Clump
of isolated
Fig. 37.-Three
condensed
clumps of isolated
Fig. 38.-Isolated ( x 4500).
chromosomes
Figs. 39-51 Aceto-Orcein Fig. 39.-Undisrupted cytoplasm and partial
chromosomes
chromosomes and fragments
( x 4500).
and nuclei ( x 4850). in material
held in phosphate
buffer
too long
Stain cell in material subjected visualization of the nuclei.
to sonic vibration, showing early clearing of Fragments of walls and chromosomes in field.
Figs. 40P42.--Upper section: Disrupted cell showing emergence of nuclei from husk. Chromosomes mainly in filamentous form. Lower section: Clump of freed nuclei and chromosomes. Remnants of cell walls are present in field. Fig. 43.-Disrupted cell. Nuclei contained Nuclei appear to be in condensed phase. Experimental
Cell Research 16
in persistent
“protoplast”,
but shorn
of cell wall.
Cyfological and chemical analysis of bacferinl nucleus
25
26
1
127
.~28
; :
29.
643
E. D. DeLamater
644
Fig. 44.-Clumps of isolated filamentous chromosomes. Many chromosomes appear double. Fig. 45.~-Isolated clump of filamentous ribbonlike chromosomes. Fig. 46.-Large clump of nuclei in condensed divisional stages. Fig. 47.-Partially disrupted cell showing clear metaphase with polar centrioles and spindles in focus and chromosomes aligned on plate. Fig. 48.--Undisrupted cell showing anaphase with chromosomes separating toward centrioles. Fig. 49.-Isolated anaphase e@valcnt to Fig. 4X. Figs. 50, 51.--Clumps of isolated chromosomes. Many of the chromosomes are filament,ons, some ribbonlike.
If the isolated nuclear material is allowed to remain in M/l5 phosphate for any protracted period, it not only loses its stainability progressively, but also, if in the filamentous form, becomes more filamentous and delicate (Figs. 28, 29, 38). It may also become more obviously headed, as seen in Figs. 28 and 29 and Fig. 50. Occasionally configurations suggesting spiral structures, as seen in Figs. 30 and 31 are to be observed. buffer
Experimental
Cell Research 16
Cytological and chemical analysis of bacterial nucleus
DISCUSSJON
Methods have been evolved which have permitted the isolation of what appear to be bacterial nuclei and their constitutive chromosomes. These structures can be readily observed in both unstained and stained preparations. The configurations are strongly reminiscent of those described in intact cells by appropriate cytologic means by the author and his associates in the past [6, 71. The observations presented here appear to support the contention that the bacterial nucleus is a complex apparatus, including not only chromosomes but an organized divisional apparatus as well, and that the divisional mechanism is mitotic. The clearing eflects produced by sodium glycocholate on cells of B. rnqaferium, cytologically at least, simulate those produced by the action of when this enzyme acts to remove cytoplasmic (primarily) RS,4> leaving the nuclear areas relatively more clearly stainable. The same is true for the action of cold TCB and cold PCA. In each of these cases the stained nuclei, whether stained by Feulgen, azure A-SO,, or Giemsa, in a general way simulate those observed here. Likewise, visualization of the nuclei by the May-Griinwald method, in which the nuclei and cytoplasm are simultaneously stained but in different colors, produces a picture strongly suggestive of the gross picture seen here. That the bile salt may be producing coagulation artifacts is a possibility that cannot at present be excluded. However, the over-all cytologic similarities, disregarding for the moment the details of interpretation, support the view that we are dealing with nuclei and their intrinsic components. That the results are not at the present time in complete accord with those of Spiegelman ef ab. [16] and Fitz-James [9] who isolated their structures from protoplasts, may depend upon two things. In the first place, the metho are w-holly dissimilar, a fact which may largely account for the differences. Secondly, these authors have isolated their structures from protoplasts which are maintained of necessity in strongly hypertonic solutions. Tbe cells from which the protoplasts are derived are probably bi- or multinucleate, as can be seen in many of Fitz-James’s photographs, and because Qf the hypertonicity of the suspending medium, the nuclei may be aggregated or clumped, causing confusion. Fitz-James [9] recognizes these possibilities. The binuclear condition of Fitz-James’s protoplasts is obvious even in the disaggregated state. The electron photomicrographs they present [9, 161 of their isolated structures likewise are not interpretable in terms Qf the present Experimental
Cell Research 16
E. D. DeLamafer findings. The significance of the long streamers of material thought to be DNA is also not clear, nor do we find any evidence of the bipartite structure consisting of a core and chromatin, which they maintain exists. It is perhaps unfortunate that so much emphasis is placed upon this supposed bipartite structure of the nuclei of B. megaterium. This interpretation reflects other as yet unconfirmed studies on these and other organisms [15] currently derived from the same laboratory. It is felt that both these workers and ourselves have, from completely different approaches, evolved methods which will ultimately aid in the resolution of the controversy concerning the nature of the bacterial nucleus. To date the study of the bacterial nucleus by means of the electron microscope, whether on whole cells or thin sections, has not yet produced a sufficiently complete picture to afford opportunity for comprehensive interpretation. By isolation of these structures, opportunity is made for a possibly more constructive and enlightened approach with this instrument. Analysis of the isolated nuclei and chromosomes both by chemical means and by electron microscopy is proceeding, and the results will be presented at a later time. Likewise, a study of the effect of glycocholate on nuclear material is in progress and will be presented at a later time.
SUMMARY
The clearing action of sodium glycocholate on cells of B. megaterium is described. This bile salt also renders the organism more fragile and amenable to rupture by sonication or mechanical (Mickle) disruption, permitting the liberation and subsequent isolation of the nuclei and chromosomes. These have been studied by means of routine cytologic and cytochemical procedures and by phase contrast microscopy. The results indicate that the bacterial nucleus is a complex structure containing chromosomes, granules suggesting centrioles, and a spindle-like element. The stages observed suggest a mitotic cycle, and conform to those previously described by other means in intact cells. The significance of the findings is discussed.
The author would like to acknowledge the assistance of Dr. Edward J. Minsavage, who likewise contributed measurably in the discussion of results. He would also like to acknowledge the more recent participation of Mr. Patrick Echlin, who as part of his orientation training in the laboratory, undertook the task of repeating independently the procedures and observations presented here. Experimental
Cell Research 16
Cytological and chemical analysis of bacterial nucleus REFERENCES 1. BIRCII-AXDERSEN, A., S. Gen. Microbial. 13, 327 (1955). 2. BIRCFI-ANDEKSEN, A., MAALBE, 0. and SJ~STRAXD, F. S., Biochim. ei Riophys. AC& 42: 395 (1953). 3. BRADFIELD, J. FL G., Nature 173, 184 (1954). 4. CHAPMAN, G. B., and HILLIER, J., J. Bucteriol. 66, 362 (1953). 5. DELAMATER, E. D., Sfain Technol. 26, 199 (1951). 6. -Rend. Ist. Super. Snnitd 108 (1953). 7. -in Bacterial Anatomy. Cambridge Univ. Press, Cambridge. 1956. 8. DELAMATER. E. D., and MINSAVAGE, E. J.. Racferiol. Proc. 1, 4% (1957). 9. FITZ-JAM&P. C., J. Racteriol. 75. 369 (1958). 0. and 10. MAALBE, bridge, 1956.SMIRCH-ANDERSEN, A., in Bacterial Anatomv, Cambridge. Yniv. Press, Cam11. 12. 13. 14. 15. 16.
M~RSHAK, A., Expf!. Cell Research 2, 243 (1951). -Hiochim. ef Biophys. Acta 18, 140 (1955). REKARSKI, 6. and GIESBRECHT, P., Nafurwissensclzuffen 4, 1 (1955). PIEKAKSKI, G. and POXTIERI, G. M., Zentr. Bakteriol. Parasifenk. Ros~xow, C. I:., Hacteriol. Rev. 20, 207 (1957). SPIEGELMAK, S., ARONSON, A. I. and FITZ-JAXES, P. C., j. Bacferiol.
42 - Lw3’io.3
Qrig. 165, 212 (1956). 75 102 (1958).
Experimenful
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