unit, gave to biology a simple, uniform working principle Valentin, in It was the great botanist, Schleiden (1838),

Transcription

1 A STUDY IN BACTERIAL MORPHOLOGY JAMES F. McDONALD, JOHN T. LITTLE AND ERNST RUCKENSTEINER School of Medicine, Creighton University, Omaha, Nebraska Received for publication July 15, 1926 The great generalization formulated as the cell theory grew entirely out of the work of the botanists. The pioneer work in relation to this theory was already under way in the seventeenth century. The cellular structural pattern of cork and green plants was noted by the botanists Robert Hooke (1665), Malpighi (1675), and Nehemia Grew (1682). The cell nucleus was discovered by Robert Brown, the botanist, in The cell nucleolus was discovered later, also by a botanist, Gabriel Valentin, in It was the great botanist, Schleiden (1838), who about this time proved that plant tissue is composed of, and developed from, groups of cells. It was he who gave the anatomist and physiologist, Schwann, the suggestion that led the latter to the observations which enabled him to extend the cell theory also to the animal world. The conception that all living matter, however varied and diverse the gross morphology of individual organisms, is built up from the same type of microscopic unit, gave to biology a simple, uniform working principle of far-reaching influence. The cell theory clearly formulated in Schwann's classic monograph in 1839 opened the way for the extraordinary developments in biological and related sciences that have continued in progressive acceleration since that time. Side by side with the development of botany and animal biology in the seventeenth and eighteenth centuries, the science of bacteriology grew and developed as a corollary of the cell doctrine. The vision of Kirchner, the Jesuit, and of Lieuwenhock, the linen-draper, with a passion for perfection in lens grinding, resulted two and a half centuries ago in the revelation of a teeming sub-visible world. The brilliant Abbe Spallanzani 255

2 256 J. F. McDONALD, J. T. LITTLE AND E. RUCKENSTEINER proved, some two generations later, that bacteria, like higher living organisms, can come only from similar preexisting forms. Upon the foundations established by these and other early workers was built the real science of bacteriology, by Pasteur, the pioneer in preventive inoculation, and by Koch, the developer of the correct theory of specific infectious diseases. It is interesting that of the five makers of bacteriology named above, only one, Koch, was a physician. On account of the intimate relation of bacteria to human life, and health, and of its economic as well as scientific interest the science of bacteriology is being pursued with great vigor in all parts of the civilized world. Many problems in this field still await solution. One of these unsolved problems concerns the intimate morphology of bacteria. All biologists agree that bacteria are cells, but there is much disagreement as to the internal structure of these cells. Does the bacterium contain nucleus, cytoplasm and cell-wall etc., organized as in the cells which make up higher plants and animals? Upon this question investigators are in sharp disagreement. A critical summary of the views of various authorities has been given recently by Park and Williams (1924) as follows: 1. Bacteria have a definite morphological, more or less centrally situated nucleus (Feinberg, Nakanischi, Shottelius, Swellengrebel and others). 2. Bacteria have no nucleus or differentiated nuclear material (Fischer, Migula, Massert and others). 3. The whole organism, except the membrane which is a delicate layer of cytoplasm, is a nucleus (Butschli, L6wit, Boni and others). 4. The nuclear material is in the form of chromatin granules distributed throughout the cytoplasm (Hertwig, Schaudinn, Guilliermond, Zettnow and others). 5. A variety of the fourth view is that bacteria possess both the chief elements of a cell, namely, cytoplasm and karyoplasm, but that these are so finely mixed that they cannot be morphologically differentiated (Weigert, Mitrophanow, Gotschlich). 6. Another view advanced, which is a variation of views 3, 4, and 5, is that the bacterial cell is a relatively simple body-a cytode in Haec-

3 A STUDY IN BACTERIAL MORPHOLOGY 257 kel's sense, or the plasson of Van Beneden-which possesses both chromatin and plastin, in the relative amounts of these chief substances corresponding more nearly to the amounts found in the nuclei of higher cells than to that of their cytoplasm (Ruzicka, Ambroz). The last two authors call attention to the fact that both nucleus and cytoplasm in the higher cells are composed of a mixture of chromatin and plastin and that the chief difference between the two mixtures is one of amount and not of kind. Park and Williams go on to say, Our studies of bacteria lead us to agree with the views expressed in nos. 4 and 6 of the above summary, that is, bacteria possess both chief elements of a cell, namely chromatin and plastin, and according to the stage of growth and division (varying with the species) the chromatin is in the form of morphological granules or may be so finely divided and mixed with the plastin as to be indistinguishable from it. Gutstein (1924; 1925), Schumacher (1922), and Laszlo (see Moeller, 1925) in separate recent contributions conclude that the bacterial cell is differentiated into several distinct structures with different chemical characteristics, for which Gutstein gives special terms. Dobell ( ) stated that all bacteria contain a nucleus, which is variable in form in different bacteria, and at different periods in the life cycle of the same species. The preceding summary exhibits clearly the utter lack of concurrence among the authorities in regard to the question of the intimate internal structure and organization of bacterial cells. In this preliminary article we are submitting evidence which bears emphatically upon the question of the internal structure of bacteria. Several micro-photographs are presented which show clearly much of the internal morphology of several varieties of bacteria. Wright's stain was used for the organisms in all photographs. The lenses used for the photography in connection with this work were as follows: 1. Ordinary high power, which gives an approximate magnifica-

4 s.'k 258 J. F. MCDONALD, J. T. LITTLE AND E. RUCKENSTEINER tion of 450. The objective was 4 mm., and the eye piece 1OX. (Used in taking first photograph (fig. 1).) 2. Ordinary oil immersion, which magnifies about 1000 times. The objective was 1.9 mm., and the eye piece 1OX. (Used in taking second photograph (fig. 2).) 3. Special high power which gave a magnification of about The objective was of special make, apochromatic in type, 1.3 mm. The eye piece was 25 X. The condenser was 12 mm., and aplanatic in type. These three parts were all of...~~~~~~.-x J * 'j ;..v..'~~~~~~~~~~~~~~~~~~~~~~~~ 0.. t< FIG. 1. WRIGHT'S STAIN OF STAPHYLOCOCCUS PYOGENES-AUREUS, MAGNIFIED 450 TIMES, (ORDINARY HIGH POWER) SHOWING CLUSTER FORMATION, BUT No DETAIL OF INTERNAL STRUCTURE OF THE INDIVIDUAL CELLS Bausch and Lomb manufacture. (Used in taking all photographs excepting figures 1 and 2.) Figure 1 is a photograph of a smear of Staphylococcus pyogenes-aureus. The tendency of the organism to group in clusters is evident. The individual cells at this magnification, which is that of the ordinary high power, or about 450, are not very clearly discernible. Much less is it possible at this magnification to distinguish their internal morphology. At the ends and corners of this photograph there appear tiny, somewhat blurred, black circles with light centers. These

5 A STUDY IN BACTERIAL MORPHOLOGY 259 are not true representations of the bacteria, but distortions due to the fact that the organisms in these portions of the photograph are out of focus. There are also a few somewhat similar black circles with light centers scattered over the more central areas of the photograph. These are clear-cut as compared to the blurred figures toward the ends. The clearer, dark rings, with lighter centers, are caused by irremovable particles on the eye piece, as could easily.~~ i_.:u S ~. 4; i;.& r t FIG. 2. STAPHYLOCOCCUS PYOGENES-AUREUS. WRIGHT'S STAIN, MA'NIFICATION ABOUT 1000 (ORDINARY OIL IMMERSION) The individual cells are more clearly evident than in figure 1, but there is little intracellular detail evident in the photograph. However, someinternal structural detail may be seen directly under the microscope at this magnification. be shown by rotating the eye piece. The masses of staphylococci at the center of the picture which are correctly represented at this magnification appear only as tiny dots, with no internal morphological differentiation apparent. Figure 2 is a photograph of the same field as was shown in figure 1. The magnification is that of the ordinary oil immersion, or about The individual cocci are more clearly apparent, yet the internal structural detail of each coccus is scarcely evident. A certain amount of structural detail of the

6 260 J. F. McDONALD, J. T. LITTLE AND E. RUCKENSTEINER organism may, however, be made out at this magnification by direct microscopic study. The reader must not misinterpret the blurred dark rings with the light centers at the periphery of the picture and the few scattered more clearly distinct dark circles with light centers located toward the center. The reason for these has already been explained in figure 1. The third photograph (fig. 3) tells an entirely different story. FIG. 3. WRIGHT'S STAIN. STAPHYLOCOCCUS PYOGENES-AUREUS AS MAGNIFIED BY THE BAUSCH AND LomB APOCHROMATic LENS SYSTEM. MAGNIFICATIO-N ABOUT 2300 It shows the heavy-staining dense, outer periphery enclosing a light staining, cytoplasm-like area which in turn surrounds a central darkly-staining, small, roundish, nucleus-like spot. Under the high magnification here employed, about 2300, much of the internal structure of the Staphylococcus pyogenes-aureus is clearly evident. Each coccus presents a dark, round'ish, nucleus-like center. Surrounding this is a lighter-staining, cytoplasm-like material; and at the periphery is a dense, darklystaining membrane. Figure 3a is a diagram which illustrates the plan of structure of the individual coccus. of the type shown in figure 3. The peripheral cellwall-like outer membrane is shown at x. The cyto-

7 A STUDY IN BACTERIAL MORPHOLOGY plasm-like body inside the cellwall is labelled y. The nucleus-like center is indicated by the reference line z. Figure 3b is a tracing of a group of cocci shown in figure 3. Figure 3c is a free-hand enlargement of the cluster of staphylococci traced in figure 3b. Figures 3, 3a, 3b, and 3c illustrate the general plan of this staphylococcus, which exhibits clearly what appear to be nucleus, cytoplasm and cellwall. K 261 3a ( 3b 3c 3a. This figure is a diagram illustrating the general plan of these cocci: x, cellwall-like periphery; y, cytoplasm-like body; z, nucleus-like central structure. 3b. Tracing of a cluster of staphylococci seen in figure 3. 3c. Free hand enlargement of the cluster of cocci traced in figure 3b. The next photograph (fig. 4) of the same organism presents a variation in external shape. Each coccus of the different groups tends to take the form of a polygon, apparently on account of the pressure upon it of neighboring cocci. To this extent, therefore, these organisms when in clusters are more polygonal in outline than they are conventionally described. Figure 5 is a photograph of the same organism with the same magnification which shows even more clearly the nuclear-like central body in several of the cells. This body stands out especially

8 262 J. F. MCDONALD, J. T. LITTLE AND E. RUCKENSTEINER FIG. 4. STAPHYLOCOCCUS PYOGENES-AUREUS, MAGNIFIED 2300 TIMES. WRIGHT'S STAIN The polygonal outline of the cocci, which shows well in an upper cluster of this photograph, is due to pressure of these cells upon each other. FIG. 5. STAPHYLOCOCCUS PYOGENES-AUREUS. WRIGHT'S STAIN. MAGNIFICATION 2300, AS IN FIGURES 3 AND 4 Structural plan of coccus identical with that already seen in Staphylococcus pyogenes-aureus with same magnification. Note the central nucleus-like, round dark spot, the peripheral membrane, and the intervening lighter, cytoplasmlike area. well in some of the cells located near the summit of the cluster. Here is shown also very clearly the deeply-staining outer rim of the cocci. The outer dark staining periphery is generally much more clearly evident when seen in the microscope (2300)

9 than in the picture. A STUDY IN BACTERIAL MORPHOLOGY 263 The outer wall shows deeper in this picture owing perhaps to the longer time of exposure to the stain. Figure 6 is a photograph of Staphylococcus pyogenes-citreus. The individual organisms of the different groups exhibit the same structural morphology as those already described in figures 3, 4, and 5. In each cell there is a dark, nucleus-like center, a lighter surrounding cytoplasm-like body, and outside of this an outer limiting membrane. The outer membrane is seen much FIG. 6. CL-USTERS OF STAPHYLOCOCCUS PYOGENES-CITREUS. WRIGH-T'S STAIN. MAGNIFICATIo-N 2300 Structuiral plan as in other cocci already-studied at same magnification. Note nucleus-like center, outer heavy membrane, and intervening light-colored cytoplasm-like area. more clearly through the microscope than in the photograph. The Staphylococcus pyogenes-ci'treus therefore exhibits a morphological differentiation similar to that already described for the Staphylococcus pyogenes-aureus. The large, heavy, dark areas near the center do not represent bacteria. In the seventh photograph (fig. 7) is shown Micrococcus tetragenus. Several four-cell groups with some larger clusters are here presented. In the individual cells of each group may

10 264 J. F. MCDONALD, J. T. LITTLE AND E. RUCKENSTEINER be seen a central, small, round, dark nucleus-like spot. At the periphery of the cell is a dark, heavy, cell-wall-like membrane. Between these parts is a lighter-colored cytoplasm-like area, forming the thickest portion of the bacterium. The single cocci of each group present, therefore, a structural plan similar to that of the cocci already described. Figure 8 is a photograph of a bacillus, the bacillus subtilis. It presents structural characteristics apparently comparable in FIG. 7. MICROCOCCUS TETRAGENUS. MAGNIFIED 2300 TiMES. WRIGHT'S STAIN Several four-cell groups with some larger clusters may here be seen. This coccus presents the same plan of structure as those cocci already studied. In each may be found a central round, dark, nucleus-like dot, and at the periphery a heavy dark, cell-wall-like outer membrane. Between these parts is a thicker, lighter, cytoplasm-like area. The plan of structure of the Micrococcus tetragenus is therefore similar to that shown in the cocci already considered. general to those of the cocci just described. The bacilus shows an outer dark-staining membrane, an inner, axially located, dark-staining, slender, rod-shaped nucleus-like body, and between the outer membrane and the inner slender,' axial rod is a more lightly staining, cytoplasm-like structure g'ivi'ng body to the bacillus. The black spots in the photograph are not bacilli. Figure 8a is a tracing of the bacillus subtilis, shown in fig. 8. The parts which appear to be cellwall, cytoplasm, and nucleus, are marked respectively x, y, and z, as in the cocci in figure 3a.

11 A STUDY IN BACTERIAL MORPHOLOGY 265 In recounting the detail of these bacteria we have for descriptive purposes used such terms as nucleus-like, cytoplasm-like, and cell-wall-like for the differentiated portions of the cells on account of the obvious structural similarity of these parts to the FIG. 8. BACILLUS SUBTILIS. WRIGHT'S STAIN. MAGNIFICATION 2300 This is a photograph of Bacillus subtilis. Like the cocci already described, this bacillus presents an outer limiting membrane, an inner, slender, rod-shaped, nucleus like axially located body, and an intervening cytoplasm-like structure. The black spots in the picture are not bacilli. Figure 8a is a tracing of the bacilli in figure 8. The parts which appear to be cell-wall, cytoplasm, and nucleus, are numbered respectively x, y, and z. In connection with the photograph is a tracing of the same baoillus (fig. 9a). The organism presents intracellular features apparently comparable to those described above for the coccus. There are (x) dark staining periphery enclosing (y) a light staining cytoplasm-like body, and this in turn surrounds (z) the rod-shaped, nucleus-like central structure. nucleus, cytoplasm, and cell-wall of the cells of higher plants and animals. This structural similarity is evident and striking, yet final conclusions upon the exact identity of the parts so designated with the known parts of the cells which compose higher organisms would be premature at this stage of our investigation.

12 266 J. F. McDONALD, J. T. LITTLE AND E. RUCKENSTEINER SUMMARY AND CONCLUSIONS A study was made of the internal structure of several micro6rganisms. 1. As seen by the ordinary high-power objective (magnification about 450), no detail of internal structure of the organisms so studied was apparent. Each coccus appeared in the photograph as a tiny round dot. 2. The individual organisms, seen under the ordinary oil immersion (magnification about 1000) stand out more clearly, but their internal structure is little, or not at all, in evidence in the photograph; however, some details of internal structural differentiation can be made out at this magnification directly under the microscope. 3. Several types of staphylococcus and the Bacillus subtilis are pictured, with magnification of 2300 in which much detail of the plan of cell structure and organization is clearly evident. It might be said that in general there is for each organism a dark-staining, cell-wall-like periphery, an inner dark-staining nucleus-like, centrally located structure, and an intermediate cytoplasm-like body consisting of more lightly staining material. The central body and the outer membrane of the coccus takea a heavy blue-black shade. The cytoplasm-like material was a bluish-pink. The bacilli took with the same stain a dark color for the central body and the peripheral membrane. These, like the nucleus-like center and cell-wall-like periphery of the coccus, were bluish-black. The cytoplasm-like material of the bacillus stained like that of the coccus a light bluish-pink. The different component parts of the bacilli stained much lighter than those of the cocci. 4. The facts presented in connection with these various microorganisms support the first of the several theories summarized above, namely, that bacterial cells, like those of higher plants and animals are composed of different kinds of structural material, definitely separated from each other in the organization of the cell. This morphological characterization brings bacteria into

13 A STUDY IN BACTERIAL MORPHOLOGY definite structural homology with the cells of higher plants and animals. We are studying other micro6rganisms, and with varied technique, in an effort to determine the extent of this structural homology in different types of bacteria, and to define more exactly the homology of these structural features with known structures of the cells of higher plants and animals. We wish to tender thanks to Dr. Francis M. Duffy of the department of Bacteriology for his co6peration in supplying us with bacterial cultures for this study. We also wish to give sincere thanks to the Bausch and Lomb Optical Company for their interest and assistance in supplying us with the special lenses, apochromatic objective, compensating eye-piece, and aplanatic condenser, which made possible the prosecution of this research. We are especially thankful for the personal assistance of Mr. H. W. Hollister of the Bausch and Lomb Company, who rendered us every courtesy and help in the initiation of our work. REFERENCES 267 GUTSTEIN 1924 Zbl. f. Bakt., Abt. I., Orig., 93, 233. GUTSTEIN 1925 Zbl. f. Bakt., Abt. I., Orig., 94, 145, and 95, 1. DOBELL, C. CLIFFORD Quarterly Jour. Microscop. Sci., 56, 395. LASZLO 1925 Diss. Budapest. Cited by A. Moeller, D. m. W. 51, PARK AND WILLIAMS 1924 Pathogenic Microorganisms, p. 31. SCHLEIDEN, MATTHIAS JOSEPH 1838 Phytogenese. Beitrage zur Phytogenese, Muller's Arch., Berlin, SCHUMACHER 1922 Zbl. f. Bakt., Abt. I., Orig., 88, 362. SCHWANN, THEODOR 1839 Mikroskopische Untersuchungen uiber die Verbereinstimmung in der Struktur und dem Wachstum dem Tiere und Pflanzen, Berlin.