THE TAXONOMY OF THE GENUS BACILLUS. Received for publication February 5, 1940

Transcription

1 THE TAXONOMY OF THE GENUS BACILLUS I. MODES OF SPORE GERMINATION CARL LAMANNA Laboratory of Bacteriology, College of Agriculture, New York Cornell University, Ithaca, Received for publication February 5, 1940 A major problem of taxonomy is to discover and to learn how to apply successfully characteristics that are of significance in the differentiation of groups of organisms, and species. In general, morphological characters have been looked upon with great favor, so that classifications down to genera usually depend upon morphology. If a morphological character like spore germination can be applied to delimit groups or species, the taxonomy of the genus Bacillus will be thereby aided. A review of the literature reveals considerable disagreement as to the correctness of using spore germination in taxonomy. The early investigators, especially Arthur Meyer's students (Gottheil, 1901; Neide, 1904), utilized this character as they considered it constant for any one species. Yet a perusal of species descriptions reveals a number of contradictory statements as to the type of germination that characterizes a given species. For example, in Marshall's "Microbiology" (1921) Bacillus megatherium is illustrated as germinating equatorially, while de Dary (1887), who originally described the species, claimed absorption of the spore coat. Gay's (1935) text records confusion in regard to the method which Bacillus anthracis exhibits. Grethe (1897) studied the question using five cultures of as many species and concluded that spore germination has no differentiating value. Ford et al. (1916) came to a similar conclusion. Current text books disagree; Salle (1939) believes germination to be generally constant while Henrici (1934) does not. Knaysi 347

2 348 CARL LAMANNA (1938) in his recent review of the cytology of bacteria takes the sensible view that it may characterize some organisms and not others. The present study, over a period of two and one-half years, has thrown some light on the subject. The length of time over which the observations were made allows an opinion as to the constancy of germination. The number of cultures of diverse origins studied permits some understanding of the taxonomic significance of spore germination. CULTURES A total of 105 cultures were studied, of which 77 were isolated by the author in 1936, 27 were received as named species and one as an unknown in They are listed under the species names assigned to them after a morphological, physiological, and serological (spores as antigen) study. The number of the culture in our collection is followed by a statement as to source. Those numbered with the letters C and S were supplied respectively by H. J. Conn of Geneva, New York, and M. H. Soule of the University of Michigan. I. Bacillus subtilis S8 Bacius subtilis, Ford. 72 Soil. 58 Soil 400C.1 43 Soil 450C. 41 Soil 500C. 78, 79 Horse manure 370C. 60 Horse manure 450C. 44, 45, 64, Horse manure 500C Air. 47 Creek. 61 Pasteurized whole milk. 70 Soil from a soy bean field 500C. 1 Temperature after source indicates the temperature of incubation at which isolations were made; otherwise the incubation temperature was 3035oC.

3 TAXONOMY OF GENUS BACILLUS II. Bacillus agri 13, 14, 15 Pasteurized skim milk Pasteurized skim milk Pasteurized skim milk 450C. 80 Soil. III. Bacillus vulgatu8 C4 Bacillus subtilis (Marburg strain) by Conn. C5 Bacillus vulgatus by Conn. C8 Bacillus arternimus by A. H. Robertson. C01 Bacillus leptosporus, Klein. Pribram collection. Si Bacillus mesentericus, Kral. From Kral by Novy, S9 Bacillus subtilis, no. 1 aus Strassenkehricht. Obtained from Grasberger, Wien, S12 From eye infection. Isolated by Foster, Denver, Large intestine of frog, Rana palkstrus. 20 Pasteurized skim milk , 69, 71 Cinders. IV. Bacillus mesentericus C7 Bacillus mesentericus by Conn. S2 Bacillus mesentericus by Ford. Slo Bacillus subtilis No. 4 from Preisz, Budapest, Air. 23 Pasteurized skim milk 400C. 51 Soil from a soy bean field. 54 Soil. 59 Horse manure 450C. 62, 63 Soil from a soy bean field V. Bacillus cereus C3 Bacillus cereus by Conn. C12 Bacillus petroselini from Pribram collection. C13 Bacillus goniosporus from Pribram collection. C14 Bacillus sessilis, Klein from Pribram collection. C16 Bacillus subtilis, Seitz, pathogen from Pribram collection. S4 Bacillus cereus from Rettger S6 Bacillus subtilis, Koch, from Germany by Novy, S7 Bacillus subtilis, Kral, from Kral, 1890.

4 350 CARL IAMANNA 25, 34 8, 12, 31, 52, 55 16, 35, 37, 38, ,2,4,5, 6 C , 48, Cl Clo , 27 7, 9, 32, 76, 77 C6 C9 17 Sl1 Creek. Soil. Pasteurized skim milk. Pasteurized skim milk, 400C. Air. Cinders. Moss. Soil from a soy bean field. Large intestine of frog, Rana palastrus. VI. Bacilus mycoides Bacillus mycoides by Conn. BaciUus mycoides, University of Michigan collection. Pasteurized skim milk. Soil. Moss. VII. Bacillus megatherium Bacilus megatherium by Conn. Bacillus niger by A. H. Robertson (lacks black pigment). Bacillus megatherium, University of Michigan collection. Bacillus megatherium, Cornell University collection. Pasteurized skim milk 400C. Creek. Soil. VIII. Bacillus simplex Bacillus simplex by Conn. IX. Bacillus albolactis Bacillus albolactis by Conn. Pasteurized skim milk. X. Unidentified Bacilus subtilis, Str. Askanar, from Grumbach, Zurich, 1922.

5 29 Horse manure 500C. 46 Creek 49, 53 Soil. 57 Horse manure 370C. TAXONOMY OF GENUS BACILLUS TECHNIQUE OF OBSERVATION A slide culture method developed by Dr. Georges Knaysi of this laboratory was used to observe the method of bacterial spore germination. The technique is as follows: 1. A clean slide is sterilized in the bunsen flame and placed in a sterile petri dish. 2. No. 1 cover slips 22 mm. square are cut into 4 or 5 equal strips, flamed, and dipped into a sterile petri dish containing a shallow depth of sterile melted paraffin. The excess paraffin is allowed to run off. 3. Two of these strips are placed 15 to 16 mm. apart on the sterile slide parallel to each other and to the width edges of the slide. The melted paraffin solidifies and attaches the strips to the slide. 4. a. A clean no mm. square cover slip is flamed and placed on top of the strips. The warm cover slip melts some of the paraffin which resolidifies, and attaches the cover slip. A small quantity of a spore suspension is placed at the edge of the cover slip next to the space between the cover slip and slide. By capillarity the spores are drawn into the space; or, b. A drop of spore suspension is placed on a clean sterile no mm. square cover slip. The suspension is spread evenly and allowed to dry. The result is a thin layer of spores attached to the surface of the cover slip. The slip is warmed slightly and placed, spore layer facing the slide, on top of the strips. 5. By capillarity, agar at 450C. is introduced into the space between cover slip and slide. The above procedure gives a film of agar with bacterial endospores at its surface right beneath the cover slip. As the agar film is only slightly thicker than a no. 1 cover slip the microscope can be used to full advantage. The slide may be incubated and examined at intervals under the oil immersion objective, or placed in a stage incubator and germination examined continuously. 351

6 352 CARL LAMANNA If any difficulty is experienced in initially focusing the spores it has been found advantageous to mark the cover slip on the same side as the spores with a china marking pencil. Then by focusing upon the china crayon mark the focus is on the same plane as the spores. By merely shifting the field the spores come under observation. In these experiments, 3 per cent agar was found superior to 1.5 per cent agar as often the latter held sufficient water to allow Brownian movement, which makes observation more difficult. The nutrients were 0.2 per cent beef extract, 1 per cent peptone, and 0.05 per cent glucose. Each culture was observed at least twice on two separate occasions and as many as five times for some cultures. The age of the spore was not found to affect the type of germination. Spores from cultures 1 to 16 days old were utilized, depending on the material at hand. OBSERVATIONS Before the work had proceeded far it was realized that often the type of germination was not a simple matter of absorption of the spore coat or of polar or equatorial germination. Important deviations were noted which are discussed under the headings of the particular type of germination. For a given culture, the particular type of germination proved constant. The observations convince one that germination is taxonomically significant, if we first properly catalogue the modes of germination, and then consider each mode separately. The following is a separation of the modes of germination observed. All have been recorded on one occasion or another by various authors (de Bary, 1888; Grethe, 1887; Gottheil, 1901; Chester, 1904; Neide, 1904). I. Spore germination by shedding of the spore coat. The characteristics of this method are three: a. Spore does not expand greatly in volume previous to the germ cell breaking through the spore coat. The limit of volume increase of the spore may be considered to be twice its original volume.

7 TAXONOMY OF GENUS BACILLUS b. Spore coat does not lose all of its refractive property previous to germination. c. After the second division of the germ cell, giving a chain of three organisms, the original spore coat, remaining attached to the cells, is visible for a long time after germination. It is persistent. A. Equatorial germination. 1. Spore coat does not split along the transverse axis. The germ cell forces its way through the coat at an angle approaching 900 to the longitudinal axis of the spore (figs. 1 to 4). The cell usually grows perpendicular to the longitudinal axis (fig. 4), but often the growth may continue parallel to that axis (fig. 5). So-called oblique germination occurs when the germ cell is at an angle of about 450 to the longitudinal axis when it first breaks through (fig. 6). In cultures showing this type of equatorial germination some spores could always be found with oblique germination. A few cultures that showed a greater number of spores with oblique germination also have many at an angle truly approaching 900. Therefore it is to be concluded on the basis of the cultures studied that an organism may exhibit both types of germination mentioned in this paragraph, but not any other type. Example: Bacillus subtilis. Cultures 42, 43, and 44 show both this type and polar germination. Cultures 45 and 65 exhibit polar germination in the great majority of instances. However, these five cultures have many spherical, and barely oval shaped spores, for which it would be difficult to distinguish equatorial from polar germnation. 2. Spore coat splits along the transverse axis (figs. 7 to 11). The growing germ cell forces its way through the wall and the spore coat splits along its transverse axis except for a narrow bit directly behind the growing cell. An analogous picture would be one of an egg split open and the two halve shells remaining attached in only one slight place. Example of such germination is Bacillus vulgatus. B. Polar germination. The germ cell breaks through at an angle of 900 to the transverse axis of the spore (figs. 12 to 16). In the cultures designated Bacillus agri polar germination was recorded upon original isolation. During the intervening years, 353

8 354 CARL LAMANNA 6I Fii.2 F*1.3 ~ Fig. 4 Fig. ~~~~~~~~~~~~. 1.. ~~ Flq. 7 Fij,8 Figp 9 Fig. 10 L.A. LONGITUDINAL AXIS T.A. T1kANVVEME AXIS FIG. 11 FIGs. 1 To 6. EQUATORIAL GERMINA- FIGS. 7 TO 11. EQUATORIAL GERMINA- TION WITHOUT SPLITTING ALONG TION WITH SPLITTING ALONG TRANSVERSE AXs TRANSVERSE Axs however, the picture changed slightly. The spores no longer remained refractive during germination. In this respect absorption of the spore coat is approached.

9 .12 F,. 13 Fic. 14. Figy i5 Fig. 17 FiT.18 Fij. 16 Figl 19 Fig. 20 FIGS. 12 TO 16. POLAR GERMINATION 355 5q.21 FIGS. 17 TO 21. COMMA SHAPED EXPANSION

10 356 CARL LAMANNA C. Comma shaped expansion (figs. 17 to 21). The growing germ cell splits the spore coat in half along the entire transverse axis. The result is that two halves of the spore coat remain at either end of the growing cell which then shows two polar caps. As the cell grows, it becomes comma shaped in the majority of cases. It acts as though a thin capsule still held the two halves of the spore coat together and the pressure exerted by the growing germ cell causes a buckling of the whole resulting in the comma shaped appearance. This method of germination seems to be a development from the equatorial germination where the coat is split only in part along the transverse axis. Further evidence for this view will be presented in a future paper where it will be shown that serologically both types of spores are related. It also seems to be a move in the direction of absorption of the spore coat, as many of the spores lose much of their refractiveness previous to germination. Example: Bacillus mesentericus. II. Spore germination by absorption of the spore coat (figs. 22 to 27) The characteristics of this method are three. a. The spore expands greatly during germination. A tripling or greater increase of the original volume occurs (fig. 24). b. The spore loses its characteristic refractiveness during germination so that it is difficult to say when the spore has disappeared and the germ cell appeared. c. After the second division of the germ cell, even if a thin capsule originally remains, all traces of the spore coat are gone. Examples: Bacillus cereus, Bacillus megatherium, Bacillus mycoides. Some strains germinating by absorption regularly show a thin capsule remaining about one end of the growing cell. This would appear as a polar germination (fig. 28). In other cases equatorially located capsules are seen (fig. 29). Yet in all instances the spore is considered to germinate by absorption inasmuch as the three characteristics of the method are still adhered to. Then again, the capsule, polar or equatorial, is not a constant feature. Many cells from the same culture will not show any. Both polar and equatorially located capsules are found in any one culture

11 TAXONOMY OF GENUS BACILLUS357 though one or the other may be preponderant. These so-called capsules, though probably originating as a part of the spore coat, F55.22 'p FIT2 Fig.23 Fsc.24 Downloaded from Fqc. 23 IC Fi.26 Fig f9g. 29 C - CAPSULE.. FIGs. 22 TO 29. SPoRT GERMINATION BY ABSORPTION are to be distinguished from the spore coat of the ungerminated spore in that their refractive property seems more nearly to on March 7, 2019 by guest

12 358 CARL LAMANNA approach that of the cell wall of the germ cell than the refractive quality of the original spore coat. Therefore, germ cells with these capsules are not to be confused with those showing true polar and equatorial germination. Of great significance is the fact that all of the organisms that Gottheil (1901) and Neide (1904) listed as having both polar and equatorial germination may be regarded as absorbing their coat, as they fulfill the three characteristics listed for this method. It may also be added that the greatest disagreement in the literature is to be found for the large-spored organisms (Bacillus megatherium, Bacillus cereus, Bacillus mycoides) whose method of germination is absorption as defined here. The remaining observation to be recorded is that all the smallcelled species studied showed coat-shedding types of germination, while all of the large-celled species showed absorption of the spore coat. S3UMMARY When the methods of spore germination are clearly defined and separated they have taxonomic value. Spore germination of a given type is constant for any one culture and species. The modes are summarized as: I. Shedding of spore coat: small-celled species. A. Equatorial. Not splitting along the transverse axis: Bacillus subtilis. Splitting along the transverse axis: Bacillus vulgatus. B. Polar: Bacillus agri. C. Comma shaped expansion: Bacillus mesertericus. II. Absorption of the spore coat: large-celled species. The above outline does not claim to be complete. Rather it is hoped that it contains sufficient truth to be used as a convenient framework around which to build or from which to subtract according to the dictates of further investigation. BIBLIOGRAPHY CHESTER, FREDERICK D A review of the Bacillus subtilis group of bacteria. Zentr. Bakt. Parasitenk., Abt. 2, 13, DE BARY, A Comparative Morphology and Biology of the Fungi, Mycetozoa, and Bacteria. Oxford.

13 TAXONOMY OF GENUS BACILLUS 359 FoRD, W. W., LAWRENCE, J. S., LAUBACH, C. A., RIcE, J. L Studies on aerobic spore bearing non pathogenic bacteria. J. Bact., 1, , GAY, FREDERICK P Agents of Disease and Host Resistance. Chas. C. Thomas. GOTTHEIL, Botanische Beschreibung einiger Bodenbakterien. Zentr. Bakt. Parasitenk., Abt. 2, 7, 430-5, , , , , , GRETHE, G Ueber die Keimung der Bacteriensporen. Fortschr. Med. 15, 43-51, 81-88, HENRICI, A. T The Biology of Bacteria. D. C. Heath & Co. KNAYSI, GEORGES 1938 Cytology of Bacteria. Botan. Rev., 4, MARSHALL, CHAs. E Microbiology. 3rd Edition. P. Blakiston's Son & Co. NEIDE, E Botanische Beschreibung eineger sporenbildener Bakterien. Zentr. Bakt. Parasitenk. Abt. 2, 12, 1-32, , 337, 352, 539, 554. SALLE, A. J Fundamental Principles of Bacteriology. McGraw Hill Book Co. Downloaded from on March 7, 2019 by guest

14 360 CARL LAMANNA PLATE 1 PHOTOMICROGRAPHS OF ACTIVELY GERMINATING SPORES OBSERVED IN COVERED SLIDE CULTURES FIGS. 1 TO 4. Bacillus vulgatus. Successive stages in equatorial germination with splitting along the transverse axis. X1100 FIG. 5. Bacillus subtilis. Early stages of equatorial germination without splitting along the transverse axis. X1100 FIG. 6. Bacillus subtilis. Late stages of equatorial germination. Remnants of spore coat persist. X1100 FIGS. 7 TO 10. Bacillus megatherium. Successive stages in germination by absorption of spore coat. X1100

15 JOURNAL OF BACTERIOLOGY, VOL. XL PLATE 1 2.~~~~~~~~S & ~ w¾11,.pen' Downloaded from on March 7, 2019 by guest I p,...<:s.. (Carl Tamanna: Taxonomy of Genus Bacillus)