minds of many investigators. Matzuschita (1902) mentioned

CYTOLOGICAL CHANGES DURING THE FORMATIONOF THE ENDOSPORE IN BACILLUS

MEGATHERIUM'STANHOPE BAYNE-JONES AND ALEXANDER PETRILLI

Department of Bacteriology, School of Medicine and Dentistry, University ofRochester, Rochester, New York

Reviewers of our knowledge of the mode of formation of endo-spores in bacilli usually present the evidence under one or more ofthree theses: (1) the endospore is formed by the growth of aspecialized granule in the bacterial protoplasm; (2) the endosporeis formed by the fusion of a number of granules, some of whichmay be nuclear material; (3) the endospore is formed by gradualcondensation of cell-substance, possibly due to dehydration.These notions have been derived from the study of both stainedand unstained bacilli. Although the origins of these conceptionsare obscure, they can be associated with names of distinguishedbacteriologists. The first of these opinions was expressed byKoch (1876) in his description of the growth of the spore from agranule in Bacillus anthracis. de Bary (1887) noted the growthof the spore from a granule in Bacillus megatherium and Ward(1895) described the same process in Bacillus ramosus. Ernst(1888-1889) and Babes (1889) are the sponsors of the secondtheory of spore-formation and their view of the origin of the sporein the fusion of granules, composed chiefly of nuclear material,has been corrected and extended in modified form by Schaudinn(1902) and Dobell (1908, 1909, 1911). The third conception ofspore-formation by protoplasmic condensation has been in theminds of many investigators. Matzuschita (1902) mentionedit definitely and lately von DarAnyi (1927, 1930) has empha-sized it as a consequence of his observations on the effects of col-

1 Presented at the thirty-third annual meeting of the Society of AmoricanBacteriologists in Baltimore, Maryland, December 28, 1931.

261

262 STANHOPE BAYNE-JONES AND ALEXANDER PETRILLI

loidal dehydration in bacteria. Probably most bacteriologistsaccept the opinion of Koch and de Bary and would agree withCook (1932), who in his recent paper on bacterial spores, statesthat de Bary's account of the growth of the spore from a granulecould not be bettered. A review of the original reports of observa-tions on unstained bacilli, however, arouses some doubt as towhether the methods of periodic visual observations and stainingprocedures used in those studies were actually capable of yieldingsufficient information to support the conclusions drawn fromthem. Furthermore, any attempt to watch the random behaviorof granules in bacilli during spore-formation will excite bothbewilderment and skepticism in the observer. An additionalbasis for doubting the opinion that the spore grows from a granuleor granules is furnished by the recent work of Wyckoff and TerLouw (1931), who have reported that even in photographs madewith ultraviolet light they were unable to detect a precedentgranule in the sporogenous area in Bacillus subtilis. As the obser-vations of Wyckoff and Ter Louw were not continuous from thecommencement to the end of spore-formation, their work cannotbe said to settle the question. While it is not beyond the rangeof possibility that all these three modes of endospore-formationmay occur in a single or different species of bacilli, it seems prob-able that so morphologically definite a phase in the life-cycle ofsome bacteria should occur according to a uniform process. Atpresent, however, the force of the published observations doesnot compel the acceptance of any one of the three prevalent con-ceptions of the process of endospore-formation.The formation of a spore in a bacillus is a cellular process com-

posed of both slow and rapid phases. Neither can be followed bythe eye. Continuous visual inspection of a slow cellular processover long periods is impossible on account of fatigue of the eye.Periodic records of visual impressions are uncertain and may leadto false conclusions because of unobserved changes taking placein the intervals between observations. Periodic photographyovercomes many of these difficulties, as far as the optical resolu-tion of objects and the registration of images will permit. Thismethod becomes especially useful when the intervals between

FORMATION OF THE ENDOSPORE IN B. MEGATHERIUM 263

the photographs are shortened by means of motion picture appar-atus. The record upon the film, when projected rapidly, presentsa dynamic reproduction of the process occurring in a selected cellor cells. When used for the study of individual frames, projectedat convenient magnification as single pictures upon a screen,this record gives material suitable for analysis by measurementand by prolonged and repeated study of details. Time intervalsbetween stages can be accurately determined from the film.This kind of record presents, finally, the consecutive changesoccurring in an unstained microorganism, undisturbed by manipu-lations, and growing under as natural conditions as an opticallyserviceable culture chamber will permit. Whenever motionphotomicrography can be applied to the investigation of a processor cyclical stage in a minute organism, it would seem to us to bethe method of choice.For these reasons, since 1928 we have been investigating the

process of endospore-formation in Bacillus megatherium by themethod of motion photomicrography. We have not investigatedthe cytological changes by means of staining reactions. Thedescription and analysis of the record obtained upon the films, tobe presented in this paper, confirm the observations of some ob-servers, are definitely opposed to the opinions of others and seemto us to add somewhat to the knowledge of the cytological changesoccurring during this process.

METHODS AND MATERIALS

The apparatus for motion photomicrography used by us inthese studies was essentially the same as that devised by Bayne-Jones and Tuttle (1927). It has been modified by driving thecamera by means of a synchronous motor through a set of adjust-able accurate gears and by the insertion of a pentaprism in theview finder. Exposures were made at the rate of 7 per minute.The film used was 16 mm. panchromatic reversal film. Thesource of light was a tungsten ribbon filament 6 volt, 108 wattlamp, set up 1 meter from the mirror of the microscope and ad-justed to give critical illumination. Between the lamp and themirror were interposed a Br8 green Wratten filter (transmission


wavelengths 460-600mM), neutral density filters from 0.6 to 0.9,and a glass cell 5.5 cm. thick containing a saturated aqueous solu-tion of quinine bisulfate to absorb heat and some of the shorterwavelengths of light. The intensity of the light falling upon thepreparation was still further reduced by the substage diaphragmof the microscope to a degree just suitable for sharp visual observa-tion through the view finder. The microscope objectives usedwere either a 1.8 mm. fluorite objective (N.A. 1.3) or a 3 mm.apochromatic objective (N.A. 1.4). The 1.8 mm. fluorite objec-tive proved to be the more serviceable. With a X3 negativeocular, this gave a magnification of X300 on the film, with suffi-cient resolution and sharpness of focus to allow the use of a 25fold enlargement of single frames for measurements.The microscope was enclosed in an incubator with temperature

regulation at 370 ±0.10C.The shutter of the Model A Cine-Kodak which we used requires

that the illumination be continuous. This would have its dis-advantages if it were necessary to use bright illumination for along time, since we have seen that the growth of yeast and bacteriais inhibited by intense light in this spectral region. But longexperience with our apparatus showed us the limits of the effectsof our illuminant and we have never seen any deleterious actionproduced by the relatively dim light focussed upon the prepara-tions photographed for these studies.The culture chamber consisted of a cell 0.5 mm. deep. It was

made by cementing a glass ring, 17 mm. in diameter, upon a clearmicroscope slide 1 mm. thick. A mixture of beeswax, paraffinand rosin was used as the cement, as this remain hard at 370C.The slide, ring and cover glass were sterilized in a flame. A thinlayer of the melted wax was placed upon the bottom and top bord-ers of the 0.5 mm. ring. The ring was then attached to the slide.In the center of the ring a small cone of agar-peptone medium wasbuilt up by placing, with a sterile capillary pipette, drops of mol-ten agar in this position until the top of the cone reached theupper level of the ring. When the agar hardened, the top of theagar cone was inoculated with bacteria by means of a fine platinumneedle. A No. 1 cover glass was then laid over this, pressed down


and cemented to the ring. Usually a small space for the passageof air was left open on one side between the under surface of thecover glass and the top edge of the ring. When the cover glasswas cemented in place, the top of the agar cone was flattened,giving a circle 3 to 4 mm. in diameter. In this area, the bacterialay in a shallow layer, usually the depth of the thickness of a singleorganism. They grew in this layer, apparently without compres-sion or obstruction, and spread over the flat surface of the mediumin a single layer. Hanging drops of agar were tried at times, buttheir optical properties were not as satisfactory as those of theshallow agar cone and they tended to dry too quickly.

Detailed descriptions of the apparatus, culture method andmethod used in measuring the images on the fims are given byBayne-Jones and Adolph (1932).We have used several cultures of Bacillus megatherium during

the course of our studies. The strain employed for the observa-tions reported here was a Kral Collection culture of Bacillusmegatherium, sent to us by Dr. P. B. Cowles in August, 1930.Its morphological and cultural characteristics were essentially thesame as those listed for Bacillus megatherium, de Bary, on page396 in the 1930 edition of Bergey's Manual of Determinative Bac-teriology, except that its endospores were formed nearer theterminal portions of the rods than in the central area. In germin-ating, the spores became round, then elongated and opened atone end, showing a type of polar germination.

FACTORS INFLUENCING SPORE-FORMATION

The conditions influencing the production of endospores byBacillus megatherium were discovered to be very precise. It wasnecessary in the culture cell, to provide for an apparently optimalbalance of oxygen tension, moisture and nutriment. We haveaccumulated a considerable amount of information upon the fac-tors influencing spore-formation in this and other species of bac-teria. While we shall present here only those details which beardirectly upon spore-formation under the conditions imposed byour photographic arrangements, we know from experiments withlarger cultures in test-tubes that factors influencing spore-forma-


tion in a layer of growth upon an agar cone below a coverslip are ingeneral the same as those affecting spore-formation under otherconditions of cultivation. With Bacillus megatherium, it seemedto us that an adequate supply of oxygen in proportion to theamount of growth was more important than the exhaustion of themedium. Wund (1906) showed that Bacillus megatherium failedto produce spores on an adequate medium when the oxygen con-centration of the gas over the culture was reduced below 1.13 percent. It is probable, also, that loss of water from the mediumtends to induce spore-formation at a stage of growth when anexcess of moisture might inhibit or delay spore-formation.

It was always necessary to provide an initially adequate med-ium for the growth of the vegetative cells, and we noted, in gen-eral, that an unfavorable environment inhibited spore-formation.We have seen appearances in the celLs of Bacillus megatherium

indicating that endospore-formation has a sort of momentum.Cells from fourteen- to sixteen-hour cultures, containing clearedareas, which we have learned to recognize as the sites of spore-formation, proceed to complete endospore development whentransferred from old to fresh medium. In such instances, spore-formation, once initiated, continued to completion, although thecell was removed from waste products and provided with a newsupply of food-stuffs. When these cells in the early stages ofspore-formation failed to complete the process on fresh medium,they remained in that stage. We have never seen a cell in the pre-liminary stage of spore-formation reverse the process and resumeits vegetative multiplication.

Since the bacterial spore serves to perpetuate the organismthrough hardships, it has been assumed that the spore develops inresponse to unfavorable environmental conditions. Our observa-tions on the factors influencing the formation of the spore areopposed to this view. Conditions close to optimum conditionsfor multiplication must be provided in order for the organism toprogress to the stage of spore-formation. We agree with Cook(1932) that the teleological argument does not give a true indica-tion of the primary function of the spore.


CONDITIONS PROVIDED FOR GROWTH AND SPORE-FORMATION

When planted upon a cone of medium composed of 2 per centagar, 2 per cent peptone, 0.5 per cent NaCl, with a pH of 6.8 to7.4., Bacillus megatherium grew abundantly under the cover slipbut few or none of the cells developed endospores. We assumedthat the organism used oxygen faster than it could diffuse into thegrowth, preventing the maintenance of an oxygen tension suitablefor spore-formation. On the same agar medium in a thin hangingdrop in the culture cell, spore-formation occurred in fourteen tosixteen hours in the cells growing between the cover glass and theagar. In this case it seemed that there was an adequate diffusionof oxygen through the thin layer of medium. This type of prepa-ration, however, was optically poor and subject to excessive dry-ing. Finally, we found that reduction of the peptone content ofthe agar medium to 0.05 per cent gave dependable preparations.Growth on the 0.05 per cent peptone, 2 per cent agar cone, be-neath the cover glass proceeded in a normal manner but came toan end before the available area of the medium was too denselycrowded with bacteria. This might be called a "starvation"medium. Nevertheless, it did not seem to cause abnormalities inthe culture. In the early stages of growth the celLs were largeand hyaline, similar to those occurring on a medium composed of2 per cent peptone and 2 per cent agar. In the late stages, thecells were the same size as, or slightly smaller than, cells measuredby Adolph and Bayne-Jones (1932) from motion photomicro-graphs of the growth on 2 per cent peptone agar. The effect ofthe reduced peptone content was to shorten the period of vegeta-tive multiplication so that the cells reached the age appropriatefor spore-formation at a time when the environmental conditionswere still favorable for spore-formation. Growth almost reachedits end in 16 to 19 hours, although some cells, which were not pro-ducing spores, continued to enlarge twenty-one hours after inocu-lation. We assumed that the retardation of growth on 0.05 percent peptone agar in the culture cell occurred before all theavailable oxygen was used or that the diffusion of oxygen couldkeep pace with the less abundant growth, permitting the main-


tenance of an oxygen tension suitable for spore-formation. Theoutcome of the experiments accorded with this prediction, but didnot, of course, prove the correctness of the assumption. Un-doubtedly, the conditions are complex. We did not make thequantitative measurements of each factor which would be neededto evaluate its separate influence.

OBSERVATION ON CYTOLOGICAL CHANGES

The cellular changes occurring during the final stages of spore-formation are shown in the motion picture frames reproduced inplate 1, figures 1, 2, 3 and 4. A description of the process is asfollows:

After the inoculation of the 0.05 per cent Difco peptone, 2 percent agar cone with Bacillus megatherium, the organisms underthe cover slip do not change during the next thirty to ninetyminutes. After this period of lag, growth proceeds rapidly.The cells are at first large and hyaline. Fine refractile granulesappear in the cells about 3 hours later. Occasionally one or morelarge refractile granules appear at this time. As the culture be-comes older, round granules of various sizes become very numer-ous in the cells. These granules are in rapid random motion.Some of the granules dart rapidly from one end of the cell to theother. There is no evidence of a streaming of granules to indi-cate a flowing of the protoplasm. In some cells, granules half aslarge as a mature spore appear and become surrounded by a clearzone. Two of these large granules may appear in a single cell.They have no connection with the formation of the spore, sincethey remain as granules in the cell after the development of thespore. The activity of the movement of the granules and theirrapid transit at times from end to end of a cell seem to indicatethat the protoplasm of the cell at this stage is quite fluid, with lowviscosity. When the cells become old, or die or begin to disinte-grate after spore-formation, the movement of these granulesceases.From the eighth to the thirteenth hour after inoculation, when

multiplication has stopped in most of the cells and when growth iscontinuing only slowly in others, the ends of the rods begin to


lose their granules. It is difficult to count and trace the granulesin these cells because moving granules drop out of focus, and newgranules may be formed. It is our opinion, however, that theends of the cells become clear of granules by a displacement of thegranules from that region and not by a liquefaction or fusion ofthe granules previously visible there, or by the growth of a specialgranule in this area. The clear area at the end of the cell fluc-tuates in size and gradually comes to occupy a cylindrical volumeequal to approximately one-quarter of the volume of the cell.The material in this cleared area appears to be denser than therest of the protoplasm of the cell. Granules in violent motion inthe remainder of the cell strike against the edge of this clearedarea but never penetrate it. This large cleared area is the regionin which the spore appears.The clear area at the end of the cell keeps a roughly cylindrical

form for three to four hours. Its density increases gradually andit gradually assumes an almost spherical shape (plate 1, fig. 1).The completion of the formation of the endospore, after the

cleared dense area of protoplasm begins to take on a definite form,occurs with astonishing rapidity. The rest of the process is fin-ished within the next thirty minutes. A stage which we havecalled the "prespore" appears as a dense refractile region in thiscleared area at the end of the cell. Its volume is smaller than thatof the original clear area but often twice that of the final spore.Within six to twelve minutes, this nearly spherical "prespore"contracts to an elongated ellipsoid and has a density, as judgedby its refractile property, as great as that of the mature spore.This is shown in cell A in plate 1, figure 3. The optical section ofthe focus used makes these bodies appear black. Neither theynor the spores were stained in these preparations. Within sixminutes or less, this elongated ellipsoidal spore contracts to thesomewhat shorter, but apparently no broader ellipsoidal spore,characteristic of this organism.During these terminal stages of spore-formation and after the

development of the spore, the movement of the granules becomesless and less in the remaining protoplasm of the cell. Neverthe-less, some of the granules may now pass into the area near the end


of the cell, along the side of the spore, into a region that was toodense to admit them before the contraction of the sporogenousarea.We have not seen the development of a membrane around the

material of the spore.Measurements of the dimensions and volumes of 12 cells with

contained endospores are presented in table 1. The time inter-vals of the visible stages of spore-formation are shown for cells Ato F. The measurements were made by projecting the singleframes of the film on a screen at an enlargement of 25 times, tak-ing the lengths and breadths of cells and spores with calipers andmeasuring these distances on a millimeter rule. The magnifica-tion of the images of the cells on the film was X300. This 25fold enlargement gave a magnification of X7500. The pictureswere sharp enough to be measured with an error within 10 percent. We think that these measurements reduced to micra areaccurate to within =0.1 micron. With a film of pictures freefrom distortion due to shifts in the focus of the microscope, wehave obtained more consistent and apparently more accuratemeasurements of bacteria than we have by the use of a filar micro-meter or ocular micrometer.

Since the spore of Bacillu8 megatherium does not swell its spor-angium, no local enlargement of the cell was to be expected.The measurements and calculations show that there was little orno increase in length or breadth of the cell and no significantchange in volume while the spore was developing.The volume of the cell was calculated as for a cylinder, using

the formula: volume = w/4 X length X square of breadth.The spores were regarded as an ellipsoid of rotation around thelong axis, with volume = r/6 X length X square of breath.The volumes of the cells containing endospores varied from 6.9

to 12.5 cubic micra. The volumes of the contained spores were0.55 to 0.97 cubic micron, with an average of 0.84 cubic micron.The average ratio of spore-volume to cell volume was 0.09. Butthis average obscures some considerable differences in this ratio,which varied from 0.06 to 0.13. Spores of the average size wereformed in both large and small cells.

FORMATION OF THE ENDOSPORE IN B. MEGATHERIuM 271

TABLE 1Dimensions and volumes of cells and contained endospores at different intervals

during spore-formation

CELL SPORECELNUBERTIMEAFTERSTGINOCULATION

Length Breadth Volume Length Breadth Volume

hours min- Ag tty cu

A Fig. 1 14 0 4.6 1.5 8.1 1.6 1.3 1.41 Prespore6 4.6 1.7 10.3 1.5 1.2 1.13 Prespore

Fig. 2 12 4.6 1.7 10.3 1.7 1.2 1.28 Prespore18 4.6 1.7 10.3 1.6 1.1 1.03 Prespore

Fig. 3 24 4.6 1.7 10.3 1.5 0.9 0.64 SporeFig. 4 30 4.6 1.7 10.3 1.5 0.9 0.64 Spore

36 4.6 1.6 9.3 1.6 0.9 0.68 Spore

B Fig. 1 14 0 3.5 1.5 6.2 1.5 1.1 Clear area6 3.6 1.5 6.3 1.5 1.1 0.97 Prespore

Fig. 2 12 3.6 1.5 6.3 1.5 1.2 1.13 Prespore18 3.5 1.5 6.2 1.5 1.1 0.97 Prespore

Fig. 3 24 3.5 1.5 6.2 1.5 1.1 0.97 PresporeFig. 4 30 3.5 1.5 6.2 1.5 1.1 0.97 Prespore

36 3.5 1.5 6.2 1.5 1.2 1.13 Prepsore

C Fig. 1 14 0 5.3 1.6 10.6 1.7 1.3 Cleararea6 5.5 1.7 12.5 1.6 1.3 Clear area

Fig. 2 12 5.3 1.7 12.0 1.3 1.3 Clear area18 5.5 1.7 12.5 1.7 1.5 Clear area

Fig. 3 24 5.5 1.7 12.5 1.5 1.3 1.32 PresporeFig. 4 30 5.5 1.7 12.5 1.6 1.3 1.15 Prespore

36 5.5 1.7 12.5 1.6 1.3 1.15 Prespore

D 15 45 5.3 1.3 7.0 1.9 1.3 Clear area16 3 5.3 1.3 7.0 2.0 1.2 Clear area

9 5.3 1.3 7.0 2.6 1.2 1.96 Prespore15 5.3 1.3 7.0 1.9 1.2 1.43 Prespore21 5.3 1.3 7.0 1.5 1.1 0.97 Prespore

17 33 5.3 1.3 7.0 1.3 1.2 0.98 Prespore45 5.3 1.3 7.0 1.3 0.9 0.55 Spore

19 27 ? ? ? 1.3 0.9 0.55 Spore21 33 ? ? ? 1.3 0.9 0.55 Spore

E 15 15 4.5 1.3 6.0 1.3 1.2 0.98 Prespore21 4.5 1.3 6.0 1.7 1.1 1.10 Prespore27 4.5 1.3 6.0 1.6 0.9 0.68 Prespore51 4.5 1.3 6.0 1.7 0.8 0.57 Prespore

16 21 4.5 1.3 6.0 1.5 0.8 0.50 Prespore


CELL NUMBER

E-Con-cluded

F

G

H

I

J

K

L

TABLE I-Concluded

TIME AFTER

hours min-

455157

19 321 33

15 152127

17 915

18 919 3

9152127

21 33

16

18

18

18

18

18

CELL SPORE

Length Breadth IVolumeLength Breadth Volume

4.

4.54.54.54.5

6.06.06.06.06.06.06.06.06.06.06.06.0

0 4.1

30 4.4

30 3.9

30 4.3

30 6.0

30 6.0

Is

1.31.31.51.5

1.41.41.41.41.41.51.51.51.51.51.51.5

1.5

1.5

1.5

1.5

1.5

1.5

cu ;s

6.06.07.97.9

9.29.29.29.29.210.610.610.610.610.610.610.6

A

1.51.51.51.51.5

1.21.31.51.51.51.51.61.61.71.51.51.5

7.3 1.5

7.8

6.9

7.6

10.6

10.6

1.5

1.5

1.5

1.5

1.5

A.

0.90.90.90.90.9

1.31.31.51.21.21.21.21.11.10.90.90.9

0.8

0.9

0.8

1.1

0.9

1.1

cu IA

0.640.640.640.640.64

1.771.131.131.131.211.041.100.640.640.64

0.50

0.64

0.50

0.97

0.64

0.97

STAGE

PresporeSporeSporeSporeSpore

Clear areaClear areaPresporePresporePresporePresporePresporePresporePresporeSporeSporeSpore

We have measured the lengths and breadths and calculated thevolumes of 75 free spores in a 6 weeks old culture of this strain ofBacillus megatherium for comparison with the sizes of the endo-spores contained in the rods of culture 14 to 21 hours old. Someof the sets of measurements of free spores with a filar micrometeror ocular micrometer showed considerable variations. The mostconsistent series showed that the average length was 1.6ki, averagebreadth 0.8M and average volume of free spores 0.6 cubic micron.

INOML&TIONI IL14 IU U L l.A


These are approximately the dimensions and volume of the con-tained endospores. In some of our measurements and calcula-tions the dimensions and volumes of free spores were larger and ageneral average of all measurements of free spores gave length,1.7,u, breadth l.l1u and volume 1.08 cubic micra. Differences inconditions of growth as well as differences in method of measure-ment may account for these variations.

SUMMARY AND CONCLUSIONS

Endospore-formation in Bacillus megatheribum was studied bymeans of motion photomicrography. The organisms were grownon a cone of 2 per cent agar containing 0.05 per cent Difco peptoneunder a cover glass. Under these conditions spores were formednormally within fourteen to eighteen hours after the inoculationof the medium. Pictures, taken at the rate of 7 per minute,were used by projection and enlargement to investigate the cyto-logical changes occurring during this process and to measure thecells and the spores at various stages.Some of the factors influencing spore-formation are described

and discussed. We are of the opinion that oxygen tension is amore important factor than exhaustion of the medium or accumu-lation of waste products. Apparently certain optimum condi-tions are required for spore-formation. Since unfavorable condi-tions inhibit spore-formation, it seems incorrect to regard thespore as a structure developed for the purpose of resisting anunfavorable environment.The process of spore-formation in these unstained preparations

of Bacillus megatherium took place by an apparent condensationof protoplasm in the ends of the cells and a rapid contraction of thematerial in this region. Cleared areas of dense material in theends of the cells persisted for two to three hours and then con-tracted within six to twelve minutes into the highly refractilespore.

Granules in active brownian movement throughout the cellswere gradually displaced from these dense clear areas and wereunable to penetrate the region in which the spore was forming.There was no visible evidence of the development of the sporefrom a granule.

JOURNAL OF BACTERIOLOGY, VOL. XXV, NO. 3


REFERENCES

ADOLPH, E. F., AND BAYNE-JONES, S. 1932 Jour. Cell. and Compar. Phys., 1,407-427.

BABES, V. 1889 Ztschr. f. Hyg., 5, 172-190.DE BARy, A. 1887 Lectures on bacteria. Translation of 2nd German edition,

1886. Translated by H. E. F. Garnsey. Revised by I. B. Balfour.Clarendon Press, Oxford, 193 pp. See p. 16.

BAYNE-JONES, S., AND ADOLPH, E. F. 1932 Jour. Cell. and Compar. Phys., 1,387-407.

BAYNE-JONES, S., AND TUTrLE, C. 1927 Jour. Bact., 14, 157-173.BERGEY, D. H. 1930 Manual of Determinative Bacteriology. Third edition,

p. 396. Williams and Wilkins Co., Baltimore.CooK, R. P. 1932 Bacterial spores. Biol. Rev., 7, 1-23.,v. DAmiNn, J. 1927 Centralblt. f. Bakt., etc., II Abt., Orig., 71, 353-357.v. DARNY, J. 1930 Centralblt. f. Bakt., etc., I Abt., Orig., 117, 543-547.DOBELL, C. C. 1908 Quart. Jour. Microscop. Sci., 52, 121-138.DOBELL, C. C. 1909 Quart. Jour. Microscop. Sci., 53, 579-596.DOBELL, C. C. 1911 Quart. Jour. Microscop. Sci., 56, 395-506.ERNST, P. 1888 Ztschr. f. Hyg., 4, 2546.ERNST, P. 1889 Ztschr. f. Hyg., 5, 428-486.KocH, R. 1876 Cohn's Beitrage z. Biol. d. Pflanzen, 2, 277.MATZUSCHITA, T. 1902 Arch. f. Hyg., 43,267-376.SCHAUDINN, F. 1902 Arch. f. Protistenkunde, 1,306-343.WARD, H. M. 1895 Proc. Roy. Soc., London, 58, 265-B48.WUND, M. 1906 Centralblt. f. Bakt., etc., I Abt., Orig., 42, 673-688.WYCKOFF, R. W. G., AND TER Louw, A. L. 1931 Jour. Exp. Med., 54, 449-451.

PLATE 1PRINT FROM SINGLz FRAMES OF FiLms No. XX, MADiE FEBRUARY 5,1931, SHOW-ING STAGES IN FORMATION OF ENDOSPORE IN Bacillus megatherium. MAG-

NIFICATION X 3000FIG. 1. Fourteen hours after inoculation. Prespore in end of cell A. Clear

areas in the ends of cells B and C.FIG. 2. Fourteen hours twelve minutes after inoculation. The prespore in

cell A has become denser. The clear area in the end of cell B has become as denseas the prespore in cell A in figure 1. The clear area in the end of cell C has begunto show a definite contour. The pattern of the granules in these cells has changedduring the first twelve minutes, but no granules entered the cleared areas.

FIG. 3. Fourteen hours twenty-four minutes after inoculation. The spore incell A has a definite elongated ellipsoidal form and is dense and refractile. Theprespore in cell B has increased in density. The shape of the cleared area in cellC has changed. Positions of granules in all these cells have changed.

FIG. 4. Fourteen hours thirty minutes after inoculation. In the six minutesbetween figures 3 and 4, the spore in cell A has contracted to its final form, ap-proaching a sphere, though still ellipsoidal. The prespore in cell B has becomedenser and the cleared area at the end of cell C has changed its shape somewhat.The pattern of the granules has been altered by movement of the granules in thecells, but no granules have entered the cleared sporogenous areas.

JOURNAL OF BACTERIOLOGY. VOL. XXV

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Documents

minds of many investigators. Matzuschita (1902) mentioned