THE FORMATION AND DEVELOPMENT OF LARGE BODIES INPROTEUS VULGARIS OX-19

II. COMPARATIVE CYTOLOGY OF BACILLI AND LARGE BODIES'

HENRY STEMPEN2 AND W. G. HUTCHINSON

Laboratory of Microbiology, Department of Botany, University of Pennsylvania, Philadelphia4, Pennsylvania

Received for publication December 15, 1950

The appearance of diverse morphological forms in Proteus vulgaris OX-19(Stempen and Hutchinson, 1951) stimulated interest in a cytological investiga-tion of the organism to compare the chromatinic structures in the large bodiesand other "atypical" forms with those in the "normal" rods. Cytological studieson large bodies of Proteus have been previously reported by Klieneberger-Nobel (1949), Tulasne (1949b), and Fleming et al. (1950). These reports dealwith large bodies induced by exposure to low temperature or penicillin. Thestrain of Proteus used in the present investigation produces large bodies insufficient numbers under normal cultural conditions for it not to be necessaryto resort to the use of an abnormal environment.

METHODS

The strain used in this investigation was the same one prepared from a single-cell isolate and described in the preceding paper. The chromatinic bodies of therods and the large bodies were studied by fixed smears and by dark phase con-trast microscopy of living cells, as previously described (Stempen, 1950). Aninoculum from an 18- to 24-hour culture in brain heart infusion broth wasspread over the surface of nutrient agar enriched with 5 per cent normal horseserum. Squares of agar cut out at hourly intervals with a sterile scalpel wereexposed to the vapors of a 2 per cent solution of osmium tetroxide (Robinow,1942). Impression films prepared from these were allowed to air-dry for 15 sec-onds and then placed in 70 per cent alcohol. These films were hydrolyzed in NHCl for 7 minutes at 60 C, rinsed in tap water, stained for 10 seconds in a filtered0.2 per cent aqueous solution of basic fuchsin, and mounted in tap water.

Parallel studies were carried out with the use of Schaudinn's fixative. Thefixative was prepared by mixing 2 parts of saturated aqueous mercuric chloridewith 1 part of 95 per cent ethyl alcohol and by adding 1.25 ml of glacial aceticacid to every 30 ml of fixing solution. Impression films prepared from excisedblocks of agar were immediately placed in Schaudinn's fixative at 45 C for 5minutes. The films were then removed to 50 per cent alcohol for 5 minutes, to70 per cent alcohol (containing sufficient iodine-alcohol to give it a light brown

I This investigation was supported by a fellowship granted by the Philadelphia LagerBeer Brewers' Association.

2 Present address: Department of Bacteriology and Immunology, Jefferson MedicalCollege, Philadelphia 7, Pennsylvania.

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tinge) for 10 minutes, to 50 per cent and then 30 per cent alcohol for 20 secondseach, and finally to a distilled water rinse. From this point, hydrolysis andstaining were performed as already described. Osmium- and Schaudinn-fixedfilms were also treated by Rafalko's (1946) modification of the Feulgen reactionand by perchloric acid extraction for varying lengths of time at 2 C (Cassel,1950). The bacteria were examined with a 10X eyepiece and a 95 X achromaticobjective in conjunction with Wratten G (yellow) and H (blue) filters. Livingcells were studied in slide cultures (Stempen, 1950) in order to follow the pro-gressive changes which occur in the chromatinic material during growth. ASpencer 95X oil immersion dark M phase contrast objective was used with a10X eyepiece and a Wratten E (orange) filter.

RESULTS

The appearance of chromatinic bodies in osmium-fixed, hydrolyzed, andstained cells from the surface of 5 per cent serum agar differs depending uponthe time at which impression films are made. About 1 hour after inoculation ofthe plate, most of the cells have two oval or dumbbell-shaped chromatinicbodies, whereas a few have three or four (figure 8). By the end of 3 hours and forthe next 5 to 7 hours, many of the rods contain chromatinic bodies that appearas granules at the margin of the rod (figures 9, 11, and 26). Faintly stainingstrands often connect opposite granules to give a "stretched" dumbbell or aV-shaped structure. A variety of other morphological forms also occurs in thistime period. Such forms include rods as found after 1 hour's incubation; en-larged cells with chromatinic granules more or less filling the cell (figures 11 to13); rod, spindle, and fusiform cells in which the chromatinic material is con-centrated in a small portion of the cell (figures 14 to 20); and large bodies. Thechromatinic material in the large bodies does not always present the same pat-tern. A small number contain a single heavily staining mass of chromatinicmaterial (figures 22 and 23). Usually the chromatinic bodies are more nu-merous (figures 24 to 27). It is difficult to determine the exact number ofthe chromatinic bodies because the dark masses are often connected by fainterstrands. The difficulty arises in determining whether each dark mass is to beconsidered as a separate unit or whether two or more dark masses connectedby strands are to be considered as a single chromatinic body. In one case, thechromatinic material appears as overlapping filaments with localized, moredensely staining portions (figure 28).

After about 11 hours, the majority of the cells are smaller and again containoval or dumbbell-shaped chromatinic bodies. Large bodies and the other formsdescribed above are still found. By the end of 13 hours, the rods are still smallerand contain two round chromatinic bodies per cell (figure 10). A few bizarreforms are still present.

This time study shows that a cyclic change in the appearance of the chroma-tinic material occurs in the course of the growth of an agar surface culture.Additional information on how the transformation from a bacillus with a pairof chromatinic bodies to one with several chromatinic bodies occurs can be ob-tained by observation, with the aid of the dark phase contrast microscope, of

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the development of a single cell in a slide culture (figures 1 to 6). With thismicroscope, the chromatinic bodies appear as light areas and the cytoplasm

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Figures 1 to 6. Changes in the chromatinic bodies occurring during the growth of a singlecell as revealed by the dark phase contrast microscope. The chromatinic bodies appear aslight areas and the cytoplasm as dark areas. Culture is 1-, 24, 3, 34, 34, and 4 hours old;X 3,100.

Figures la to 6a. Drawings corresponding to figures 1 to 6. The chromatinic bodies aredrawn black and the cytoplasm is white.

Figure 7. Microcolony of bacilli produced by the growth and division of the bacillusshown in figure 1. The culture is 22 hours old. The bacilli appear more or less homogeneous,with no apparent differenitiation into cytoplasm and chromatinic bodies; X 3,100.

Figures 8 to 10. Representative cells from impression films fixed and stained after 1, 6,and 13 hours' incubation to indicate the cyclic change that occurs in the appearance ofthe chromatinic material during growth; X 3,100.

appears dark (Tulasne, 1949c; Stempen, 1950). The increase in number of thechromatinic bodies at different stages in the growth of a single rod can be fol-lowed with some certainty, although the details of their division cannot be

1951] 339

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determined. An interpretation of the chromatinic bodies in figures 1 to 6 isshown in a corresponding series of drawings (figures la to 6a). The latter showsthe bodies as dark forms and the cytoplasm as light areas. In this series, it can

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to divide at the time of fixation; X 3,100.Figure 28. A large body with chromatinic material in a filamentous form. The deeply

staining swellings on the filaments are quite distinct; X 3,100.Figures 22 to 28 represent cells from osmium-fixed and fuchsin-stained preparationis

made from 6-hour-old cultures.

be seen that the chromatinic bodies increase in number as the rod enlarges,and some of the bodies are situated at the periphery of the cell by the time of thefirst division. At later stages of growth the interior of the rod commonly as-

Figures 11 to 13. Enlarged forms, some branched, filled with chromatinic material; X3,100.

Figures 14 to 16. Fusiform cells with a tendency for the chromatinic material to accumu-late in the swollen portion leaving the ends of the cell clear. In figure 16 a kidney-shapedlarge body lies to the left of the fusiform cell; X 3,100.

Figure 17. A crescent-shaped form in which the chromatinic material assumes a fila-mentous appearance. Note the presence of darkly staining nodules on the filaments; X 3,100.

Figure 18. A spindle-shaped cell with the chromatinic bodies concentrated in the centralportion. In the lowest portion of the chromatinic material there is a suggestion of shrtfilaments or rods with darkly staining swellings; X 3,100.

Figures 19 and 20. Bacilli with centrally located chromatinic material; X 3,100.Figure 21. An oval cell with centrally placed chromatinic material; X 3,100.Figures 11 to 21 represent cells from osmium-fixed and fuch3in-stained preparations made

from 6-hour-old cultures.

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HENRY STEMPEN AND W. G. HUTCHINSON

sumes a mottled appearance so that the behavior of the bodies is difficult tofollow. The rods appear homogeneous after cell division ceases (figure 7). Thelack of differentiation in these cells does not necessarily mean that the chroma-tinic bodies are no longer present but more likely that sufficient phase differ-ences in the light passing through the cells do not exist. The cytoplasm andchromatinic bodies, therefore, cannot be differentiated.With Schaudinn's fixative, the chromatinic bodies appear essentially the same

as when similar cells are fixed by osmium tetroxide vapors. Fixation with theformer, however, results in more shrinkage of the cells. Also, some of the largebodies appear to have empty centers, with the chromatinic material concen-trated in granules at the periphery. Otherwise the large bodies resemble thosedescribed above.The chromatinic bodies of the bacilli and large bodies are Feulgen-positive.

Treatment of fixed cells with 10 per cent perchloric acid for 48 hours at 2 C re-sults in the removal of cytoplasmic basophilia. The rods and large bodies appearas after warm acid hydrolysis. In both the Feulgen and the perchloric acidreactions, Schaudinn's fixative gives more uniform results than osmium tet-roxide.

DISCUSSION

The appearance of the chromatinic bodies during the first 2 hours of growthcorresponds to that of those illustrated for Proteus by Robinow (1944) andKlieneberger-Nobel (1947). In the later stages, presumably corresponding tothe logarithmic phase of growth, the majority of bacilli contain chromatinicbodies that appear as darkly staining granules at the margins of the cells. Manyof the granules are connected with one or two others on the opposite side of thecell by faintly staining strands. The chromatinic bodies at this stage resemblein appearance those shown in Clostridium welchii by Klieneberger-Nobel (1945)and in A. fischeri by Johnson and Gray (1949). Still later the chromatinic bodiesappear as they did during the first two hours of incubation; and after 24 hours'incubation the cells are very small, with two polar chromatinic bodies. The ap-pearance of the chromatinic bodies in the bacilli and in some of the fusiformand oval-shaped cells, as well as in one of the large bodies, indicates the possiblefilamentous nature of the chromatinic material. If this is true, the dense chro-matinic bodies of cells not in active division would represent a condensed massof filaments; in those cells actively dividing, the chromatinic bodies themselvesare in various stages of division and more readily exhibit their filamentous na-ture. When the culture enters the stationary phase and the phase of decline,cell division is no longer actively occurring and the chromatinic bodies resume amore condensed form.Johnson and Gray (1949), also using osmium-fixed, hydrolyzed, and stained

films in A. fischeri, found large bodies, most of which contained chromatinic ma-terial in a weblike filamentous form though some contained a rounded, deeplystaining body. Dienes and Smith (1943) with a different method (fixation

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through agar w^ith Bouin and staining with Giemsa) in Bacteroides found largebodies with one or more round, oval, or irregular chromatinic bodies. In thepresent investigation the fixed and stained large bodies of Proteus resemblethose above in only a few cases. Large bodies with a single deeply staining bodyor a filamentous netw-ork occur only rarely. The majority of the large bodiescontain several to many round, oval, and V-shaped bodies, which in general re-semble the chromatinic bodies in cells after 1 to 2 hours' incubation on a serumagar plate. These large bodies resemble closely those induced in Proteus bypenicillin (Tulasne, 1949b). As mentioned previously in the phase contraststudies of living cells (Stempen and Hutchinson, 1951), the large bodies arisingby the fusion of two bacilli appear to contain fewer chromatinic bodies thanthose arising from a single bacillus. An examination of stained films shows largebodies with only a few chromatinic bodies as well as others with many bodies.Those containing only a few chromatinic bodies may represent fusion products,but one cannot be certain since the origin of fixed cells is difficult to determine.Another explanation could be that those with a few chromatinic bodies areyounger than others with more of the bodies.The rod and spindle-shaped forms that have their chromatinic material more

or less concentrated in the central portion of the cell have, oddly enough, notbeen recognized in slide cultures. Their formation and subsequent developmentin this strain of Proteus, therefore, are uncertain. The central accumulation ofchromatinic material in rod forms has been interpreted as nuclear fusion (Bis-set, 1949). This conclusion is based on results obtained from fixed, hydrolyzed,and stained smears with no supporting evidence obtained from living cells. Forthe present, therefore, it is perhaps better to consider them as interesting cellsof unknown significance. The filaments with a swollen portion resemble formsintermediate in the formation of a Pettenkofer body (Kuhn 1924) or a largebody in Proteuis under the influence of penicillin (Tulasne, 1949a) and in B.funduliformis and S. moniltiformis (Dienes, 1942). One large body in the presentinvestigation was seen to form as the result of a central swelling of a filament.Thus there is some evidence to indicate that these fusiform structures may rep-resent a stage in large body formation.

SUMMARY

The appearance of the chromatinic bodies in fixed, hydrolyzed, and stainedcells from an agar plate culture depends upon the time at which they are re-moved. For the first 2 hours, bacilli with oval and dumbbell-shaped chromatinicbodies are found. For the next 6 to 8 hours, many of the bacilli contain chroma-tinic bodies that appear as granules at the margin of the cell. Faintly stainingstrands often connect opposite granules. Also found in this time period are cellsas seen during the first 2-hour period; enlarged cells filled with chromatinicbodies; rod, spindle, and fusiform cells wvith their chromatinic material con-centrated in the central or sw^ollen portions; and large bodies. Trhe largebodies may contain one large chromatinic body or a number of smaller ones.

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After 11 hours the cells again resemble those during the first 2 hours of incu-bation, and after 13 hours the cells are smaller with a pair of polar chromatinicbodies.

REFERENCESBISSET, K. A. 1949 The nuclear cycle in bacteria. J. Hyg., 47, 182-187.CASSEL, W. A. 1950 The use of perchloric acid in bacterial cytology. J. Bact., 59,

185-187.DIENES, L. 1942 The significance of the large bodies and the development of L type of

colonies in bacterial cultures. J. Bact., 44, 37-74.DIENES, L., AND SMITH, W. E. 1943 Chromatin structures suggesting a nuclear apparatus

in the large bodies of B. funduliformis. Proc. Soc. Exptl. Biol. Med., 53, 195-196.FLEMING, A., VOUREKA, A., KRAMER, I. R. H., AND HUGHES, W. H. 1950 The morphology

and motility of Proteus vulgaris and other organisms cultured in the presence of peni-cillin. J. Gen. Microbiol., 4, 257-269.

JOHNSON, F. H., AND GRAY, D. H. 1949 Nuclei and large bodies of luminous bacteria inrelation to salt concentration, osmotic pressure, temperature, and urethane. J. Bact.,58, 675-688.

KLIENEBERGER-NOBEL, E. 1945 Changes in nuclear structure, particularly during sporeformation. J. Hyg., 44, 99-108.

KLIENEBERGER-NOBEL, E. 1947 Morphological appearances of various stages in B. proteusand coli. J. Hyg., 45, 410-412.

KLIENEBERGER-NOBEL, E. 1949 Origin, development and significance of L forms in bac-terial cultures. J. Gen. Microbiol., 3, 434-443.

KUHN, P. 1924 Weitere Einblicke in die Entwicklung der A-Formen (Pettenkoferiafor-men). Zentr. Bakt. Parasitenk., I, Orig., 93, 280-288.

RAFALKO, J. S. 1946 A modified Feulgen technic for small and diffuse chromatin ele-ments. Stain Technol., 21, 91-93.

ROBINOW, C. F. 1942 A study of the nuclear apparatus of bacteria. Proc. Roy. Soc.(London), B, 130, 299-324.

ROBINOW, C. F. 1944 Cytological observations on Bact. coli, Proteus vulgaris, and vari-ous aerobic spore-forming bacteria with special reference to the nuclear structures.J. Hyg., 43, 413-423.

STEMPEN, H. 1950 Demonstration of the chromatinic bodies of Escherichia coli andProteus vulgaris with the aid of the phase contrast microscope. J. Bact., 60, 81-87.

STEMPEN, H., AND HUTCHINSON, W. G. 1951 The formation and development of largebodies in Proteus vulgaris OX-19. I. Bright phase contrast observations of living bac-teria. J. Bact., 61, 321-335.

TULASNE, R. 1949a Cytologie des Proteus et ses modifications sous l'effet de la p6nicil-line. Compt. rend. soc. biol., 143, 286-288.

TULASNE, R. 1949b Existence of L forms in common bacteria and their possible impor-tance. Nature, 164, 876-877.

TULASNE, R. 1949c Mise en 6vidence du noyau chez les bact6ries vivantes grace au dis-positif A contraste de phase. Compt. rend., 229, 561-563.

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