CYTOLOGICAL CHANGES IN AGING BACTERIAL CULTURES

B. R. CHATTERJEE AND ROBERT P. WILLIAMSDepartment of Microbiology, Baylor University College of Medicine, Houston, Texas

Received for publication March 1, 1962

ABSTRACT

CHATTERJEE, B. R. (Baylor University Collegeof Medicine, Houston, Texas) AND ROBERTP. WILLIAMS. Cytological changes in agingbacterial cultures. J. Bacteriol. 84:340-344.1962.-Morphological changes occurring in agingbroth and agar cultures of Bacillus anthracis wereobserved with the phase-contrast microscope.In 4 to 5 days, in liquid culture, cells lost theirnormal shape and become fusiform or spherical.Chromatin bodies were clearly visible inside thecells at this time. Spontaneous conversion intoprotoplast-like, spherical cells was observed onthe 6th or 7th day. These cells were sensitive toosmotic changes, but could be preserved by theaddition of sucrose to the medium. Budding andatypical multicellular forms also were observedat this stage. In agar cultures, the conversioninto spherical forms was observed with moreregularity; the presence of sucrose in the mediumfavored the process. In very late stages of growth,the spherical cells disintegrated into conglomer-ated masses of cellular debris with the discretechromatin bodies enmeshed in them. At thisstage, secondary colonies appeared, superimposedon the primary ones, and on transfer to suitablemedium gave rise to unstable L colonies.

Various morphological changes occur in thecells of "old" bacterial cultures. These cells areusually atypical and have been termed involu-tional forms. Knaysi (1951) observed irregularoutlines (budding or branching) in cells fromold cultures of Bacillus megaterium. However,no publications have appeared detailing sequen-tial changes occurring in aging bacterial cultures.We wish to report some observations on cellularchanges in aging cultures of B. anthracis.

MATERIALS AND METHODS

A smooth variant of the Vollum strain of B.anthracis was grown in a medium composed ofTryptone (Difco), 10 g; sodium citrate, 2 g;

sodium chloride, 5 g; glucose, 3 g; made up to 1liter with double-distilled water. For agarcultures, 11 g of agar (Difco) were added perliter, and the concentration of glucose waslowered to 1.3 g. The pH of the media was 7.2.This medium delayed, and at times suppressed,sporulation. When necessary, the broth mediumwas supplemented with 15% sucrose. Mediumfor L-form growth was composed of PPLOSerum Fraction (Difco), 20%; sodium chloride,5%; crystalline penicillin (Pfizer), 1,000 IU perml; in a Brain Heart Infusion Agar (Difco) base.

Cultures were examined with a Zeiss phase-contrast microscope equipped with a Ph-3Neofluor objective. Liquid cultures wereexamined as cover-slip preparations. Forexamination of agar cultures, a square block ofagar, containing colonies, was cut out with aplatinum loop and pressed under a cover slip ona slide. Photographs were taken through a greenfilter on Kodak Tri-X Pan film with a Bauschand Lomb microphotographic camera. Forchromatin staining of cells, the modified Feulgentechnique of DeLamater (1951) was used withoutthe posthydrolysis freezing step.

RESULTS

Morphological changes in liquid cultures.Figure 1, from a culture grown for 5 days inbroth, shows long chains of bacilli, with variouschanges occurring in individual cells. Some cellsstill retain their normal appearance while othershave increased in width. A number of cells havecompletely lost their typical rod form and haveassumed spherical shape. Inside each individualcell there are one or more spherical, highlyrefractile bodies which have been identified aschromatin bodies (Chatterjee and Williams,1962). In contrast to spores, which stain uni-formly with Feulgen or acid-Giemsa stains, thesebodies show only peripheral staining under thesame conditions.At 6 or 7 days, the individual cells in a chain

fall apart, and a large number of spherical,340

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CYTOLOGICAL CHANGES IN AGING CULTURES

protoplast-like bodies appear. The osmoticsensitivity of these spherical bodies varies. Someare very unstable and lyse quickly while underexamination. Lysis can be prevented by sup-plementing the medium with 15% sucrose.Figure 2 shows two preserved spherical bodiesalong with two intact bacilli.

Other cell changes occur simultaneously withthe spherical transformation. Budding or branch-ing sometimes appears from the side of cells. Twobudding cells are shown in Fig. 3. The appearanceof these buds suggests the ballooning out ofmaterial from the side of the cell. The buds werenever observed to divide separately from theparent cell.

Figure 4 shows a cell from a 7-day-old culture.Multiple, crisscross walls are seen which ap-parently divide the cell into small compartments.Such atypical cells as this with an odd number ofsepta were found rarely. However, multicellularforms with an even number of septa similar tothe one shown in Fig. 5 were found frequently.

Mforphological changes in agar culture. Althoughthe changes occurring in agar cultures areessentially the same as those described for brothcultures, the pattern is more uniform and regular.Figures 6a-d illustrate sequential changes thattake place in the morphological character ofcells as they grow for a period of days on thesame agar culture. The appearance of the cultureafter 24 hr of growth is shown in Fig. 6a. Thepictures in Fig. 6b, c, and d were taken on the3rd, 4th, and 6th days, respectively. After the3rd day, almost half of the cells have assumedspherical shape and the long chains have brokenapart. Not a single bacillary form can be foundafter 4 days of growth (Fig. 6c). These sphericalcells can be induced to appear earlier and aremaintained for longer periods if, as in brothcultures, the medium is supplemented with 15%sucrose. Figure 6d shows the appearance of thecells after 6 days of incubation. Very few intactcells are present, but there are delicate outlinesof disintegrated spherical cells. Enmeshed withinthe disintegrated cells are small, spherical,discrete bodies which show the same stainingreactions as the chromatin bodies we havedescribed (Chatterjee and Williams, 1962). After6 days, changes also occur in the colonial mor-phology. Secondary, mucoid colonies frequentlyappear on top of the old colonies. The secondarycolonies appear to contain a mixture of sphericaland bacillary cell forms, although it is difficult

to examine only cells from the secondary colonybecause they are easily admixed with bacteriafrom the primary colony underneath.

Growth of unstable L forms from the secondarycolonies. When sequential, daily transfers weremade to L-form medium, the secondary, mucoidcolonies showed the successive growth changesillustrated in Fig. 7a-c. On the first transfer,after 24 hr of growth, the bacillary forms aretransformed into spherical cells of different sizes(Fig. 7a). The irregularity in spherical shape wasprobably due to the relatively hard agar, whichprevented the cells from assuming an evenspherical shape. When suspended in suitableliquid medium, the cells were regularly spherical.

Figure 7b shows the appearance of the growthafter the second daily transfer. The large sphericalbodies and distorted bacillary forms seen earlierhave been totally replaced by highly refractile,discrete, spherical bodies very similar in appear-ance to the chromatin bodies referred to earlier(Chatterjee and Williams, 1962). Many of thesebodies gave the characteristic peripheral stainingreaction of the chromatin bodies when stainedby the Feulgen technique. However, a con-siderable number of them were destroyed by thestaining procedure. On the third or fourthtransfer, these tiny, spherical bodies are nolonger visible. Figure 7c illustrates this phase ofgrowth. It seems that all the formed and organ-ized cellular elements are disintegrating into aconglomerated mass of debris. Figure 8 shows theappearance of the colony on the L-form mediumat this stage. The colony closely resemblestypical L-form colonies. However, the sharp andalmost spherical edge of a typical L-form colonyhas not developed. These colonies could not bemaintained for more than seven daily transfers.Toward the end of this period, the colonies eitherreverted to those showing the original bacillaryforms or gradually disappeared completely. TheL-form type of colony could not be maintainedeven by increasing the concentration of penicillinin the medium. Although the older, secondary,mucoid colonies would grow as described forseveral transfers in medium containing penicillin,young agar or broth cultures of B. anthraciswould not grow in the same medium.

DISCUSSION

The changes described are brought about byconditions created by the growing bacteriathemselves and not by outside influences. In

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addition to exhaustion of nutrients, which couldbring about degenerative changes in older cells,bacteria often elaborate autolytic enzymes intothe medium (Nomura and Hosoda, 1956; Mitchelland Moyle, 1957). Certain of these enzymes fromsporulating cultures will cause protoplast forma-tion in the homologous strain. Although sporula-tion was not observed in the cultures we em-ployed, similar enzymes might have effected thechange from bacillary to spherical form.

Atypical, multicellular forms such as the oneshown in Fig. 4 have not been reported. Bisset(1956) described an example of multicellularityin bacteria and referred to the pictures of Newton(1955) to support his discussion. In this type ofmulticellularity the cell is divided into compart-ments which are multiples of two. Bisset attrib-uted this situation to an asynchrony betweencell fission and cell division, in which fission lagsbehind division. Thorough examination of Fig. 4demonstrates that at least seven compartmentsare present in the right-hand cell and three in theleft. Division of cells into an odd number ofcompartments does not agree with Bisset'sexamples of multicellularity in bacteria. Theorigin of multicellular forms with an odd numberof compartments is unexplained. As shown in

Fig. 5, we have also observed multicellularforms divided into compartments which aremultiples of two. These cells are frequently foundin old cultures.The changes in agar cultures illustrated in

Fig. 6d are similar to those occurring during thetransitional phase of bacterial conversions to Lforms. Dienes and Weinberger (1951) describedthe development of L forms by spontaneousautolysis of the parent culture. The developmentof unstable L-form colonies that we have de-scribed could have resulted from similar spon-taneous autolysis.

ACKNOWLEDGMENT

This investigation was supported by researchgrant E-1535 (CRI) from the National Institutesof Health, U.S. Public Health Service.

LITERATURE CITED

BISSET, K. A. 1956. Cellular organization inbacteria. Symposium Soc. Gen. Microbiol.6:1-18.

CHATTERJEE, B. R., AND R. P. WILLIAMS. 1962.Cytological observations on the chromatinbodies of two Bacillus species. J. Bacteriol.83:1112-1118.

DELAMATER, E. D. 1951. A new staining and

FIG. 1. A 5-day-old broth culture showing long chains of bacilli and morphological differences of individualcells. Arrows point to a spherical cell and a constellation of three chromatin bodies in a cell. All pictureswere taken through a phase-contrast microscope at a magnification of X 3,500 except for Fig. 8.

FIG. 2. A 6-day-old broth culture showing two spherical and two normal cells.FIG. 3. Same culture as in Fig. 2, showing, by arrows, two budding forms.FIG. 4. A 7-day-old broth culture showing an atypical, multicellular unit. Arrow points to the multiple,

crisscross septa which are odd in number.FIG. 5. Same culture as in Fig. 4, showing a typical multicellular unit with an even number of septa.FIG. 6a. Agar culture after 24 hr of growth, showing parallel chains of bacilli.FIG. 6b. Same culture as shown in Fig. 6a, on the Srd day. The long bacillary chains have fallen apart,

with many individual bacilli converted into spherical cells.FIG. 6c. Agar culture from the same transfer as in Fig. 6a and 6b. The medium has been supplemented

with 15% sucrose to preserve the photoplast-like cells. The picture was taken on the 4th day and shows onlyspherical cells.

FIG. 6d. Same culture as in Fig. 6a and 6b after 6 days of growth, showing disintegrating spherical cellswith faint outlines. Arrow points to the discrete chromatin bodies in one such cell.

FIG. 7a. Culture of the secondary, mucoid colonies on L-form medium. The picture was taken 24 hr aftertransfer, and shows (by arrows) a large, irregularly spherical cell, distorted bacillary forms, and small,discrete, refractile, spherical chromatin bodies inside and outside of the cells.

FIG. 7b. Picture taken 24 hr after the second daily transfer on L-form medium of the culture shown inFig. 7a. Only large and small refractile bodies are seen. Arrows point to interconnected chromatin bodies.

FIG. 7c. Picture showing development of the culture 24 hr after the third daily transfer on L-form medium.Only a few intact elements as seen in Fig. 7b are visible. Most of the bodies are disintegrating (shown byarrow).

FIG. 8. Appearance of the colony of the same culture as in Fig. 7c. X 63.

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KNAYSI, G. 1951. Elements of bacterial cytology,2nd ed., p. 289-291. Comstock Publishing Co.,Inc., Ithaca, N.Y.

MITCHELL, P., AND J. MOYLE. 1957. Autolyticrelease and osmotic properties of 'Proto-

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