(Sackett) Bergey et al. var. phaseolicola Burkholder. A convoluted

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1 HETEROMORPHIC COLONIES ASSOCIATED WITH RING FORMATION K. W. KREITLOW Department of Animal and Plant Pathology, The Rockefeller Institute for Medical Re8earch, Princeton, New Jersey Received for publication May 10, 1941 A striking phenomenon was observed in heavily seeded dilution plates of the bean-halo-blight organism, Phytomonas medicaginis (Sackett) Bergey et al. var. phaseolicola Burkholder. A convoluted variant of this species (plate I, fig. 1) characterized by colonies with rubbery consistency, rolled margins, and depressed centers was isolated from a sector occurring in a smooth colony. Broth cultures of the variant produced no colonies resembling the original smooth form, but gave rise to two distinct kinds of convoluted colonies on agar plates. Colonies of one form occupied an irregularly shaped circular area that varied in diameter from several centimeters to almost the width of the plate. The outer edge of this area consisted of colonies that were greatly enlarged so that a raised ring was formed. The other colony-form occupied the entire area outside the raised ring. Colonies within the ring were translucent, semi-fluid, and lobed, as though by renewed growth from points on their peripheries. Those that made up the ring itself were translucent, semi-fluid, and greatly enlarged. Colonies outside the ring were white in color and rubbery in consistency. Microscopically, the colonies inside the ring were translucent, colorless, and composed of elongated, thin, non-capsulated cells. Those outside the ring were opaque, light brown, and composed of short, plump, capsulated cells. Bacteria from all colonies reacted negatively to Gram's stain. A study of the ring showed that, while it usually developed near the center of the plate, it sometimes occurred towards one side. 237

2 238 K. W. KREITLOW In all cases it followed the curvature of the bottom of the plate, which controlled the thickness of the medium. The first noticeable sign of its formation was manifested after a 24- to 48-hour incubation by a swelling of the colonies on a certain area of the plate. The ring was usually well-formed in from 48 to 72 hours after the plate was poured. Typical rings are shown in plate I, figures 2 to 4. A striking correlation was found between colony-form and ability of the bacteria to grow when transferred to fresh culture media. Organisms from the ring or the area within the ring initiated growth readily at any time when transferred to fresh media. Bacteria from outside the ring failed to grow in fresh media, no matter how long they were incubated. This phenomenon differs from the concentric "ring formation" caused by periodic swarming in species of the Proteus group. The Proteus phenomenon was first reported by Hauser (1885) and later studied by Moltke (1929), Russ-Miinzer ( ), and Knoll (1939). Ring formation by Phytomonas medicaginis var. phaseolicola appeared to differ also from the "Bakterienniveaus" first reported for liquid media by Beijerinck in 1893, later studied by Jegunow (1896), Lehmann and Curchod (1905), and Eisenberg (1919), and more recently studied in solid media by Williams (1939). A search of the literature revealed no further references that might indicate a previous description of the phenomenon reported here. The regular occurrence of heteromorphic colonies and their surprising correlation with viability stimulated further investigation to determine the conditions responsible for their origin and peculiar behavior. EXPERIMENTAL PRODUCTION OF RINGS Experiments were conducted to test the effect of thickness of medium on ring formation. Difco nutrient agar to which 1 per cent glucose had been added was used throughout the studies to be reported. Except as otherwise stated, the medium was adjusted to neutrality and tubed in amounts of approximately 8 ml. per tube. Dilution series were made in sets of 3 nutrientagar tubes by the following procedure: Three loopfuls of bacteria

3 HETEROMORPHIC COLONIES AND RING FORMATION 239 from a 48-hour broth culture were transferred to the first melted and cooled agar tube of each dilution series. Three loopfuls of material were then transferred successively from tube to tube through the series of 3 tubes. Each tube was rolled between the palms of the hands to insure thorough dispersion of the bacteria before a transfer was made to the next tube. Each dilution tube was then poured in a plate containing a layer of solidified nutrient agar. The plates were inverted for incubation at 28 C. Bacteria in heavily seeded deep-agar plates behaved differently from those in ordinary poured plates. Ring formation either failed to occur or occurred only rarely. The colonies in deep agar appeared to break down and partially liquefy only at the edge of the plate where the medium had dried out and shrunk away from the glass. Colonies at the outer edge of the plate swelled and grew on the side walls where a thin film of medium adhered to the glass. Breakdown and liquefaction of the colonies was not so extensive as in ordinary plates and was confined to colonies immediately adjacent to the periphery of the plate. As a further test of the hypothesis that thickness of the medium influenced ring formation, a number of watch glasses of different sizes were placed in various parts of Petri plates and sterilized. A layer of nutrient agar was then poured into the plates, so that all but a small portion of the top of each watch glass was immersed in agar, leaving a small rounded dome of glass projecting out of the medium. Dilution tubes were prepared as before and the watch glasses completely covered, the medium being thinnest directly over the highest part of each watch glass. After suitable incubation, a ring formed around each watch glass and close examination showed that colonies within the ring broke down first over the highest part of each watch glass where the medium was thinnest. The same type of experiment was repeated, using a sterile microscope slide in each Petri plate in place of a watch glass. The ring then followed the shape of the slide and colonies were found to break down over the surface of the medium above the slide where the agar was thinnest. The manner in which a ring followed the outline of a slide is shown in plate II, figure 5. When agar dilution plates were poured and the plates tilted so

4 240 2K. W. KREITLOW that the medium solidified in a layer of progressively increasing thickness from one side of the plate to the other, no rings formed. Breakdown of the colonies occurred on that side of the plate where the medium was thinnest. The semi-fluid colonies were separated from the white, convoluted colonies in the thicker agar by a straight raised line. The same phenomenon was observed on agar slants. The breakdown of the colonies occurred at the upper end of each slant where the medium was thinnest. The swollen, cleared growth was sharply demarcated from the growth on the lower portion of the slant in the earlier stages of breakdown. Later, the semi-fluid character of the upper growth caused it to flow down over the rest of the slant, thus giving the entire slant a semi-fluid appearance. All of these experiments demonstrated that ring formation was influenced appreciably by thinness of the medium. EFFECT OF H-ION CONCENTRATION OF THE MEDIUM ON VISCOSITY OF THE COLONIES AND ON RING FORMATION Difco nutrient agar plus 1 per cent glucose was adjusted with 0.1 N NaOH and HC1 to ph 6.2, 7.6, 8.6, and 9.7. The medium was placed in tubes and used in dilution series to determine whether or not hydrogen-ion concentration would influence breakdown of the colonies. Dilution plates at the various ph's revealed that an alkaline reaction favored breakdown and that clear and sharply pronounced rings were produced only in plates at ph 6.2. Bacteria grown at ph 6.2 produced the typical white, convoluted colony-form in 48 to 72 hours with breakdown of colonies in the thinner portions of the plates. The colonies which developed at ph 7.6 retained their convoluted form but were translucent and partially fluid throughout their entire growth period. The colonies which developed at ph 8.6 and 9.7 were more hyaline and fluid as the alkalinity of the medium was increased. Material from semi-fluid colonies grown on alkaline agar was plated in dilution series at ph 6.2. The resulting growth formed typical white, convoluted colonies and rings. These results showed that an acid reaction of the culture medium favored the

5 HETEROMORPHIC COLONIES AND RING FORMATION 241 manner of growth conducive to colony breakdown- and ring formation. CHANGES IN THE H-ION CONCENTRATION OF THE MEDIUM ON WHICH RINGS APPEARED Heavily seeded Petri plates showing typical ring formation were tested for reaction of the medium by placing drops of bromthymol blue or cresol red indicator in various portions of each plate. Drops of bromthymol blue placed within a ring turned intensely blue, indicating an alkaline reaction of the medium. Drops of this indicator placed outside the ring became yellow, demonstrating acidity of the medium. Cresol red as an indicator likewise gave a red color within each ring and a change to yellow outside the ring. That ring formation had resulted in a change of reaction in the medium was further confirmed by placing strips of bromthymol blue or cresol red paper on the surface of the medium across the entire plate. Bromthymol blue paper turned intensely blue over the area within each ring. The ring itself gave a blue reaction at its inner margin and green at its outer edge. The part of the indicator paper extending from the outer edge of the ring to the walls of the Petri plates remained bright yellow in color. Figures 6 to 8 of plate II show the changes which took place in the indicator paper. Dilution plates containing well-separated colonies that had broken down to a translucent, semi-fluid state also displayed an alkaline reaction of the medium in the imediate vicinity of each colony. On the other hand, white convoluted colonies that showed no signs of breaking down did not cause the medium to become more alkaline. The exact nature of the material causing the alkaline change in the medium has not been determined. The white, convoluted colonies were, however, easily broken down by placing them in 0.1 N sodium or potassium hydroxide for a few minutes. The solution obtained was quite clear but viscid. Addition of a small quantity of acid caused the liquid to gel immediately into a turbid, slimy mass which was redissolved by addition of alkali. Nutrient broth adjusted to ph 7.8 to 8.0 was capable of dissolving the

6 242 K. W. KREITLOW capsular material of the white, convoluted colonies overnight. Nutrient broth at a ph below 7.6 caused no dissolution of the capsular material. VIABILITY OF THE COLONIES ON PLATES SHOWING RING FORMATION The relationship between ring formation and ability of the bacteria to grow on sub-transfer was studied in detail. Typical well-separated colonies first appeared hyaline, moist, and raised. They reached their maximum size after 48 to 72 hours, at which time they were white and convoluted with raised margins and depressed centers. If ring formation took place, colonies at and within the ring remained hyaline and did not mature into the convoluted form; only those outside the ring became white and convoluted. When the colonies outside each ring were streaked on agar slants or placed in nutrient glucose broth, no growth took place even after prolonged incubation. On the other hand, when the partially broken-down colonies within a ring were transferred to agar slants or broth, growth readily took place, giving rise once more to the typical convoluted form. Bacteria that made up the ring itself likewise grew readily on agar or in broth. Heavily seeded plates were incubated for 24 hours, at the end of which time no visible rings had formed. Portions of agar were transferred from the center and outer areas of the plates to tubes of broth. Growth in the tubes showed that the bacteria were viable in all portions of the plates. An additional 24-hour incubation gave definite rings in some plates. Transfers were made of agar blocks from the center and outer areas of plates showing ring formation. Growth occurred only in those tubes which received blocks from inside the rings. No growth was obtained from material outside the rings or from heavily seeded 48-hour plates showing no ring formation. This indicated that the bacteria in heavily seeded plates lost their ability to continue growth unless rings were formed. In lightly seeded plates, those colonies separated a millimeter or two from each other often matured after 72 hours into typical white, convoluted colonies which failed to break down and like-

7 HETEROMORPHIC COLONIES AND RING FORMATION 243 wise failed to grow in nutrient broth or on fresh agar slants. Frequently, however, individual colonies or groups of colonies broke down into the hyaline form from which growth was readily obtained on transfer to broth or agar. Plates that contained fewer than 50 colonies and in which the colonies were several millimeters apart offered favorable conditions for long-continued growth. The cells in these colonies remained cultivable longer than 72 hours, but eventually they too matured and failed to grow when transferred to fresh culture media unless breakdown took place before sub-transfer. ATTEMPTS TO INDUCE BACTERIA FROM OUTSIDE THE RINGS TO GROW Since microscopic examination of the organisms in colonies outside the rings suggested they might be viable but in a dormant state, preliminary attempts were made to rid the cells of their gelatinous matrix and test whether this might induce growth. Aliquots of nutrient glucose broth were adjusted to ph 7.6, 8.6, and 9.7. Colonies from outside typical rings were then placed in the broth tubes and incubated at 28 C. Examination of the tubes revealed that the capsular material surrounding the cells was broken down at ph 8.6 and 9.7; no growth, however, occurred in the tubes. Sub-transfers to fresh nutrient broth at the same ph as the original tube or to broth at ph 7.0 also gave no growth. The bacterial cells were next treated by placing them in solutions containing a buffer salt at various concentrations. Tubes of 1 M K2HPO4 were diluted with distilled water in such a way that each tube represented one-half the concentration of the preceding one. The first tube of the series contained 1 MK2HPO4, while the tenth tube contained M K2HPO4. The buffer tubes were sterilized and later inoculated with material from outside the rings. Capsule dissolution took place at different rates depending on the concentration of K2HPO4. The inoculum was sub-transferred from buffer to broth, care being taken to make the transfer as soon as the capsular material was dissolved. One ml. of material was transferred from each tube to a tube of fresh nutrient broth at ph 7.0. Material from the first tube was trans-

8 9AA 2K. W. KREITLOW ferred after 2 minutes and material from the third tube after 15 minutes. The sixth tube of -K2HPO4 represented a M concentration and required an overnight treatment before the colonies were sufficiently broken down to warrant transfer to broth. No breakdown of the capsular material occurred at any greater dilution. Subsequent examination of the broth tubes revealed that no growth took place on sub-transfer, regardless of the treatment. Mechanical separation of the cells from their confining matrix was next tried. Nutrient glucose broth in 50 ml. portions was placed in 125 ml. flasks containing a number of 5 mm. glass beads. The flasks were steam-sterilized at 15 pounds pressure for 20 minutes and then heavily inoculated with material from typical convoluted colonies which had not broken down. The flasks were shaken by hand until turbidity of the broth indicated that pulverization of the material had taken place. No growth occurred, however, in any of the flasks. The possibility that a cold treatment might stimulate growth was considered. Petri plates containing 5-day-old colonies which had not broken down were placed in a refrigerator at 5 C. for 24 hours. The plates were then kept at for 24 hours, and material from various portions of the plates was transferred to broth. No growth was obtained by this method. A preliminary attempt was made to extract the alkaline material from Petri plates showing ring formation. It was thought that this material incorporated in broth might stimulate the cells and cause them to grow. Petri plates displaying well-developed rings were used. The agar within the rings was cut out of the plates, macerated, and placed in 20 ml. of nutrient broth for 30 minutes. The broth was then filtered through coarse filter paper to remove the agar and the filtrate passed through a Jena "5 auf 3" sintered glass filter. The filtrate was incubated 24 hours to insure sterility and then dispensed aseptically in 1 ml. portions to small sterile test tubes. These tubes were inoculated with material from plates showing no ring formation and incubated at No growth resulted from this treatment. Colonies from outside the rings were also inoculated into young bean plants, since the possibility existed that plant juice might be a more favorable medium than nutrient broth. No lesions

9 HETEROMORPHIC COLONIES AND RING FORMATION 245 developed, however, and it was concluded that the bacteria probably had not grown. Since many of the above described treatments were of a preliminary nature, no definite conclusions were reached as to whether the bacteria outside the rings were living or dead. DISCUSSION The significance of ring formation and its relation to colony viability is not yet understood. The marked change in culture medium reaction within the rings, correlated with the change in consistency of the colonies, indicated that the organism had altered its metabolism in some manner. This may have been brought about by its ability to change the reaction of the medium within the rings to a ph level favorable for continued growth. The fact that colonies broke down first where the medium was thinnest supported this conclusion. The buffering system in thin areas of culture medium would be easier to change than in areas where the culture medium was thicker. Colonies outside the rings, where the medium was deep, would then become dormant or die because of inability to change the reaction of the medium during the active stage of growth. On the other hand, the organism may act on the culture medium to form a toxic substance which kills or inhibits growth of the cells. The amount of this substance formed would be proportional to the density of bacterial growth and the quantity of medium available. The amount of toxic substance formed would then be greater where the agar was thickest. The quantity produced within the ring where the agar was thin would not be sufficiently great to cause death of the organism, particularly if the bacteria were capable of altering their metabolism and changing the culture medium reaction. The change in metabolism might permit the cells within the ring to continue growth. Preliminary culture experiments for the purpose of determining the status of the bacteria outside the rings indicated that they might be dead. Microscopic examination, however, revealed that the cells appeared to be in better condition than those actively growing inside the rings. This suggested a third possibility, that preparation for a long

10 246-2K. W. KREITLOW dormant period was adequate only in a deep medium and that under certain conditions this dormancy might be necessary for survival in nature. SUMMARY 1. A gummy, convoluted colony type of Phytomonas medicaginis (Sackett) Bergey et al. var. phaseolicola Burkholder was observed to produce two different kinds of colonies in heavily seeded dilution plates. The two colony-forms were separated by rngs, one form occurring inside and the- other outside the rings. 2. The rings were usually irregularly shaped, varying in diameter from several centimeters to almost the width of the plate, and consisting of a narrow, slightly raised margin surrounding clear or partially clear central areas. 3. Ring formation was influenced by depth of medium, as was shown by imbedding slides or various-sized watch glasses in poured plates. 4. Rings formed in those portions of plates where the agar medium was thinnest. They rarely formed in plates containing a deep layer of agar. 5. A very striking change in reaction of the medium accompanied ring formation., The medium inside the rings gave an intensely alkaline reaction, while that outside the rings showed no change in reaction. 6. Studies on viability of the bacteria revealed that those inside the rings and from the rings themselves gave immediate growth when transferred to broth or agar. Bacteria outside the rings failed to grow even after prolonged incubation. 7. A number of experiments were tried to induce growth of bacteria outside the rings, but none of these met with success. REFERENCES B3uujmNcK, W. M Ueber Atmungsfiguren beweglicher Bakterien. Zentr. Bakt. Parasitenk., 14, EIsENBERG, P Ueber Niveaubildung bei aerophilen Sporenbildnern und denitrifizierenden Bakterien. Zentr. Bakt. Parasitenk., Abt. 1, Orig., 82, HAUsEm, G Ueber Faulnissbakterien und deren Beziehungen zur Septicfimie. Ein Beitrag zur Morphologie der Spaltpilse. Leipzig, 94 pp. 15 pi.

11 HETEROMORPHIC COLONIES AND RING FORMATION 247 JEGUNOW, M Bakterien-Gesellschaften. Zentr. Bakt. Parasitenk., Abt. 2, 2, 11-21; KNULL, H Untersuchungen tiber das rhythmische Schwarmen des Bacterium vulgare (proteus). Kolloid-Z., 88-89, LEHMANN, K. B., AND CURCHOD, H Beitrage zur Kenntnis der Bakterienniveaus von Beijerinck und der Bakteriengesellschaften von Jegunow. Zentr. Bakt. Parasitenk., Abt. 2, 14, MOLTKE, Untersuchungen ilber das Phanomen des Schwiirmens der Proteus-Bazillen. Zentr. Bakt. Parasitenk., Abt. 1, Orig., 111, RusS-MtNzER, A Das Schwiirmph&inomen bei Bacillus proteus. Zentr. Bakt. Parasitenk., Abt. 1, Orig., 133, WILLIAMS, J. W Growth of microorganisms in shake cultures under increased oxygen and carbon dioxide tension. Growth, 3,

12 248 K. W. KREITLOW PLATE I (Photograph by J. A. Carlile) FIG. 1. A convoluted colony type of Phytomonas medicaginis (Sackett) Bergey et al. var. phaseolicola Burkholder. X 1. FIGS. 2 TO 4. Typical rings formed in heavily seeded dilution plates.

13 JOURNAL OF BACTERIOLOGY. VOL. 43 PLATE I FIG. I FIG. 2 I FIG.4 (K. W. Kreitlow: Heteiomorphic Colonies and Ring Formation)

14 250 K. W. KREITLOW PLATE II (Photograph by J. A. Carlile) FIG. 5. Ring formed over a microscope slide imbedded in agar. FIGS. 6 TO 8. The distinct change in culture medium reaction as demonstrated by bromthymol blue paper laid on the agar surface. The ring forms a sharp line of demarcation between the inner part of the medium which is alkaline and the outer part of the medium which is acid.

15 JOURNAL OF BACTERIOLOGY, VOL. 43 PLATE I11 FIG.5 _-a FIG. 7 FIG. 8 (K. W. Kreitlow: Heteromorphic Colonies and Ring Formation)