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 con- voluted 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 occu- pied 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 re- acted 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 on April 18, 2020 by guest http://jb.asm.org/ Downloaded from
K. W. KREITLOWDepartment 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 dilutionplates of the bean-halo-blight organism, Phytomonas medicaginis(Sackett) Bergey et al. var. phaseolicola Burkholder. A con-voluted variant of this species (plate I, fig. 1) characterized bycolonies with rubbery consistency, rolled margins, and depressedcenters was isolated from a sector occurring in a smooth colony.Broth cultures of the variant produced no colonies resemblingthe original smooth form, but gave rise to two distinct kinds ofconvoluted colonies on agar plates. Colonies of one form occu-pied an irregularly shaped circular area that varied in diameterfrom several centimeters to almost the width of the plate. Theouter edge of this area consisted of colonies that were greatlyenlarged so that a raised ring was formed. The other colony-formoccupied 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 incolor 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 ofshort, plump, capsulated cells. Bacteria from all colonies re-acted 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
In all cases it followed the curvature of the bottom of the plate,which controlled the thickness of the medium. The first notice-able sign of its formation was manifested after a 24- to 48-hourincubation by a swelling of the colonies on a certain area of theplate. The ring was usually well-formed in from 48 to 72 hoursafter 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 culturemedia. Organisms from the ring or the area within the ringinitiated growth readily at any time when transferred to freshmedia. Bacteria from outside the ring failed to grow in freshmedia, 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. TheProteus phenomenon was first reported by Hauser (1885) andlater studied by Moltke (1929), Russ-Miinzer (1934-35), andKnoll (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 studiedby 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 referencesthat might indicate a previous description of the phenomenonreported here.The regular occurrence of heteromorphic colonies and their
surprising correlation with viability stimulated further investiga-tion to determine the conditions responsible for their origin andpeculiar behavior.
EXPERIMENTAL PRODUCTION OF RINGS
Experiments were conducted to test the effect of thickness ofmedium on ring formation. Difco nutrient agar to which 1 percent glucose had been added was used throughout the studies tobe reported. Except as otherwise stated, the medium was ad-justed to neutrality and tubed in amounts of approximately8 ml. per tube. Dilution series were made in sets of 3 nutrient-agar tubes by the following procedure: Three loopfuls of bacteria
from a 48-hour broth culture were transferred to the first meltedand cooled agar tube of each dilution series. Three loopfuls ofmaterial were then transferred successively from tube to tubethrough the series of 3 tubes. Each tube was rolled between thepalms of the hands to insure thorough dispersion of the bacteriabefore a transfer was made to the next tube. Each dilution tubewas then poured in a plate containing a layer of solidified nutrientagar. The plates were inverted for incubation at 28°C.
Bacteria in heavily seeded deep-agar plates behaved differentlyfrom those in ordinary poured plates. Ring formation eitherfailed to occur or occurred only rarely. The colonies in deep agarappeared to break down and partially liquefy only at the edge ofthe plate where the medium had dried out and shrunk away fromthe glass. Colonies at the outer edge of the plate swelled andgrew on the side walls where a thin film of medium adhered tothe glass. Breakdown and liquefaction of the colonies was notso extensive as in ordinary plates and was confined to coloniesimmediately 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 differentsizes were placed in various parts of Petri plates and sterilized.A layer of nutrient agar was then poured into the plates, so thatall but a small portion of the top of each watch glass was immersedin agar, leaving a small rounded dome of glass projecting out ofthe medium. Dilution tubes were prepared as before and thewatch glasses completely covered, the medium being thinnestdirectly over the highest part of each watch glass. After suitableincubation, a ring formed around each watch glass and closeexamination showed that colonies within the ring broke down firstover the highest part of each watch glass where the medium wasthinnest.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 werefound to break down over the surface of the medium above theslide where the agar was thinnest. The manner in which a ringfollowed the outline of a slide is shown in plate II, figure 5.When agar dilution plates were poured and the plates tilted so
that the medium solidified in a layer of progressively increasingthickness from one side of the plate to the other, no rings formed.Breakdown of the colonies occurred on that side of the platewhere the medium was thinnest. The semi-fluid colonies wereseparated from the white, convoluted colonies in the thicker agarby a straight raised line. The same phenomenon was observedon agar slants. The breakdown of the colonies occurred at theupper end of each slant where the medium was thinnest. Theswollen, cleared growth was sharply demarcated from the growthon the lower portion of the slant in the earlier stages of breakdown.Later, the semi-fluid character of the upper growth caused it toflow down over the rest of the slant, thus giving the entire slanta semi-fluid appearance.
All of these experiments demonstrated that ring formation wasinfluenced appreciably by thinness of the medium.
EFFECT OF H-ION CONCENTRATION OF THE MEDIUM ON VISCOSITYOF THE COLONIES AND ON RING FORMATION
Difco nutrient agar plus 1 per cent glucose was adjusted with0.1 N NaOH and HC1 to pH 6.2, 7.6, 8.6, and 9.7. The mediumwas placed in tubes and used in dilution series to determinewhether or not hydrogen-ion concentration would influence break-down of the colonies. Dilution plates at the various pH's re-vealed that an alkaline reaction favored breakdown and that clearand sharply pronounced rings were produced only in plates atpH 6.2. Bacteria grown at pH 6.2 produced the typical white,convoluted colony-form in 48 to 72 hours with breakdown ofcolonies in the thinner portions of the plates. The colonies whichdeveloped at pH 7.6 retained their convoluted form but weretranslucent and partially fluid throughout their entire growthperiod. The colonies which developed at pH 8.6 and 9.7 weremore hyaline and fluid as the alkalinity of the medium wasincreased.
Material from semi-fluid colonies grown on alkaline agar wasplated in dilution series at pH 6.2. The resulting growth formedtypical white, convoluted colonies and rings. These resultsshowed that an acid reaction of the culture medium favored the
manner of growth conducive to colony breakdown- and ringformation.
CHANGES IN THE H-ION CONCENTRATION OF THE MEDIUM ON WHICH
RINGS APPEARED
Heavily seeded Petri plates showing typical ring formation weretested for reaction of the medium by placing drops of bromthymolblue or cresol red indicator in various portions of each plate.Drops of bromthymol blue placed within a ring turned intenselyblue, indicating an alkaline reaction of the medium. Drops ofthis indicator placed outside the ring became yellow, demonstrat-ing acidity of the medium. Cresol red as an indicator likewisegave a red color within each ring and a change to yellow outsidethe ring. That ring formation had resulted in a change of reac-tion in the medium was further confirmed by placing strips ofbromthymol blue or cresol red paper on the surface of the mediumacross the entire plate. Bromthymol blue paper turned intenselyblue over the area within each ring. The ring itself gave a bluereaction at its inner margin and green at its outer edge. The partof the indicator paper extending from the outer edge of the ringto the walls of the Petri plates remained bright yellow in color.Figures 6 to 8 of plate II show the changes which took place inthe indicator paper.
Dilution plates containing well-separated colonies that hadbroken down to a translucent, semi-fluid state also displayed analkaline reaction of the medium in the imediate vicinity of eachcolony. On the other hand, white convoluted colonies thatshowed no signs of breaking down did not cause the medium tobecome more alkaline.The exact nature of the material causing the alkaline change in
the medium has not been determined. The white, convolutedcolonies were, however, easily broken down by placing them in0.1 N sodium or potassium hydroxide for a few minutes. Thesolution obtained was quite clear but viscid. Addition of a smallquantity of acid caused the liquid to gel immediately into a turbid,slimy mass which was redissolved by addition of alkali. Nutrientbroth adjusted to pH 7.8 to 8.0 was capable of dissolving the
capsular material of the white, convoluted colonies overnight.Nutrient broth at a pH below 7.6 caused no dissolution of thecapsular material.
VIABILITY OF THE COLONIES ON PLATES SHOWING RING
FORMATION
The relationship between ring formation and ability of thebacteria to grow on sub-transfer was studied in detail. Typicalwell-separated colonies first appeared hyaline, moist, and raised.They reached their maximum size after 48 to 72 hours, at whichtime they were white and convoluted with raised margins anddepressed centers. If ring formation took place, colonies at andwithin the ring remained hyaline and did not mature into theconvoluted form; only those outside the ring became white andconvoluted. When the colonies outside each ring were streakedon agar slants or placed in nutrient glucose broth, no growth tookplace even after prolonged incubation. On the other hand, whenthe partially broken-down colonies within a ring were transferredto agar slants or broth, growth readily took place, giving riseonce more to the typical convoluted form. Bacteria that madeup the ring itself likewise grew readily on agar or in broth.
Heavily seeded plates were incubated for 24 hours, at the endof which time no visible rings had formed. Portions of agar weretransferred from the center and outer areas of the plates to tubesof broth. Growth in the tubes showed that the bacteria wereviable in all portions of the plates. An additional 24-hour in-cubation gave definite rings in some plates. Transfers were madeof agar blocks from the center and outer areas of plates showingring formation. Growth occurred only in those tubes whichreceived blocks from inside the rings. No growth was obtainedfrom material outside the rings or from heavily seeded 48-hourplates showing no ring formation. This indicated that the bac-teria in heavily seeded plates lost their ability to continue growthunless rings were formed.
In lightly seeded plates, those colonies separated a millimeteror two from each other often matured after 72 hours into typicalwhite, convoluted colonies which failed to break down and like-
wise failed to grow in nutrient broth or on fresh agar slants.Frequently, however, individual colonies or groups of coloniesbroke down into the hyaline form from which growth was readilyobtained on transfer to broth or agar.
Plates that contained fewer than 50 colonies and in which thecolonies were several millimeters apart offered favorable condi-tions for long-continued growth. The cells in these colonies re-mained cultivable longer than 72 hours, but eventually they toomatured and failed to grow when transferred to fresh culturemedia unless breakdown took place before sub-transfer.
ATTEMPTS TO INDUCE BACTERIA FROM OUTSIDE THE RINGS
TO GROW
Since microscopic examination of the organisms in coloniesoutside the rings suggested they might be viable but in a dormantstate, preliminary attempts were made to rid the cells of theirgelatinous 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 inthe broth tubes and incubated at 28°C. Examination of thetubes revealed that the capsular material surrounding the cells wasbroken down at pH 8.6 and 9.7; no growth, however, occurred inthe tubes. Sub-transfers to fresh nutrient broth at the same pHas the original tube or to broth at pH 7.0 also gave no growth.The bacterial cells were next treated by placing them in solu-
tions containing a buffer salt at various concentrations. Tubesof 1 M K2HPO4 were diluted with distilled water in such a waythat each tube represented one-half the concentration of thepreceding one. The first tube of the series contained 1 MK2HPO4,while the tenth tube contained 0.002 M K2HPO4. The buffertubes were sterilized and later inoculated with material from out-side the rings. Capsule dissolution took place at different ratesdepending on the concentration of K2HPO4. The inoculum wassub-transferred from buffer to broth, care being taken to makethe transfer as soon as the capsular material was dissolved. Oneml. of material was transferred from each tube to a tube of freshnutrient broth at pH 7.0. Material from the first tube was trans-
ferred after 2 minutes and material from the third tube after 15minutes. The sixth tube of -K2HPO4 represented a 0.031 M con-centration and required an overnight treatment before the colonieswere sufficiently broken down to warrant transfer to broth. Nobreakdown of the capsular material occurred at any greater dilu-tion. Subsequent examination of the broth tubes revealed thatno growth took place on sub-transfer, regardless of the treatment.
Mechanical separation of the cells from their confining matrixwas next tried. Nutrient glucose broth in 50 ml. portions wasplaced in 125 ml. flasks containing a number of 5 mm. glass beads.The flasks were steam-sterilized at 15 pounds pressure for 20minutes and then heavily inoculated with material from typicalconvoluted colonies which had not broken down. The flaskswere shaken by hand until turbidity of the broth indicated thatpulverization of the material had taken place. No growth oc-curred, however, in any of the flasks.The possibility that a cold treatment might stimulate growth
was considered. Petri plates containing 5-day-old colonies whichhad not broken down were placed in a refrigerator at 5°C. for24 hours. The plates were then kept at 2800. for 24 hours, andmaterial from various portions of the plates was transferred tobroth. No growth was obtained by this method.A preliminary attempt was made to extract the alkaline ma-
terial from Petri plates showing ring formation. It was thoughtthat this material incorporated in broth might stimulate the cellsand cause them to grow. Petri plates displaying well-developedrings were used. The agar within the rings was cut out of theplates, macerated, and placed in 20 ml. of nutrient broth for 30minutes. The broth was then filtered through coarse filter paperto remove the agar and the filtrate passed through a Jena "5auf 3" sintered glass filter. The filtrate was incubated 24 hoursto insure sterility and then dispensed aseptically in 1 ml. portionsto small sterile test tubes. These tubes were inoculated withmaterial from plates showing no ring formation and incubatedat 2800. No growth resulted from this treatment.
Colonies from outside the rings were also inoculated into youngbean plants, since the possibility existed that plant juice mightbe a more favorable medium than nutrient broth. No lesions
developed, however, and it was concluded that the bacteriaprobably had not grown.
Since many of the above described treatments were of a pre-liminary nature, no definite conclusions were reached as towhether the bacteria outside the rings were living or dead.
DISCUSSION
The significance of ring formation and its relation to colonyviability is not yet understood. The marked change in culturemedium reaction within the rings, correlated with the change inconsistency of the colonies, indicated that the organism hadaltered its metabolism in some manner. This may have beenbrought about by its ability to change the reaction of the mediumwithin the rings to a pH level favorable for continued growth.The fact that colonies broke down first where the medium wasthinnest supported this conclusion. The buffering system in thinareas of culture medium would be easier to change than in areaswhere the culture medium was thicker. Colonies outside therings, where the medium was deep, would then become dormantor die because of inability to change the reaction of the mediumduring 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 thecells. The amount of this substance formed would be propor-tional to the density of bacterial growth and the quantity ofmedium available. The amount of toxic substance formed wouldthen be greater where the agar was thickest. The quantity pro-duced within the ring where the agar was thin would not be suffi-ciently great to cause death of the organism, particularly if thebacteria were capable of altering their metabolism and changingthe culture medium reaction. The change in metabolism mightpermit the cells within the ring to continue growth.
Preliminary culture experiments for the purpose of determiningthe status of the bacteria outside the rings indicated that theymight be dead. Microscopic examination, however, revealedthat the cells appeared to be in better condition than thoseactively growing inside the rings.This suggested a third possibility, that preparation for a long
dormant period was adequate only in a deep medium and thatunder certain conditions this dormancy might be necessary forsurvival in nature.
SUMMARY
1. A gummy, convoluted colony type of Phytomonas medica-ginis (Sackett) Bergey et al. var. phaseolicola Burkholder was ob-served to produce two different kinds of colonies in heavily seededdilution 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 di-ameter from several centimeters to almost the width of the plate,and consisting of a narrow, slightly raised margin surroundingclear or partially clear central areas.
3. Ring formation was influenced by depth of medium, as wasshown by imbedding slides or various-sized watch glasses inpoured plates.
4. Rings formed in those portions of plates where the agarmedium was thinnest. They rarely formed in plates containinga deep layer of agar.
5. A very striking change in reaction of the medium accom-panied ring formation., The medium inside the rings gave anintensely alkaline reaction, while that outside the rings showedno change in reaction.
6. Studies on viability of the bacteria revealed that those insidethe rings and from the rings themselves gave immediate growthwhen transferred to broth or agar. Bacteria outside the ringsfailed to grow even after prolonged incubation.
7. A number of experiments were tried to induce growth ofbacteria outside the rings, but none of these met with success.
REFERENCESB3uujmNcK, W. M. 1893 Ueber Atmungsfiguren beweglicher Bakterien. Zentr.
Bakt. Parasitenk., 14, 827-845.EIsENBERG, P. 1919 Ueber Niveaubildung bei aerophilen Sporenbildnern und
KNULL, H. 1939 Untersuchungen tiber das rhythmische Schwarmen des Bac-terium vulgare (proteus). Kolloid-Z., 88-89, 135-144.
LEHMANN, K. B., AND CURCHOD, H. 1905 Beitrage zur Kenntnis der Bakterien-niveaus von Beijerinck und der Bakteriengesellschaften von Jegunow.Zentr. Bakt. Parasitenk., Abt. 2, 14, 449-459.
MOLTKE, 0. 1929 Untersuchungen ilber das Phanomen des Schwiirmens derProteus-Bazillen. Zentr. Bakt. Parasitenk., Abt. 1, Orig., 111, 399-403.
RusS-MtNzER, A. 1934-35 Das Schwiirmph&inomen bei Bacillus proteus. Zentr.Bakt. Parasitenk., Abt. 1, Orig., 133, 214-223.
WILLIAMS, J. W. 1939 Growth of microorganisms in shake cultures underincreased oxygen and carbon dioxide tension. Growth, 3, 21-33.
(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 lineof demarcation between the inner part of the medium which is alkaline and theouter part of the medium which is acid.
