A CULTURAL STUDY OF FILAMENTOUS BACTERIA OBTAINED FROM THE HUMAN MOUTH' BASIL G. BIBBY AND GEORGE PACKER BERRY Department of Bacteriology, School of Medicine and Dentistry, University of Rochester, Rochester, New York Received for publication October 27, 1938 In a survey of the literature dealing with filamentouss" oral bacteria (Bibby, 1935), it was found that the successful growth of such organisms has been reported on fifty-seven occasions. Most of the cultivated forms have been inadequately described; furthermore, no two descriptions are exactly alike. It is pos- sible, nevertheless, to recognize fourteen as Leptotrichia, of which there are at least two recorded types, and seven as Actinomyces. Of the latter group, none has been sufficiently well studied to establish a filamentous morphology nor an origin in the mouth. Accordingly, a systematic study of oral filamentous bacteria was undertaken. The present report is a record of the cultural findings. METHODS The material for study consisted of "materia alba," calculus, and scrapings made from the teeth, gingivae and mucous surfaces at various sites in healthy and diseased mouths of adults and children. Specimens were collected on sterile dental instruments with precautions to reduce contamination to a minimum. Inocu- lations of various culture media were made immediately after collection. 1 Based on portions of a thesis presented on October 29, 1935, by Basil G. Bibby to the University of Rochester in partial fulfillment of the requirements for the degree of Doctor of Philosophy. The study upon which this report is based was supported in part by grants from the Rockefeller Foundation and the Carnegie Corporation of New York. 263 on August 27, 2020 by guest http://jb.asm.org/ Downloaded from
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A CULTURAL STUDY OF FILAMENTOUS BACTERIAOBTAINED FROM THE HUMAN MOUTH'
BASIL G. BIBBY AND GEORGE PACKER BERRYDepartment of Bacteriology, School of Medicine and Dentistry, University of
Rochester, Rochester, New York
Received for publication October 27, 1938
In a survey of the literature dealing with filamentouss" oralbacteria (Bibby, 1935), it was found that the successful growthof such organisms has been reported on fifty-seven occasions.Most of the cultivated forms have been inadequately described;furthermore, no two descriptions are exactly alike. It is pos-sible, nevertheless, to recognize fourteen as Leptotrichia, of whichthere are at least two recorded types, and seven as Actinomyces.Of the latter group, none has been sufficiently well studied toestablish a filamentous morphology nor an origin in the mouth.Accordingly, a systematic study of oral filamentous bacteriawas undertaken. The present report is a record of the culturalfindings.
METHODS
The material for study consisted of "materia alba," calculus,and scrapings made from the teeth, gingivae and mucous surfacesat various sites in healthy and diseased mouths of adults andchildren. Specimens were collected on sterile dental instrumentswith precautions to reduce contamination to a minimum. Inocu-lations of various culture media were made immediately aftercollection.
1 Based on portions of a thesis presented on October 29, 1935, by Basil G. Bibbyto the University of Rochester in partial fulfillment of the requirements for thedegree of Doctor of Philosophy.
The study upon which this report is based was supported in part by grants fromthe Rockefeller Foundation and the Carnegie Corporation of New York.
In addition to Douglas' agar, rabbit-blood agar, and meat-extract agar, we employed the special media used by previousinvestigators. These included various enriched media, mediamade selective by the addition of inhibitory substances, andmedia of low nutritive value designed to favour the growth offungus-like organisms. Incubation at 370C. was carried outaerobically, and anaerobically, using Brown's (1922) modificationof M'Intosh and Fildes' anaerobic method. To favour thedevelopment of fungus-like organisms, a few cultures were madeat room temperature. For certain purposes a bent glass rodwas employed to inoculate solid media. Early subcultures weremade on the media which had been used in the original isolation,but thereafter, where growth was satisfactory, stock media weresubstituted.
Isolations were made under a binocular microscope at a mag-nification of 14 diameters. The separation of colonies wasgreatly facilitated by the use of a sharp-edged platinum spadeabout 1.5 mm. wide and 0.2 mm. thick.
Ascitic agar slopes containing 2 per cent of carbohydrate wereused for fermentation tests. The acidity produced was estimatedcalorimetrically in 10-day cultures in Douglas' broth containing5 per cent of glucose. Tests for diastatic activity were madein 0.3 per cent starch plates. Peptone water and Douglas'broth were used for determining the formation of indol.
CULTURAL OBSERVATIONS
Using morphology and colonial form as a basis for classifica-tion, it was possible to recognize a number of distinct groupsof filamentous, pleomorphic, non-spore-forming, non-motileorganisms. These groups are described below.
Group IOrganisms of Group I were found regularly in anaerobic cul-
tures, in which they comprised from 0.5 to 2 per cent of thecolonies. The individual elements were from 3 or 4 to 100 ormore , long and about 1 M& wide. Thread forms predominatedin both liquid and solid media. The filaments were unsegmented.
straight or curved and sometimes intertwined in tangled masses.Ends were square or rounded, commonly resembling a thickbacillus from which a filament appeared to grow (fig. 1). Irregularelements occurred (fig. 2). Short and round forms were mostprominent in smooth colonies; branched, forked, swollen, andirregular elements in intermediate colonies; and filamentoussquare-ended forms in rhizoid colonies. Young elements wereuniformly gram-positive. In old cultures, irregular staining gavea coarsely granular or banded appearance. Completely de-colorized "shadow" forms were not uncommon. The Ljubinskystain showed round granules in all elements (fig. 3). Iodin didnot give the blue "granulose" reaction.
Colonies of freshly isolated strains appeared as greyish whitepin points after forty-eight hours on blood agar. These reacheda size of 1 or 2 mm. in from 4 to 7 days. Mature colonies werecircular, having a slightly raised smooth center and a flat periph-eral portion, across which tangled filamentous processes radiatedto give a rhizoid appearance (fig. 4). The colonies were toughand cartilaginous and emulsification was impossible. Asciticand Douglas' agar, but not meat-extract agar, gave satisfactorygrowths. In subculture, atypical colonies often appeared (fig. 5),especially when cultivation was aerobic or when platings weremade from old Douglas' broth cultures. A study of four strainsduring thirty "generations" showed that, although all strainsdid not react in quite the same way, anaerobic conditions andsubculture on alkaline media tended to stabilize the rhizoidcolonial type, whereas aerobic cultivation and the use of liquidmedia tended to produce smooth and unstable intermediatevariants. In Douglas' broth, growth was slow, a scanty granularsediment appearing after a week. The granules were from 0.25to 2 mm. in diameter and settled to the bottom or adhered firmlyto the walls of the tube, leaving the medium clear. In subcul-tured strains, a heavy finely-granular growth often occurred.
Subcultures grew readily under atmospheric conditions. Slowgrowth took place at room temperature. A pH of from 7.0 to7.3 was optimal, but growth occurred between pH 6.0 and 8.5.The organisms remained viable for four months in the incubator
or refrigerator, but did not survive drying in air. They werekilled in five minutes at 650C.With a few exceptions, the fifty-three strains tested produced
acid, but no gas, from glucose, levulose, maltose, sucrose, raffinose,and dextrin after from two to six days' aerobic or anaerobicincubation. Lactose, galactose, inulin, dulcitol, and mannitolwere not attacked. Milk was not changed. Seven of thirty-four strains hydrolysed starch. The average pH produced bythirty-five strains after twelve days' growth was 5.1. On severaloccasions, the final reaction waspH 4.8. Gelatin and blood serumwere not liquefied. Indol, ammonia, or hydrogen sulphide werenot formed. Nitrates were reduced to nitrites. A comparisonof seven rough and smooth strains did not reveal significantdifferences in the rate of acid-production or the final acidity at-tained. Injections of freshly isolated and of subcultured roughand smooth strains into guinea pigs, rabbits, and mice failed toproduce any progressive pathological reaction.
Group IIFilaments of Group II showed some resemblance to those of
Group I. They were observed in only a few cultures. Butthreestrains were successfully subcultured. Individual organismsvaried in length from 2 to 200,g and in width from 0.8 to 1.8 ,(fig. 6). The filaments were generally twisted and sometimesshowed septation. Branching occurred frequently. The endswere blunt or rounded and terminal clubbing was seen on a fewoccasions. The reaction to the gram stain was positive, butsome threads decolorized completely and others only in parts.The Ljubinsky stain gave a granular appearance like the organismsof Group I. The iodine stain was negative.
After twenty-four hours, colonies were greyish white andfrom 1 to 5 mm. in diameter. The small colonies were convex,the larger ones (fig. 7) raised, with an even rounded edge fromwhich extended fine filamentous outgrowths. The surface wasfinely granular or fibrous. The consistency was soft and emulsi-fication easy. Sometimes "daughter" colonies with a smoothsurface and even outline appeared. Growth occurred on blood,
ascitic, Douglas' and meat-extract agars. In Douglas' b)roth,a slight stringy sediment was formed.Good growth was observed under both aerobic an(l anaerobic
conditions. Biochemical tests were completed with only twostrains. One of these was actively saccharolytic, fermentingglucose, levulose, maltose, sucrose, lactose, and mannitol withoutthe production of gas. Inulin was not fermented. The otherdid not ferment any sugar tested. Neither strain changed starchor milk. Gelatin and blood serum were not liquefied nor wereindol or hydrogen sulphide produced. Both strains reducednitrates.
Group III
The organisms of Group III (fig. 8) were straight or slightlycurved rods and filaments from 5 to 100,4 long and from 0.7 to1.0 / thick. The ends were blunt, 1)ut occasionally rounded ortapered. Irregular swollen forms were encountered. The re-action to the gram stain was uncertain, most elements being com-pletely decolorized. The Ljubinsky and iodin stains weresometimes weakly positive. After from seven to ten days,mature colonies (fig. 9) were dull grey or colorless, rough, slightlymoist and irregularly shaped, having a slight central eminencefrom which interlacing ridges radiated to extremely filamentousedges. The colonies were adherent, 1)ut gelatinous and easilyemulsified. Except in first transplants, growth occurred onlyon media containing blood and under strictly anaerobic condi-tions. In enriched Douglas' broth, a white coherent membranoussediment appeared which on shaking formed stringy floccules.Three strains produced acid without gas from glucose, maltose,levulose, and sucrose; one strain also fermented lactose, galactose,inulin, and raffinose and produced a pH of 4.2. Gelatin andblood serum were not liquefied. Nitrites and hydrogen sulphidewere not produced. Indol was formed.
Group IV
The organisms of Group IN' were from 7 to 50 / long andbetween 0.7 and 1 y broad. The ends were generally pointed,
but when in tandem formations, square ends occurred (fig. 10).Sometimes one end was swollen and the other pointed. Swellingsreaching a diameter of 2.5 / were found (fig. 11). The reactionto the gram stain was positive, but when grown on gentian-violetmedia (used for isolation), the organisms retained the gramstain but slightly. The Ljubinsky stain revealed no granules.A positive iodin reaction occasionally occurred in organismsgrown on media containing sugar. After five days on bloodagar, colonies were 1 or 1.5 mm. in diameter, colorless, umbonate,with an uneven surface and small tangled ridges running to theedge, where they formed uneven outgrowths (fig. 12). Thecolonies were soft and emulsified easily. Growth was good onascitic and Douglas' agar. In Douglas' broth, a slight cloudingof the medium developed after two days and a greyish-whitesediment was formed. Growth was most satisfactory underanaerobic conditions, although it sometimes took place underlow oxygen tensions. Acid was produced from glucose, maltose,sucrose, and dextrin by the sixteen strains tested and only onefailed to ferment levulose and lactose. Inulin, mannitol, anddulcitol were broken down by a few strains. Acid was producedin milk without clotting. Only a single strain of the six testedhad diastatic properties. Of the variable acidities produced,pH 4.5 was the lowest. Gelatin and blood serum were notliquefied, indol and hydrogen sulphide were not formed, andnitrates were not reduced. The single strain tested was killedin five minutes at 650C. Several strains survived storage in theincubator for five months.
Group VOrganisms of Group V (fig. 13), of which only a few strains sur-
vived subculture, were from 15 to 50 /. in length and about 0.8 ,uin diameter, the longer forms occurring in subcultures. Theywere straight or curved, with blunt, rounded, and occasionallytapered ends. The reaction to the gram stain was positiveexcept in old and "gentian-violet" cultures. The Ljubinskyand iodin stains were negative. The original colonies on gentian-violet blood agar were less than 1 mm. in diameter, having
inconstant, leaf-like, or ameboid shapes (fig. 14). They werecolorless with a stippled surface and a butyrous consistency.Subcultured colonies generally had long rhizoid outgrowths anddiameters of up to 8 mm. In Douglas' broth, growth was in-definite. A slight flocculent precipitate sometimes appeared.The organisms were strict anaerobes. None of three strainyielding satisfactory growth fermented carbohydrates and onlya single strain reduced nitrates.
Group VIA sixth group of organisms was clearly set apart by colonial
type, morphology, and staining reaction. These organisms havebeen described elsewhere (Knighton and Bibby, 1933) and iden-tified as Fusiformis polymorphus (Hine and Berry, 1937). Thereis no need for further mention here.
Group VIIAn indefinite seventh group of filaments was created to embrace
heterogeneous types which did not fit in any of the other groups.These organisms, of which only single strains were found, showedvariable colonial form, morphology (figs. 15 to 17) and biochemicalproperties. Until more strains can be studied, detailed de-scriptions will serve no useful end.
DISCUSSION
The principal interest of the present work lies, perhaps, inthe fact that it establishes the existence of a considerable varietyof filamentous bacteria in the human mouth and indicates thatthere are further types which have not yet been cultivated. Thegroups recognized can be correlated with certain morphologicaltypes easily distinguishable in smears prepared directly fromthe mouth (Bibby and Berry, 1939) and with various organismsdescribed by previous investigators. Table 1 presents a sum-mary of these relationships.Of the genera Leptotrichia, Fusiformis, or Actinomyces to which
the oral filaments might be assigned, Groups I, II, III, and Vseem to be acceptable as Leptotrichia, if Bergey's (1934) definition
be accepted. Since it is only in Group I that the constituentstrains show uniform cultural and biochemical properties, thesuggestion of a specific name is warranted for this single grouponly. For these organisms, we propose the name Leptotrichiabuccalis, making this recommendation in full cognizance ofBergey's use of the name for another species. This suggestionis made because, as has been pointed out elsewhere (Bibby,
TABLE 1Relationships between the cultivated mouth filaments reported herewith (Groups I
to VI), morphologically identified filaments, and previously cultivatedfilamentous oral bacteria
* Growth characteristics different from those reported herewith.t Biochemical characteristics different from those reported herewith.t Morphological characteristics different from those reported herewith.
1935), we consider the organisms of the species in question,Leptotrichia buccalis (Robin) Trevisan, to be bacilli which haveno place in the genus Leptotrichia.The classification of the organisms in Group IV presents
another difficult problem. Their properties embrace some ofthose considered to be characteristic of both the genera Lepto-trichia and Fusiformis. Because present systems of classificationoffer no significant points of distinction between these genera
(although they are assigned to different bacterial families, Actino-mycetaceae and Mycobacteriacae respectively), there seem to beno cogent reasons for preferring either genus.The failure to find a significant difference between the occur-
rence of filaments in healthy and diseased mouths suggests thatthese organisms are not important in oral disease. The highconcentration of acid formed by some of the strains in carbo-hydrate-containing media, however, supports the possibility thatthese filaments may have an important part in the causation ofdental caries (Kligler, 1915). Furthermore, the adherent prop-erties of the colonies of the organisms in Group I, which fix themtenaciously to the side walls of a glass culture-tube, points tothe possible importance (Bulleid, 1924, Bibby, 1935) of thistype in the formation of salivary calculus. The characteristicarrangement of filaments on the surfaces of the teeth suggeststhat they may exert a determining influence on the flora there.Thus the possibility of their playing an indirect but significantr6le in the mouth, by determining the basic nature of the oralflora as a whole, cannot be disregarded.
SUMMARY
By the use of anaerobic methods and of a wide variety of cul-ture media, a large number of strains of filamentous bacteria havebeen isolated from the mouth. On the basis of a study of themorphology and the characteristics of growth of the eighty-threestrains which were successfully carried in subculture, a tentativeworking division into seven groups is proposed. Of these, sixgroups have distinct characteristics. Only two, however, canbe identified with organisms which have been isolatedbypreviousinvestigators, although organisms showing some resemblance toa third group have been described. Four of the groups of fila-ments have the characteristics of the genus Leptotrichia, onethose of the genus Fusiformis, and one those of both genera. Ofall the groups, only one embraced strains all of which were alikein biological properties. For these organisms we suggest thename Leptotrichia buccals8 because they represent the mostcommonly isolated of the culturable oral filaments.
The authors are happy to acknowledge the help given to themby Dr. H. T. Knighton during the early part of the investigationwhen he isolated and studied some of the organisms describedin the present report.
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