- Email: [email protected]
Actinomycetes and other pleomorphic organisms recovered from pigmented carious lesions of human teeth
Oral
Pathology
A~TINOlKYCETES AND OTHER PLltW?EUW~IC OR RECOVERED FROM PIGMENTED CARb’ifls LRSIONS OF HWMAN TEETH* MASAO ONISI,
D.M.S.,”
TOKYO, JAPAN, AKD JAMES NUCKOLLS, D.D.S.t
F THE many studies of the bacterial flora of caries, few if any have been based on representative samples. The difficulty arises in part from the fact that there can be no real relation between the material mass or volume of a substance and its bacterial population. For example, a small mass o-f dense bacterial population can contaminate a large mass which is lightly populated. Another difficulty arises from the fact that, when the sample is subjected to the new environment, the flora may change. Inoculation should follow sampling as soon as is feasible, lest one or more subordinate species multiply in the sample to such an extent as to become predominant. This is equivalent to contamination with a species which overgrows after sampling. Substances used in connection with the sampling procedure may be toxic for some forms, leading to results qualitatively similar to those obtained in overgrown samples, although the total count may be reduced. Another type of due contamination, referred to by Harrison,l is an adventitious contamination to the accidental presence of bacteria in the material from which samples art’ taken. The researcher cannot control true adventitious contamination. But, more important, he cannot even recognize it unless other types of contamination have been controlled. A review of the literature illustrates many failures to appreciate the Goadby made no mention of the importance of representative samples. Although Kligler and Gies3 collected samples from tooth surfaces matter. aseptically, their method of collection did not prevent contamination of material taken from caries with surface material from the edge of the lesion. Hartzell and Henric? pointed out the problem in their 1917 review. Gies and Kramer,5 who attempted to extend Kligler’s investigation to the flora of caries, used only a slight modification of the latter’s methods. In Kligler’s work the problem did not require separation of surface material from carioux debris, but such a separation was required in the work of Gies and Kramer.
0
*This work was supported in part by a grant from the Sugar Research Foundation. **Present address : Department of Microbiology, Tokyo Medico-Dental TJniversity. Yn3-chome, Tokyo, Japan. TDeceased. Former Professor of Oral Pathology and Chairman of the Division of Frcclinical Sciences, University of California.
shima
629
630
MASAO
ONISI
AND
JAMES
NUCKOLLS
Howe and Hatch6 made a number of informative mistakes in their work, so that it may be worth while to discuss it in some detail. These workers isolated the lesions which were to be studied either with “inert” or “slightly antiseptic” fillings for varying periods of time: up to six months in the case of the inert fillings but only a period of days to weeks with the “slightly antiseptic” fillings. The selective effect of isolation must be considered in those cases where inert fillings were placed. Undoubtedly the samples collected from the isolated lesions were not representative of normal lesions because of the dramatic shift in conditions resulting from the placing of the filling and the great decrease in interchange with the environment which must have been a consequence. Probably the samples contained only those forms able to survive for long periods of time in the isolated lesions. This may seem more or less obvious, but the point is that isolation from the environment, whether by filling or extraction, may be a major factor in determining the results. The use of antiseptic fillings confuses results further, since it superimposes the effects of antiseptics on those of isolation. At the time of sampling, the teeth were isolated with rubber dam and painted with iodine to prevent contamination of samples by bacteria from tooth surfaces. However, no experiments were conducted to test the effectiveness of this procedure, and evidence, to be presented below,13 indicates that the method is not very effective. It has the twofold drawback that it may not kill all the potential contaminants while killing some of the bacteria in the sample.’ From this work, some idea is gained of what ought not to be done in collecting samples of carious material for study. McIntosh, James, and Lazarus-Barlow7 merely flamed t,he extracted teeth from which they collected samples and apparently made no effort to Clarke* and McLean9 dipped extest the effectiveness of this procedure. tracted teeth in boiling water for a moment, a method which was probably not completely effective, but they made no tests of the degree of sterilization achieved. OkumuralO and Jay and Voorhees’l ignored the problem; at least they made no mention of it in their publications. Morishita12 separated teeth to be sampled from adjacent teeth and gums with rubber dam and then painted iodine tincture on the teeth, subsequently removing it with ethanol. Again no t,est of the effectiveness of Samples were then taken with a drill. this elaborate procedure was reported, but the work of Tunnicliff and Hammond13 indicates that it is not very effective. Tunnicliff and Hammond,13 in order to demonstrate the presence of bacteria in the pulps of teeth which were intact by clinical standards, had to show that samples collected from these teeth for cultivation had not been contaminated from the tooth surface. They, therefore, made a study of the sterilization of the surfaces of extracted teeth. Of six teeth dipped in alcohol and then flamed, only two had sterile surfaces when tested in 0.2 per cent glucose-brain broth at pH 7. When 12 teeth were exposed to the alcohol flaming procedure repeated five times, only two of the teeth had sterile Of 22 teeth treated with tincture of iodine surfaces by their method.
PLEOMORPHIC
ORGANISMS
FRO11
PIGMENTED
CbRIOI-S
LESIONS
Ci!l I
followed by two alcohol rinses and then flamed. only I1 or 50 per cent were* sterile. When 34 teeth were exposed to 8X per (>ent phenol for fif’t~ll minutes, followed by fifteen minutes in ethanol, and then flamed, only 2;) teeth, or 73.5 per cent, were sterile by the method of tIesting. Thus it is cleal that sterility of the tooth surface is very difYicult to achieve. Hut, one map ask, Why is it so important to have the tooth surface sterile! The count,s 01 h’ligler and Gies” indicate that tooth surfaces may have from a few hundretf thousands to several million bacteria per rnillig~~n~~z by cultural methods 01% even hundreds of millions by mic~roscopic counting methods. Thus, a ver\small surface contamination of a piece of dentine in which caries is ()~-IJ~ beginning may completely alter the flora of that sample either when microscopic counts are made, such as those of Kibby and tIitlr,‘4 or when (*ulturiIi enumerations are made. Because of the difficulty of obtaining surface sterility, Tuunicliff ant1 IIammondl”~ I6 simply washed tooth surfaces in soap and water before taking samples in their later studies without making any check of the effectiveness of this method. isolated teeth to be sampled with Canby and Rernierl’ rubber dam and used in that sequence: 7 per cent iodine in ethanol, 95 per cent phenol, and ‘i0 per cent ethanol. After each sterilizing procedure had been carried out, samples were taken. Rut no tests of the effectiveness of thrl method were reported. Harrison,‘” aware of the work of Tunnicliff and Hammond,l:’ in a st,udy of caries in rats, attempted to solve the problem by a tenfold washing or samples in sterile saline solution. This procedure should reduce the count of bacteria which may be readily washed from tooth surfaces but mart’ adherent forms will not be so removed. Therefore. this method does not solve the problem. Noncarious teeth used as cont,rols indicated that, this method reduced surface contamination but did not eliminate it. (‘aunts from carious teet,h and from noncarious controls overlapped each other to a ccfnsiderable degree. Recently Burnett and Scherp 19,2o have published the results of investigations of the flora of caries. While these workers are much more conscious of the problem of collecting representative samples than earlier workers, their met,hods do not rule out the possibility of surface contamination of the material collected for study. Further, in spite of precautions, the carious dentine collected by them and thought to represent the advancing front of caries. may have been contaminatecl with bacteria from portions of the lesion showing a rnore advanced state of decay because the samples were taken through the outer surface of the lesion. Jt, must also be pointed out t,hat t,heir samples were taken from extracted teeth which had been stored under aseptic contlitions for varying periods of time. As indicatecl above, this amounts to i&a-. It may be argued t,hat microtion of the tooth in an artificial environment. scopic examination of ground dentine samples protects one against, mistakes This is probably not true, because of the fnc+ introduced by contamination. that microscopic counts are much greater, usually. than counts obtained by-
632
MASAO
ONISI
AND
JAMES
NUCKOLLS
methods which entail cultivation of bacteria. Thus, a sample of dentine of relatively low population which becomes grossly contaminated from the tooth surface will be even more contaminated, in an absolute sense, by microscopic methods than by cultural methods. As pointed out by Tunnicliff and Hammond,15 strong antiseptics, because of their toxic effects, cannot be used on caries that one hopes to sample. But it may be possible to use physical sterilization, and we have studied heat and ultraviolet sterilization as possible agents. Physical sterilization may also select the more resistant organisms in the sample but such procedures may achieve adequate surface sterilization, and one can then be sure that isolated forms do not represent surface contaminants. This article, then, represents a preliminary study of the sterilization of tooth surfaces and of the recovery of bacteria from the interior of the carious tooth following such sterilization.
Methods It was decided that the best approach to the problem of recovery and study of bacteria of the caries lesion would be to divide the study into two distinct parts. The first part would be concerned with the development of an acceptable bacteriologic teahnique that wduld permit the recovery of a nearly representative sample of bacteria from the lesion. The second part of the work would primarily be concerned with a tentative identification of the bacteria in caries. As physical sterilizing agents, we employed paraffin at 200° C. and a Cenco No. 71387 “Mineralite” ultraviolet light at exposure times up to Some teeth were simply dipped in the paraffin for a varying twenty minutes. number of seconds and then tested for sterility. Other teeth were rotated at a distance of 10 cm. from the source of the ultraviolet light for a suitable period of time. As a means of testing for sterility, various specimens were immersed in thioglycollate broth (Difco) or brain-heart liver broth of the following composition : Brain-heart infusion, Difco Liver extract, Lilly Distilled water
3.7 Gm. 1.5 Gm. 100 ml. PH 7
In later cultivation of samples and isolation of cultures, we; utilized brain-heart liver agar which differed from the broth only in that it was solidified by an addition of 2 am. of agar per 100 ml. of the water. The teeth used in this study were obtained from the Division of Oral Surgery, College of Dentistry, University of California. Immediately following extraction, teeth were collected in glass containers and stored in a refrigerator. The carious lesions selected for a study were ‘divided into two general types : 3.. The preclinical lesion which exhibits an unbroken but altered surface. This type of lesion generally extends through the enamel and involves the
PLEOMORPHIC
ORGANISMS
FROM
PIGMENTED
CARIOUS
LESIONS
KC:
dentine in varying degrees. The caries of both enamel and dent,ine is, for The color of the pigment varies from a light to N the most part, pigmented. deep brown. 2. The clinical lesion is one in which cavitation of: the enamel surface has occurred and involves a greater area of dentine. Cavities with minimal breakdown of the enamel were selected since they were least likely to have penetrated to the pulp. An infected pulp is a consideration whenever an att.empt is made to recover organisms from the deeper part of any caries. Other methods are discussed in connection with the particular experiments to which they pertain.
Results In order to determine whether surface contamination could be minimized without destroying the bacteria in the deeper parts of the lesion, it seemed desirable to develop a technique which would insure a margin of surface sterility. The first attempt at surface sterilization consisted in placing the extracted teeth in a bath of molten paraffin at 200’ C. for varying periods of time. Upon removal from the paraffin, they were aHowed to cool under aseptic conditions. A sterile dental burr was inserted through the thin coating of paraffin, and a portion of the enamel surface was ground away from a noncarious area. The findings were cultured in a suitable medium for several days. When there was no growth, it was assumed that the intact surface under the paraffin coating was sterile. Surface sterilization by ult,raviolet light was also tried, but this method proved ineffectual and was dis.. carded. In order further to test the efficacy of the methods, the following procedure was carried out. When the teeth heated in the paraffin bath were placed in the relatively cold broth, the thin coating of molten pa.raffin congealed and separated from the tooth surfaces. These teeth with their exposed surfaces were t,hen incubated at 37’ C. for several days to determine the sterility of the surfaces. The specimens which did not give rise to turbid cultures were considered to be free from surface contaminants. The teeth with surfaces free of contaminants were then split into parts in an aseptic environment. The splitting was accomplished with the ordinary end-cutting wire nippers which produce a clean fracture through the crown or root as desired, The fracture was made through the caries lesion. The split segments were again incubated in brain-heart liver broth or thioglycollate broth at 37O C. for several days under aerobic and anaerobic conditions. One part of the tooth fragment containing a portion of the lesion was cultured aerobically, while the remaining part was cultured anaerobically. The resu1t.s shown in Table I indicate a sigmoid relation between the number of positive cultures from the internal lesion and the various periods of immersion in the paraffin at 200° C. It may be deduced from Ta.ble I tha.t an immersion of
634
MASAO
TABLE 1. ToTAL NUMBER WHrcH MIcRooRGANISMS AT 200” C. AND AFTER
ONISI
AND
JAMES
NUCKOLLS
0~ TEETH STUDIED AND NUMBER OF SURFACES WERE GROWN AFTER EXPOSURES OF VARYING TNCUBATION IN A SUITABLE MEDIUM UNDER ANAEROBIC CONDITIONS
AND INTERIORS FROM TIMES TO PARAFFIN EITHER AEROBIC OR
___~
E&EE 0 i.5 1; 20 30 45 60
/ ;;gi
pjiiiYi~;p::;> 3
-
1 5 i 5 3 2
3
4 2 0 0 0 0 0 0
gfjYgiiigYjI:;; 100 80 50 0 0 0 0 ::
3 5 4 5 3 2 1 0 0
100
3
100 100 100 60 33 20 0 0
ii 6 5" 5 3 2
3 1 1 0 0 0 0 0 0
100
3
100
20 20 0 0 0 0 0 0
5 4 5 3 2 0 0 0
100 80 83 60 40 0 EI
fifteen seconds in 200’ C. paraffin sterilized the external surfaces of the teeth and gave approximately 50 per cent positive cultures from internal lesions. The second phase of the study consisted of the recovery of organisms from the caries lesion. In order to prevent the tooth surfaces from becoming secondarily contaminated following their immersion in hot paraffin, they were allowed to cool to room temperature under aseptic conditions. This resulted in a thin paraffin membrane which remained adherent to the tooth surfaces. The slow cooling of the teeth actually gives a longer period in which the tooth retains heat as contrasted to the immediate cooling by plunging the specimen into cold media which was done in testing for surface sterility during the first part of the experiment. Further experimentation indicated that with the longer cooling period, surface sterilization could be accomplished by immersion in paraffin at 200’ C. for one second in the case of the preclinical lesion and 2.5 seconds for the clinical lesion. Following surface sterilization and cooling, the crowns of the teeth were split from their roots with the wire nippers. The crowns were then split at the center of the lesion so as to expose the inner part of the affected area. Samples were obtained by grinding with a 1/2 round burr from the border area of the lesion. The border area was determined as being the innermost part of the affected dentine which had been exposed by fracturing with the Although hard, the area showed definitive color changes. In 6he nippers. case of the preclinical lesion, the sample wa.s usually taken from the dentine The areas from which samples in the region of the amelodentinal junction. of the border area of the clinical lesion were taken varied with the depth that The entire procedure was executed under caries had penetrated the dentine. aseptic conditions. In addition to the brain-heart liver medium, plates of tomato juice agar (Difco) were inoculated with samples which were simultaneously taken from some of the caries lesions. This was done in order to compare the results with the usual met,hod of cultivating aciduric organisms.
Initially, the specimens were incubated at 37’ (1. under micrdaerophilicd conditions in candle jars, but it was found that one type of colony which greM very slowly under these conditions grew rnlore rapitlly as an anaerobe. Et>cause of this peculiarity, the specimens were incuba.ted at Xi0 C’. for severs days under microaerophilic conditions and then for Four clays under stri(st anaerobiosis (phosphorus jar). Samples were taken from a total of 75 lesions; -14 were preclinical lesions and 31 were clinical lesions. Upon cultivation, 3:) of the samples taken from the 75 lesions failed to develop colonies (plates were examined under ii tlissetting microscope). Nineteen of the negative samples were from thr 1)~ clinical lesions, while 14 were from t,he clinican lesions (Table II). TABLE II. FREQUENCY OF ISOLATION OF PLEOMORPIIIC AND OF DEFINITE FORMS OF BACTERIA FROM CCLTIJRED SAMPLES OF CARIOUS DENTINE FROM TEETH WHOSE SURFACES WERE STEKII.IZED BY IMMERSION IN PARAFFIN AT 200" C. TOGETHER WITH THE FREQUENCY AT WHICH TIII: SAMPLES YIEI,DED POSITIVE CT-I.T<:K~:s
I~ES~lRIPTION CARIOI'S
OF
1x310~s
Preclinical Clinid
Altogether, 97 strains of bacteria were ohtainetl from the s::mpling 01’ 42 lesions. Twenty-five of the positive samples which yielded the 97 strains were from the preclinical lesions and 17 were from the clinical lesions. ‘l’abl(~ 11 summarizes the number of strains according to the type of lesion froul which they were obtained. Eleven of the strains (11 per cent of the total number of strains obtained from both preclinical and clinical lesions) ~uld be tentat.ively elassifietl on a morphologic basis. These were staphylococci, gram-positive rods, and The colonies of these strains on the plates were always few -ill Gafkya. number; there was often only one colony. The remaining 86 strains (89 pet cent) were pleomorphic. Fifty-nine pleomorphic types (90 per cent) WPW isolated from preclinical lesions and 27 pleomorphie types (84 per cent’) WTY’ recovered from clinical lesions. The 86 pleomorphic strains were tentatively classified into five types based on the similarities ant1 dissimilarities of their colonies and the morphologic characteristics of the cells at prima.ry isolations. as follows : Type I. Upon cultivation on nutrient agar. pleornorphic gram-positivtl rods; in broth, “branched chains” of cocci. (‘~~11sare shown in Fig. 1, colonial forms in Fig. 2. Type IT. Pleomorphic gram-positive cocci. The colonies were very low and terracelike in appearance (Figs. 3 and 4). Type ITT. Filaments having thickened ends, often branching and frapmentetl (l’ig. 5). The colony was rough. hard and coherent. and firmly acl-
636
MASAO
ONISI
AND
JAMES
NUCKOLLS
a, br 0th ramifying streptococcal arrangement and a packet of cocci from Fig. 1.-A (Gram stain; ca. X1,000.) re 01i Type I. Part is Central rough heaped 2.-A colony of Type I, grown on agar surface. Fig. flat, more transparent marginal frill. (X17.) SW T‘ )unde ?d by a smooth, (Gr cocci or short diphtheroids from an agar culture of Type II. Fig. 3k.-$fot;10rphic sta in : ca . 9 verv thin flat colony of Tme II. ( X17.) Fig. 4.-A UP? pleomorphic cells with clavate ends and branches from an agar Fig. 5.-Highly (Gram stain: ca. X1,000.) of T. YPe I[II. (Apar streak pl atte : ca. ordinary pulvinnte and opaque colony of Type III. Fig. %--An Xl 7. ) (Gram stain: cit. X1,000 Fig. 7.-Long threads. rods, and other cells of Type IV. .I rough colony of Type IV. (ca. X17.) Fig. 8.-A cul
tu
PLEOMORPHIC
ORGANISMS
FROM
PIGMENTED
CARIOUS
LESIONS
637
herent to the medium (Fig. 6). It was possible to identify Type III tentatively as actinomycetes because of their morphologic characteristics, whereas the identification of Types I and II was dificult. Type IV. linusually thick, short or long threads, exhibiting a tendency t)o grow into the agar medium. They st,ained gram-positive (Fig. 7). The usual colonial form is shown in Fig. 8. Upon further study, it developed that this type is a nonsporulating variant of an aerobic spore former. and it should probably be placed in the genus bacillus. Type V. Miscellaneous pleomorphic strains that were not ident,ifiable with any of the above types. It may be seen from the data presented in Table III, which differentiates between the frequencies of isolation of pleomorphic types occurring in the preclinical and clinical lesions, that Types II, III, and IV were frequently present in both bhe preclinical and clinical lesions. Type I seems to occut somewhat more frequently in open clinical lesions. TABLE III. RELATIVE FREQUENCY WITH WHICH VARIOVS TYPES 0% PLEOMORPHIC ANVD DEFINITE FORMS OF BACTERIA WERE ISOLATED FROM SAMPLES OF DENTINE TAKEN FROM PRECLINICAL AND CLINICAL CARIES AFTER SURFACE STERILIZATION OF THE TOOTH IN LIQUID PARAFFIN AT 200" C. DISTRIBUTION
DESCRIPTION OF CARIOUS LESION Preclinical Clinical
l
I
TOTAL ! ISOLATES
I
I % 6 19
OF CULTURES
ISOLATED AMONGPLEOMORPHICAND DEFINITE--FORMS OF BACTERIA --_ PLEOMORPHIC DEFINITE FORMS_--..I -__ --GRAM GAFFKYA III STAPH. II RODS % 7% % % 2 3 J
~--__
9
3 _______..__~
?, .-
Bacteriologic smears were prepared from ground dentine of the lesions. The microorganisms observed under the microscope resembled the strains from Types I, II, and III. These types seemed to occur in the deeper parts of the lesion. The thick threadlike Type IV were most frequently found in the ground dentine taken from the more superficial areas of the brown discolored lesion. The samples taken from nine lesions, which included four preelinical and five clinical lesions, were simultaneously cultured in a medium favorable to aciduric organisms, that is, tomato juice agar. These cultures were then compared with similar samples cultured in brain-heart liver media. In all nine cultures on brain-heart liver agar, colonies grew well OT luxuriantly. although in relatively small numbers, On tomato juice agar, three of the nine plates were negative as judged with the binocular dissecting microscope at x30 magnification. Growth in the other six cultures was poor in comparison to that seen on the hrdn-heart liver agar.
Attention is invited to the fact that the material collected in the study is not, comparable with that of other st,udies. Samples were collected front
638
MBSAO
ONISI
AND
JAMES
NUCKOLLS
the hard, apparently sound but pigmented, innermost part of the precliniual and clinical lesions. These samples were not collected through the outer surface but from the surfaces formed by splitting the teeth. Histologic examinations of several sections of areas of similar appearance have shown the presence of microorganisms at depths corresponding to the field from which the bacteriologic samples were taken. The dentine samples were collected only after adequate means of surface sterilization had been developed. The teeth which were sampled were refrigerated after collection and prior to examination. Other factors being under control, the organisms in the samples should represent’ the flora of the sound but altered dentine underlying the obvious carious lesions. There were, however, two factors which were not under direct experiIt is possible that heat sterilization of the surface may have mental control. killed thermosensitive forms in the interior of the lesions, and, in view of Tunnicliff and Harnrnond’~~~ results which demonstrated bacteria in the pulps of about one-third of intact, extracted teeth, contamination may have arisen from infected pulps. In respect to the first factor, it may be said that the pleomorphic forms which were isolated were not found to be heat resistant, since about 40 per cent of the lesions sampled failed to yield cultures after surface sterilization, although in the absence of such sterilization (Table I) 100 per cent of samples tested gave rise to viable cultures. Concerning the second factor, it seems unlikely that material from the pulps could have contaminated the lesions because the nippers bring about a splitting of the tooth rather than a slicing or sawing action which might spread the material from infected pulps over the carious lesion. In any event, this factor was under statistical control since infected pulps were observed by Tunnicliff and Hammond in only one-third of the teeth they examined. It may be contended that the selection for isolation of colonies which resulted from cultivation of the dentine samples was arbitrary. Although subjective factors may play a role in such procedures, the strains isolated did appear to have formed the largest numbers of colonies on the plates. True, also, different species may form similar colonies, but it seems probable, considering the large number of strains involved, that the predominating forms were isolated. Approximately equal numbers of cultures were isolated from each of the plates. If there is a bias, it is likely to have been in the direction of unusual forms. In view of these considerations, it seems likely that the pleomorphic forms isolated are truly representative of the carious lesions which were sampled. It may be significant that the organisms of “Type III” (most frcquently isolated from either type of lesion) morphologically resembled actinomycetes.
Summary In order to establish the flora of caries lesions of dentine it is essential to take measures which rule out contamination by microorganisms from tooth
PIXOMORPHIC
ORGAWISRIS
FROM
PIGJIFSTED
(‘ARIOUS
LE8IONS
639
A survey of the literat,ure surfaces of specimens taken for cultivation. indicates tha,t antiseptics may be ineffective in establishing surface sterility. Our results indicate that sterility of surfaces may be achieved by heating t,eeth in paraffin at 200’ C. for suitable periods and that under these condit,ions organisms in the interior of the tooth are not necessarily killed. Since irr this investigation we are primarily concerned with bacteria found al the innermost part of the preclinical and clinical lesions, destruction of desired organisms by the methods employed to kill undesirable ones on t,he outer surfaces does not seem to be important. So that contamination of our samples from more superficial carious material might not occur, specimen teeth were split through carious lesions with sterile nippers. Samples were then ground from the split tooth surfaces with sterile burrs. Samples so collected were used to inoculate plates OI culture media. All of these operations were carried out under aseptic conditions. In specimens from representative preclinical and early clinica. caries The form most frequently isolatt~tl pleomorphic bacteria predominated. Further studies ot‘ appeared morphologically to be possible act,inomycetes. the characteristics of these microorganisms will be reported in a subsequelll article.
References 1. Harrison, 2. 3.
4. F 3. 6. 7. 8. 9. 10.
11.
R. W.: of Dental Caries, Lactobacillus Versus Streptococci in the Etiology J. Am. Dent. A. 37: 391, 1948. Goadby, K. W.: Mycology of the Mouth, London, 1904, Longmans, Green & Co. Kligler, I. J., and Gies, W. J.: A Biochemical Study and Differentiation of Oral Bacteria With Special Reference to Dental Caries, J. Allied Dent. Sot. 10: 141, 282, 445, 1915. Hartzell, T. B., and Henriei, A. T.: The Pathogenicity of Mouth Streptococci and Their Role in Etiology of Dental Disease, J. Am. Dent. A. 4: 477, 1917. Gies, W. J., and Kramer, S. D.: Chemical Studies of the Relations of Oral Microorganisms to Dental Caries, J. Allied Dent. Sot. 13: 413,19X Howe, P. R., and Hatch, R. E.: A Study of the Microorganisms of Dental Caries, J. Med. Res. 36: 481, 1917. McIntosh, J., James, W. W., and Lazarus-Barlow, P.: An Investigation Into the Aetiology of Dental Caries, Brit. J. Exper. Path. 3: 138, 1922. Clarke, J. R.: On the Bacteriological Factor in the Etiology of Dental Caries, Rrit. J. Exper. Path. 5: 141, 1924. McLean, I. H.: The Bacteriology of Dental Caries, Iirit. D. J. 48: 579, 1927. Okumura, T.: Bacteriologic Studies of Dental Decay With Special Reference to tire Specific Parts Played by Certain Bacteria in Causation of Dental Caries, Tr. Seventh Int. Dent. Cong. 1: 369,1926. Jay, P., and Voorhees, R. S.: Bacillus Acitlol~hilus and 1)ental (.‘aries, l)ent,al Cosmos 69:
977,
1927.
Study of Dental Caries With Special Reference to Acicluric Organisms 12. Morishita, T.: Associated With the Process. I. Tsolatiou and Description of Organisms, ,I. Bact. 18: X31, 1929. R., and Hammond, C.: Presence of Bacteria in Pulps of Intact Teeth, 13. Tunnicliff, .J. Am. Dent. A. 24: 1663, 1937. B. G., and Hine, M. K.: Bacteriologic Study of Clarious Cavities, J. Am. Dent. 14. Ribby, A. 25: 1934, 1938. A Xew Streptococcus From Dental Caries, Arch, R., and Hammond, E.: 15. Tunnicliff, Path. 26: 61, 1938. in Pulps R., and Hammond, C.: Smooth and Rough Greening Streptococci 16. Tunnicliff, .T. Am. Dent. A. 25: 1046, of Intact and Carious Teeth anii in Carions Dentin, 1 WS.
640
MASAO
ONISI
AND
JAMES
NUCKOLLS
17. Canby, C. P., and Bernier, J. L.: Bacteriologic Studies of Carious Dentin, Dent. A. 23: 2083, 1936. Bacteriological Studies on Experimental Dental Caries in 18. Harrison, R. W.: III. Flora of the Advancing Carious Lesion in the Rat, J. Infect. Dis. 1940. 19. Burnett, G. W., and Scherp, H. W.: The Distribution of Proteolytic and Bacteria in the Saliva and in the Carious Lesion, ORAL SURG., ORAL MED., PATH.
4: 469,
J. Am. the Rat. 67: 106, Aciduric AND ORAL
1951.
20. Burnett, G. W., and Scherp, H. W.: Lesion, J. D. Res. 30: 766, 1951.
Bacteriologic
Studies
of the Advancing
Dentinal
Pathology
A~TINOlKYCETES AND OTHER PLltW?EUW~IC OR RECOVERED FROM PIGMENTED CARb’ifls LRSIONS OF HWMAN TEETH* MASAO ONISI,
D.M.S.,”
TOKYO, JAPAN, AKD JAMES NUCKOLLS, D.D.S.t
F THE many studies of the bacterial flora of caries, few if any have been based on representative samples. The difficulty arises in part from the fact that there can be no real relation between the material mass or volume of a substance and its bacterial population. For example, a small mass o-f dense bacterial population can contaminate a large mass which is lightly populated. Another difficulty arises from the fact that, when the sample is subjected to the new environment, the flora may change. Inoculation should follow sampling as soon as is feasible, lest one or more subordinate species multiply in the sample to such an extent as to become predominant. This is equivalent to contamination with a species which overgrows after sampling. Substances used in connection with the sampling procedure may be toxic for some forms, leading to results qualitatively similar to those obtained in overgrown samples, although the total count may be reduced. Another type of due contamination, referred to by Harrison,l is an adventitious contamination to the accidental presence of bacteria in the material from which samples art’ taken. The researcher cannot control true adventitious contamination. But, more important, he cannot even recognize it unless other types of contamination have been controlled. A review of the literature illustrates many failures to appreciate the Goadby made no mention of the importance of representative samples. Although Kligler and Gies3 collected samples from tooth surfaces matter. aseptically, their method of collection did not prevent contamination of material taken from caries with surface material from the edge of the lesion. Hartzell and Henric? pointed out the problem in their 1917 review. Gies and Kramer,5 who attempted to extend Kligler’s investigation to the flora of caries, used only a slight modification of the latter’s methods. In Kligler’s work the problem did not require separation of surface material from carioux debris, but such a separation was required in the work of Gies and Kramer.
0
*This work was supported in part by a grant from the Sugar Research Foundation. **Present address : Department of Microbiology, Tokyo Medico-Dental TJniversity. Yn3-chome, Tokyo, Japan. TDeceased. Former Professor of Oral Pathology and Chairman of the Division of Frcclinical Sciences, University of California.
shima
629
630
MASAO
ONISI
AND
JAMES
NUCKOLLS
Howe and Hatch6 made a number of informative mistakes in their work, so that it may be worth while to discuss it in some detail. These workers isolated the lesions which were to be studied either with “inert” or “slightly antiseptic” fillings for varying periods of time: up to six months in the case of the inert fillings but only a period of days to weeks with the “slightly antiseptic” fillings. The selective effect of isolation must be considered in those cases where inert fillings were placed. Undoubtedly the samples collected from the isolated lesions were not representative of normal lesions because of the dramatic shift in conditions resulting from the placing of the filling and the great decrease in interchange with the environment which must have been a consequence. Probably the samples contained only those forms able to survive for long periods of time in the isolated lesions. This may seem more or less obvious, but the point is that isolation from the environment, whether by filling or extraction, may be a major factor in determining the results. The use of antiseptic fillings confuses results further, since it superimposes the effects of antiseptics on those of isolation. At the time of sampling, the teeth were isolated with rubber dam and painted with iodine to prevent contamination of samples by bacteria from tooth surfaces. However, no experiments were conducted to test the effectiveness of this procedure, and evidence, to be presented below,13 indicates that the method is not very effective. It has the twofold drawback that it may not kill all the potential contaminants while killing some of the bacteria in the sample.’ From this work, some idea is gained of what ought not to be done in collecting samples of carious material for study. McIntosh, James, and Lazarus-Barlow7 merely flamed t,he extracted teeth from which they collected samples and apparently made no effort to Clarke* and McLean9 dipped extest the effectiveness of this procedure. tracted teeth in boiling water for a moment, a method which was probably not completely effective, but they made no tests of the degree of sterilization achieved. OkumuralO and Jay and Voorhees’l ignored the problem; at least they made no mention of it in their publications. Morishita12 separated teeth to be sampled from adjacent teeth and gums with rubber dam and then painted iodine tincture on the teeth, subsequently removing it with ethanol. Again no t,est of the effectiveness of Samples were then taken with a drill. this elaborate procedure was reported, but the work of Tunnicliff and Hammond13 indicates that it is not very effective. Tunnicliff and Hammond,13 in order to demonstrate the presence of bacteria in the pulps of teeth which were intact by clinical standards, had to show that samples collected from these teeth for cultivation had not been contaminated from the tooth surface. They, therefore, made a study of the sterilization of the surfaces of extracted teeth. Of six teeth dipped in alcohol and then flamed, only two had sterile surfaces when tested in 0.2 per cent glucose-brain broth at pH 7. When 12 teeth were exposed to the alcohol flaming procedure repeated five times, only two of the teeth had sterile Of 22 teeth treated with tincture of iodine surfaces by their method.
PLEOMORPHIC
ORGANISMS
FRO11
PIGMENTED
CbRIOI-S
LESIONS
Ci!l I
followed by two alcohol rinses and then flamed. only I1 or 50 per cent were* sterile. When 34 teeth were exposed to 8X per (>ent phenol for fif’t~ll minutes, followed by fifteen minutes in ethanol, and then flamed, only 2;) teeth, or 73.5 per cent, were sterile by the method of tIesting. Thus it is cleal that sterility of the tooth surface is very difYicult to achieve. Hut, one map ask, Why is it so important to have the tooth surface sterile! The count,s 01 h’ligler and Gies” indicate that tooth surfaces may have from a few hundretf thousands to several million bacteria per rnillig~~n~~z by cultural methods 01% even hundreds of millions by mic~roscopic counting methods. Thus, a ver\small surface contamination of a piece of dentine in which caries is ()~-IJ~ beginning may completely alter the flora of that sample either when microscopic counts are made, such as those of Kibby and tIitlr,‘4 or when (*ulturiIi enumerations are made. Because of the difficulty of obtaining surface sterility, Tuunicliff ant1 IIammondl”~ I6 simply washed tooth surfaces in soap and water before taking samples in their later studies without making any check of the effectiveness of this method. isolated teeth to be sampled with Canby and Rernierl’ rubber dam and used in that sequence: 7 per cent iodine in ethanol, 95 per cent phenol, and ‘i0 per cent ethanol. After each sterilizing procedure had been carried out, samples were taken. Rut no tests of the effectiveness of thrl method were reported. Harrison,‘” aware of the work of Tunnicliff and Hammond,l:’ in a st,udy of caries in rats, attempted to solve the problem by a tenfold washing or samples in sterile saline solution. This procedure should reduce the count of bacteria which may be readily washed from tooth surfaces but mart’ adherent forms will not be so removed. Therefore. this method does not solve the problem. Noncarious teeth used as cont,rols indicated that, this method reduced surface contamination but did not eliminate it. (‘aunts from carious teet,h and from noncarious controls overlapped each other to a ccfnsiderable degree. Recently Burnett and Scherp 19,2o have published the results of investigations of the flora of caries. While these workers are much more conscious of the problem of collecting representative samples than earlier workers, their met,hods do not rule out the possibility of surface contamination of the material collected for study. Further, in spite of precautions, the carious dentine collected by them and thought to represent the advancing front of caries. may have been contaminatecl with bacteria from portions of the lesion showing a rnore advanced state of decay because the samples were taken through the outer surface of the lesion. Jt, must also be pointed out t,hat t,heir samples were taken from extracted teeth which had been stored under aseptic contlitions for varying periods of time. As indicatecl above, this amounts to i&a-. It may be argued t,hat microtion of the tooth in an artificial environment. scopic examination of ground dentine samples protects one against, mistakes This is probably not true, because of the fnc+ introduced by contamination. that microscopic counts are much greater, usually. than counts obtained by-
632
MASAO
ONISI
AND
JAMES
NUCKOLLS
methods which entail cultivation of bacteria. Thus, a sample of dentine of relatively low population which becomes grossly contaminated from the tooth surface will be even more contaminated, in an absolute sense, by microscopic methods than by cultural methods. As pointed out by Tunnicliff and Hammond,15 strong antiseptics, because of their toxic effects, cannot be used on caries that one hopes to sample. But it may be possible to use physical sterilization, and we have studied heat and ultraviolet sterilization as possible agents. Physical sterilization may also select the more resistant organisms in the sample but such procedures may achieve adequate surface sterilization, and one can then be sure that isolated forms do not represent surface contaminants. This article, then, represents a preliminary study of the sterilization of tooth surfaces and of the recovery of bacteria from the interior of the carious tooth following such sterilization.
Methods It was decided that the best approach to the problem of recovery and study of bacteria of the caries lesion would be to divide the study into two distinct parts. The first part would be concerned with the development of an acceptable bacteriologic teahnique that wduld permit the recovery of a nearly representative sample of bacteria from the lesion. The second part of the work would primarily be concerned with a tentative identification of the bacteria in caries. As physical sterilizing agents, we employed paraffin at 200° C. and a Cenco No. 71387 “Mineralite” ultraviolet light at exposure times up to Some teeth were simply dipped in the paraffin for a varying twenty minutes. number of seconds and then tested for sterility. Other teeth were rotated at a distance of 10 cm. from the source of the ultraviolet light for a suitable period of time. As a means of testing for sterility, various specimens were immersed in thioglycollate broth (Difco) or brain-heart liver broth of the following composition : Brain-heart infusion, Difco Liver extract, Lilly Distilled water
3.7 Gm. 1.5 Gm. 100 ml. PH 7
In later cultivation of samples and isolation of cultures, we; utilized brain-heart liver agar which differed from the broth only in that it was solidified by an addition of 2 am. of agar per 100 ml. of the water. The teeth used in this study were obtained from the Division of Oral Surgery, College of Dentistry, University of California. Immediately following extraction, teeth were collected in glass containers and stored in a refrigerator. The carious lesions selected for a study were ‘divided into two general types : 3.. The preclinical lesion which exhibits an unbroken but altered surface. This type of lesion generally extends through the enamel and involves the
PLEOMORPHIC
ORGANISMS
FROM
PIGMENTED
CARIOUS
LESIONS
KC:
dentine in varying degrees. The caries of both enamel and dent,ine is, for The color of the pigment varies from a light to N the most part, pigmented. deep brown. 2. The clinical lesion is one in which cavitation of: the enamel surface has occurred and involves a greater area of dentine. Cavities with minimal breakdown of the enamel were selected since they were least likely to have penetrated to the pulp. An infected pulp is a consideration whenever an att.empt is made to recover organisms from the deeper part of any caries. Other methods are discussed in connection with the particular experiments to which they pertain.
Results In order to determine whether surface contamination could be minimized without destroying the bacteria in the deeper parts of the lesion, it seemed desirable to develop a technique which would insure a margin of surface sterility. The first attempt at surface sterilization consisted in placing the extracted teeth in a bath of molten paraffin at 200’ C. for varying periods of time. Upon removal from the paraffin, they were aHowed to cool under aseptic conditions. A sterile dental burr was inserted through the thin coating of paraffin, and a portion of the enamel surface was ground away from a noncarious area. The findings were cultured in a suitable medium for several days. When there was no growth, it was assumed that the intact surface under the paraffin coating was sterile. Surface sterilization by ult,raviolet light was also tried, but this method proved ineffectual and was dis.. carded. In order further to test the efficacy of the methods, the following procedure was carried out. When the teeth heated in the paraffin bath were placed in the relatively cold broth, the thin coating of molten pa.raffin congealed and separated from the tooth surfaces. These teeth with their exposed surfaces were t,hen incubated at 37’ C. for several days to determine the sterility of the surfaces. The specimens which did not give rise to turbid cultures were considered to be free from surface contaminants. The teeth with surfaces free of contaminants were then split into parts in an aseptic environment. The splitting was accomplished with the ordinary end-cutting wire nippers which produce a clean fracture through the crown or root as desired, The fracture was made through the caries lesion. The split segments were again incubated in brain-heart liver broth or thioglycollate broth at 37O C. for several days under aerobic and anaerobic conditions. One part of the tooth fragment containing a portion of the lesion was cultured aerobically, while the remaining part was cultured anaerobically. The resu1t.s shown in Table I indicate a sigmoid relation between the number of positive cultures from the internal lesion and the various periods of immersion in the paraffin at 200° C. It may be deduced from Ta.ble I tha.t an immersion of
634
MASAO
TABLE 1. ToTAL NUMBER WHrcH MIcRooRGANISMS AT 200” C. AND AFTER
ONISI
AND
JAMES
NUCKOLLS
0~ TEETH STUDIED AND NUMBER OF SURFACES WERE GROWN AFTER EXPOSURES OF VARYING TNCUBATION IN A SUITABLE MEDIUM UNDER ANAEROBIC CONDITIONS
AND INTERIORS FROM TIMES TO PARAFFIN EITHER AEROBIC OR
___~
E&EE 0 i.5 1; 20 30 45 60
/ ;;gi
pjiiiYi~;p::;> 3
-
1 5 i 5 3 2
3
4 2 0 0 0 0 0 0
gfjYgiiigYjI:;; 100 80 50 0 0 0 0 ::
3 5 4 5 3 2 1 0 0
100
3
100 100 100 60 33 20 0 0
ii 6 5" 5 3 2
3 1 1 0 0 0 0 0 0
100
3
100
20 20 0 0 0 0 0 0
5 4 5 3 2 0 0 0
100 80 83 60 40 0 EI
fifteen seconds in 200’ C. paraffin sterilized the external surfaces of the teeth and gave approximately 50 per cent positive cultures from internal lesions. The second phase of the study consisted of the recovery of organisms from the caries lesion. In order to prevent the tooth surfaces from becoming secondarily contaminated following their immersion in hot paraffin, they were allowed to cool to room temperature under aseptic conditions. This resulted in a thin paraffin membrane which remained adherent to the tooth surfaces. The slow cooling of the teeth actually gives a longer period in which the tooth retains heat as contrasted to the immediate cooling by plunging the specimen into cold media which was done in testing for surface sterility during the first part of the experiment. Further experimentation indicated that with the longer cooling period, surface sterilization could be accomplished by immersion in paraffin at 200’ C. for one second in the case of the preclinical lesion and 2.5 seconds for the clinical lesion. Following surface sterilization and cooling, the crowns of the teeth were split from their roots with the wire nippers. The crowns were then split at the center of the lesion so as to expose the inner part of the affected area. Samples were obtained by grinding with a 1/2 round burr from the border area of the lesion. The border area was determined as being the innermost part of the affected dentine which had been exposed by fracturing with the Although hard, the area showed definitive color changes. In 6he nippers. case of the preclinical lesion, the sample wa.s usually taken from the dentine The areas from which samples in the region of the amelodentinal junction. of the border area of the clinical lesion were taken varied with the depth that The entire procedure was executed under caries had penetrated the dentine. aseptic conditions. In addition to the brain-heart liver medium, plates of tomato juice agar (Difco) were inoculated with samples which were simultaneously taken from some of the caries lesions. This was done in order to compare the results with the usual met,hod of cultivating aciduric organisms.
Initially, the specimens were incubated at 37’ (1. under micrdaerophilicd conditions in candle jars, but it was found that one type of colony which greM very slowly under these conditions grew rnlore rapitlly as an anaerobe. Et>cause of this peculiarity, the specimens were incuba.ted at Xi0 C’. for severs days under microaerophilic conditions and then for Four clays under stri(st anaerobiosis (phosphorus jar). Samples were taken from a total of 75 lesions; -14 were preclinical lesions and 31 were clinical lesions. Upon cultivation, 3:) of the samples taken from the 75 lesions failed to develop colonies (plates were examined under ii tlissetting microscope). Nineteen of the negative samples were from thr 1)~ clinical lesions, while 14 were from t,he clinican lesions (Table II). TABLE II. FREQUENCY OF ISOLATION OF PLEOMORPIIIC AND OF DEFINITE FORMS OF BACTERIA FROM CCLTIJRED SAMPLES OF CARIOUS DENTINE FROM TEETH WHOSE SURFACES WERE STEKII.IZED BY IMMERSION IN PARAFFIN AT 200" C. TOGETHER WITH THE FREQUENCY AT WHICH TIII: SAMPLES YIEI,DED POSITIVE CT-I.T<:K~:s
I~ES~lRIPTION CARIOI'S
OF
1x310~s
Preclinical Clinid
Altogether, 97 strains of bacteria were ohtainetl from the s::mpling 01’ 42 lesions. Twenty-five of the positive samples which yielded the 97 strains were from the preclinical lesions and 17 were from the clinical lesions. ‘l’abl(~ 11 summarizes the number of strains according to the type of lesion froul which they were obtained. Eleven of the strains (11 per cent of the total number of strains obtained from both preclinical and clinical lesions) ~uld be tentat.ively elassifietl on a morphologic basis. These were staphylococci, gram-positive rods, and The colonies of these strains on the plates were always few -ill Gafkya. number; there was often only one colony. The remaining 86 strains (89 pet cent) were pleomorphic. Fifty-nine pleomorphic types (90 per cent) WPW isolated from preclinical lesions and 27 pleomorphie types (84 per cent’) WTY’ recovered from clinical lesions. The 86 pleomorphic strains were tentatively classified into five types based on the similarities ant1 dissimilarities of their colonies and the morphologic characteristics of the cells at prima.ry isolations. as follows : Type I. Upon cultivation on nutrient agar. pleornorphic gram-positivtl rods; in broth, “branched chains” of cocci. (‘~~11sare shown in Fig. 1, colonial forms in Fig. 2. Type IT. Pleomorphic gram-positive cocci. The colonies were very low and terracelike in appearance (Figs. 3 and 4). Type ITT. Filaments having thickened ends, often branching and frapmentetl (l’ig. 5). The colony was rough. hard and coherent. and firmly acl-
636
MASAO
ONISI
AND
JAMES
NUCKOLLS
a, br 0th ramifying streptococcal arrangement and a packet of cocci from Fig. 1.-A (Gram stain; ca. X1,000.) re 01i Type I. Part is Central rough heaped 2.-A colony of Type I, grown on agar surface. Fig. flat, more transparent marginal frill. (X17.) SW T‘ )unde ?d by a smooth, (Gr cocci or short diphtheroids from an agar culture of Type II. Fig. 3k.-$fot;10rphic sta in : ca . 9 verv thin flat colony of Tme II. ( X17.) Fig. 4.-A UP? pleomorphic cells with clavate ends and branches from an agar Fig. 5.-Highly (Gram stain: ca. X1,000.) of T. YPe I[II. (Apar streak pl atte : ca. ordinary pulvinnte and opaque colony of Type III. Fig. %--An Xl 7. ) (Gram stain: cit. X1,000 Fig. 7.-Long threads. rods, and other cells of Type IV. .I rough colony of Type IV. (ca. X17.) Fig. 8.-A cul
tu
PLEOMORPHIC
ORGANISMS
FROM
PIGMENTED
CARIOUS
LESIONS
637
herent to the medium (Fig. 6). It was possible to identify Type III tentatively as actinomycetes because of their morphologic characteristics, whereas the identification of Types I and II was dificult. Type IV. linusually thick, short or long threads, exhibiting a tendency t)o grow into the agar medium. They st,ained gram-positive (Fig. 7). The usual colonial form is shown in Fig. 8. Upon further study, it developed that this type is a nonsporulating variant of an aerobic spore former. and it should probably be placed in the genus bacillus. Type V. Miscellaneous pleomorphic strains that were not ident,ifiable with any of the above types. It may be seen from the data presented in Table III, which differentiates between the frequencies of isolation of pleomorphic types occurring in the preclinical and clinical lesions, that Types II, III, and IV were frequently present in both bhe preclinical and clinical lesions. Type I seems to occut somewhat more frequently in open clinical lesions. TABLE III. RELATIVE FREQUENCY WITH WHICH VARIOVS TYPES 0% PLEOMORPHIC ANVD DEFINITE FORMS OF BACTERIA WERE ISOLATED FROM SAMPLES OF DENTINE TAKEN FROM PRECLINICAL AND CLINICAL CARIES AFTER SURFACE STERILIZATION OF THE TOOTH IN LIQUID PARAFFIN AT 200" C. DISTRIBUTION
DESCRIPTION OF CARIOUS LESION Preclinical Clinical
l
I
TOTAL ! ISOLATES
I
I % 6 19
OF CULTURES
ISOLATED AMONGPLEOMORPHICAND DEFINITE--FORMS OF BACTERIA --_ PLEOMORPHIC DEFINITE FORMS_--..I -__ --GRAM GAFFKYA III STAPH. II RODS % 7% % % 2 3 J
~--__
9
3 _______..__~
?, .-
Bacteriologic smears were prepared from ground dentine of the lesions. The microorganisms observed under the microscope resembled the strains from Types I, II, and III. These types seemed to occur in the deeper parts of the lesion. The thick threadlike Type IV were most frequently found in the ground dentine taken from the more superficial areas of the brown discolored lesion. The samples taken from nine lesions, which included four preelinical and five clinical lesions, were simultaneously cultured in a medium favorable to aciduric organisms, that is, tomato juice agar. These cultures were then compared with similar samples cultured in brain-heart liver media. In all nine cultures on brain-heart liver agar, colonies grew well OT luxuriantly. although in relatively small numbers, On tomato juice agar, three of the nine plates were negative as judged with the binocular dissecting microscope at x30 magnification. Growth in the other six cultures was poor in comparison to that seen on the hrdn-heart liver agar.
Attention is invited to the fact that the material collected in the study is not, comparable with that of other st,udies. Samples were collected front
638
MBSAO
ONISI
AND
JAMES
NUCKOLLS
the hard, apparently sound but pigmented, innermost part of the precliniual and clinical lesions. These samples were not collected through the outer surface but from the surfaces formed by splitting the teeth. Histologic examinations of several sections of areas of similar appearance have shown the presence of microorganisms at depths corresponding to the field from which the bacteriologic samples were taken. The dentine samples were collected only after adequate means of surface sterilization had been developed. The teeth which were sampled were refrigerated after collection and prior to examination. Other factors being under control, the organisms in the samples should represent’ the flora of the sound but altered dentine underlying the obvious carious lesions. There were, however, two factors which were not under direct experiIt is possible that heat sterilization of the surface may have mental control. killed thermosensitive forms in the interior of the lesions, and, in view of Tunnicliff and Harnrnond’~~~ results which demonstrated bacteria in the pulps of about one-third of intact, extracted teeth, contamination may have arisen from infected pulps. In respect to the first factor, it may be said that the pleomorphic forms which were isolated were not found to be heat resistant, since about 40 per cent of the lesions sampled failed to yield cultures after surface sterilization, although in the absence of such sterilization (Table I) 100 per cent of samples tested gave rise to viable cultures. Concerning the second factor, it seems unlikely that material from the pulps could have contaminated the lesions because the nippers bring about a splitting of the tooth rather than a slicing or sawing action which might spread the material from infected pulps over the carious lesion. In any event, this factor was under statistical control since infected pulps were observed by Tunnicliff and Hammond in only one-third of the teeth they examined. It may be contended that the selection for isolation of colonies which resulted from cultivation of the dentine samples was arbitrary. Although subjective factors may play a role in such procedures, the strains isolated did appear to have formed the largest numbers of colonies on the plates. True, also, different species may form similar colonies, but it seems probable, considering the large number of strains involved, that the predominating forms were isolated. Approximately equal numbers of cultures were isolated from each of the plates. If there is a bias, it is likely to have been in the direction of unusual forms. In view of these considerations, it seems likely that the pleomorphic forms isolated are truly representative of the carious lesions which were sampled. It may be significant that the organisms of “Type III” (most frcquently isolated from either type of lesion) morphologically resembled actinomycetes.
Summary In order to establish the flora of caries lesions of dentine it is essential to take measures which rule out contamination by microorganisms from tooth
PIXOMORPHIC
ORGAWISRIS
FROM
PIGJIFSTED
(‘ARIOUS
LE8IONS
639
A survey of the literat,ure surfaces of specimens taken for cultivation. indicates tha,t antiseptics may be ineffective in establishing surface sterility. Our results indicate that sterility of surfaces may be achieved by heating t,eeth in paraffin at 200’ C. for suitable periods and that under these condit,ions organisms in the interior of the tooth are not necessarily killed. Since irr this investigation we are primarily concerned with bacteria found al the innermost part of the preclinical and clinical lesions, destruction of desired organisms by the methods employed to kill undesirable ones on t,he outer surfaces does not seem to be important. So that contamination of our samples from more superficial carious material might not occur, specimen teeth were split through carious lesions with sterile nippers. Samples were then ground from the split tooth surfaces with sterile burrs. Samples so collected were used to inoculate plates OI culture media. All of these operations were carried out under aseptic conditions. In specimens from representative preclinical and early clinica. caries The form most frequently isolatt~tl pleomorphic bacteria predominated. Further studies ot‘ appeared morphologically to be possible act,inomycetes. the characteristics of these microorganisms will be reported in a subsequelll article.
References 1. Harrison, 2. 3.
4. F 3. 6. 7. 8. 9. 10.
11.
R. W.: of Dental Caries, Lactobacillus Versus Streptococci in the Etiology J. Am. Dent. A. 37: 391, 1948. Goadby, K. W.: Mycology of the Mouth, London, 1904, Longmans, Green & Co. Kligler, I. J., and Gies, W. J.: A Biochemical Study and Differentiation of Oral Bacteria With Special Reference to Dental Caries, J. Allied Dent. Sot. 10: 141, 282, 445, 1915. Hartzell, T. B., and Henriei, A. T.: The Pathogenicity of Mouth Streptococci and Their Role in Etiology of Dental Disease, J. Am. Dent. A. 4: 477, 1917. Gies, W. J., and Kramer, S. D.: Chemical Studies of the Relations of Oral Microorganisms to Dental Caries, J. Allied Dent. Sot. 13: 413,19X Howe, P. R., and Hatch, R. E.: A Study of the Microorganisms of Dental Caries, J. Med. Res. 36: 481, 1917. McIntosh, J., James, W. W., and Lazarus-Barlow, P.: An Investigation Into the Aetiology of Dental Caries, Brit. J. Exper. Path. 3: 138, 1922. Clarke, J. R.: On the Bacteriological Factor in the Etiology of Dental Caries, Rrit. J. Exper. Path. 5: 141, 1924. McLean, I. H.: The Bacteriology of Dental Caries, Iirit. D. J. 48: 579, 1927. Okumura, T.: Bacteriologic Studies of Dental Decay With Special Reference to tire Specific Parts Played by Certain Bacteria in Causation of Dental Caries, Tr. Seventh Int. Dent. Cong. 1: 369,1926. Jay, P., and Voorhees, R. S.: Bacillus Acitlol~hilus and 1)ental (.‘aries, l)ent,al Cosmos 69:
977,
1927.
Study of Dental Caries With Special Reference to Acicluric Organisms 12. Morishita, T.: Associated With the Process. I. Tsolatiou and Description of Organisms, ,I. Bact. 18: X31, 1929. R., and Hammond, C.: Presence of Bacteria in Pulps of Intact Teeth, 13. Tunnicliff, .J. Am. Dent. A. 24: 1663, 1937. B. G., and Hine, M. K.: Bacteriologic Study of Clarious Cavities, J. Am. Dent. 14. Ribby, A. 25: 1934, 1938. A Xew Streptococcus From Dental Caries, Arch, R., and Hammond, E.: 15. Tunnicliff, Path. 26: 61, 1938. in Pulps R., and Hammond, C.: Smooth and Rough Greening Streptococci 16. Tunnicliff, .T. Am. Dent. A. 25: 1046, of Intact and Carious Teeth anii in Carions Dentin, 1 WS.
640
MASAO
ONISI
AND
JAMES
NUCKOLLS
17. Canby, C. P., and Bernier, J. L.: Bacteriologic Studies of Carious Dentin, Dent. A. 23: 2083, 1936. Bacteriological Studies on Experimental Dental Caries in 18. Harrison, R. W.: III. Flora of the Advancing Carious Lesion in the Rat, J. Infect. Dis. 1940. 19. Burnett, G. W., and Scherp, H. W.: The Distribution of Proteolytic and Bacteria in the Saliva and in the Carious Lesion, ORAL SURG., ORAL MED., PATH.
4: 469,
J. Am. the Rat. 67: 106, Aciduric AND ORAL
1951.
20. Burnett, G. W., and Scherp, H. W.: Lesion, J. D. Res. 30: 766, 1951.
Bacteriologic
Studies
of the Advancing
Dentinal