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Comparative Study of Dark-Field and Stained Smear Technics for Identification of Oral Spirochetes on the Basis of Morphologic Characteristics
3
i8
carditis. 1941.
T h e J o u r n a l o f t h e A m e r ic a n D e n t a l A s s o c ia t io n
J .A .M .A ., 117¡1870, November 29,
7 . K e e fe r , C. S., et al. : Penicillin in Treat ment of Infections. J .A .M .A ., 1 2 2 : 1 2 1 7 , Au gust 2 8 , 1 9 4 3 .
8. L o ew e , L eo, et al.: Combined Penicillin and Heparin Therapy of Subacute Bacterial Endocarditis. J .A .M .A ., 124:144, January 15, 1944. 9. D a w so n , M. H ., and H o bby , G ladys L.: Clinical Use of Penicillin. J .A .M .A ., 1 2 4 :6 i l , March 4, 1944. 10. D ubos, R. J.: Antimicrobial Agents of Biologic Origin. J .A .M .A ., 124:633, March 4, 1944.
1 1 . U l r i c h , J. L.: Blind Dental Abscess. J .A .M .A ., 6 5 :1619, 1916. 12. H artzell , T. B., and H enrici, A. T . : Study of Streptococci from Pyorrhea Alveolaris and from Apical Abscesses. J.A .M .A ., 64 :10 5 5 , 1915. 13. F raser, C. J.: Preliminary Report on Relation of Streptococcus Viridans to Peri apical Infection. Brit. D . J., 44:1350, De cember 1, 1923. 14. A ppleton , J. L. T .: Bacterial Infection, with Special Reference to Dental Practice. Ed 2. Philadelphia: Lea & Febiger, 1933. 15. R osenow , E. C.: Oral Sepsis in Rela tion to Focal Infection. J .A .D .A ., 14:1417, August 1927.
COM PARATIVE STUDY OF DARK-FIELD AND STAINED
SMEAR TECHNICS
FOR
IDEN
TIFIC A TIO N OF ORAL SPIROCHETES ON
THE
BASIS OF
M ORPHO
LOGIC CHARACTERISTICS E d w a r d G. H
am pp
* D.D.S.. M .S.. Bethesda. M d.
INTRODUCTION
U R knowledge of bacterial morph ology has been gained for the most part by systematic microscopic ob servation of pure cultures of organisms maintained in the laboratory on artificial culture media. In this respect, the stained smear and the hanging-drop preparation have been used to advantage in studies of most bacterial species; but such prepa rations are of relatively little value in morphologic studies of spirochetes. The dark-field microscope has been of great value in such work, representing the only simple method by which the details of spirochetal morphology may be eluci dated. In spite of knowledge of this technic, there persist a considerable num
O
*Fellow, Research Commission, American Dental Association; Passed Assistant Dental Surgeon (R ), National Institute of Health, United States Public Health Service. '
Jour. A .D .A ., V ol. 32, M arch 1, 1945
ber of workers who attempt to identify spirochetes by observation of fixed and stained smears. It is the purpose of this paper to compare the findings observed when pure cultures of spirochetes are studied microscopically, using fixed and stained smears observed with direct il lumination and wet preparations ob served with dark-field illumination, and to point out the fallacy of employing the former method as a means of establish ing the identity of the oral spirochetal species. The fine structural differences that exist among the various members of the group, which are the only means avail able for the differentiation of the vari ous species, may be so distorted or obscured when fixed and stained smears are used that identification of the spiral forms is difficult. This is especially true of the smaller oral treponemes whose
H a m p p — I d e n t if ic a t io n o f S p ir o c h e t e s
structure m ay be so altered in many instances that they may be confused with the intermediate forms o f the larger spirochetes o f the mouth. m a t e r ia l a n d m e t h o d s
T h e source o f the material in this study was subgingival scrapings from hum an cases o f gingivitis and V in cen t’ s stomatitis, and also from numerous pure cultures o f the smaller treponemes iso lated from oral infections. Pure cu l tures, when used as a source o f material, were examined within twenty-four hours after growth was initiated. T h e sample organisms were in all cases selected from the periphery o f the advancing edge o f growth so that young actively grow ing
crystal violet: i part o f a saturated solution o f the dye in 95 per cent a lco hol and 9 parts o f distilled water. Since m uch has been written con cern in g measurements o f the oral spiro chetes,1’ 2>3>4’ 5> 6111 n o attempt will be m ade to repeat this work, but such m or ph ologic variations as are m ade apparent by the tw o methods will be discussed. RESULTS AND COMMENT
Thirty-five pure cultures o f the smaller oral treponemes obtained from human cases o f oral infection were studied in detail. Numerous heat-fixed stained smears prepared from these cultures ex hibited organisms w ith from a m oderate to an extreme degree o f distortion, w hich
F ig. 1.-— D ark-field and stained sm ear preparation s obtained from pu re culture o f sm aller oral treponem es; fou rth passage. ( X I455-)
organisms w ould be obtained, since they are generally considered typical o f the species. W hether the material contain ing spirochetes was obtained directly from lesions in the m outh or from cul tures, the sample was suspended in sterile physiologic sodium chloride solu tion. Small amounts o f this bacterial suspension were then used to make heatfixed stained smears and, at the same time, wet preparations were m ade from the same sample. These served as co n trols fo r direct examination o f the living organisms under the dark-field m icro scope. T h e stain used fo r the dried films was
primarily affected the shape, size and spacing o f the coils. In many instances, the structure o f these organisms was so altered that they could have easily been confused with certain o f the interm edi ate forms o f the larger oral spirochetes. T h e probable explanation fo r such cellular changes m ay be based on sev eral facts. T h e act o f drying a smeared preparation in air will cause plasmolysis o f the spirochetal body.8 T h e extreme length o f the oral treponemes in co m parison with their w idth tends to cause the organisms to unfold their closely knit, symmetrically placed coils and as sume a straight position when dried in
320
T h e J o u r n a l o f t h e A m e r ic a n D e n t a l A s s o c ia t io n
air. This tendency is further accentuated by heating during fixation. T h e smaller treponemes have been fou n d to be m ore stain-resistant than other oral spirochetes. This necessitates prolonged staining and, in certain instances, m ay require steam in g o f the dye at some time during the procedure to obtain a suitable prepara tion. This further contributes to the alteration o f their structure. T h e endresult is w avy distorted filaments m od i fication o f the structure o f w hich m ay vary from a slight to an excessive de gree on com parison with the same or ganisms observed in the living state by dark-field examination. A lthough this altered structure is n ot fully evident in all the cells, it usually predom inates in
Several organisms are represented in w hich n o coils are in evidence. Such form s, w hich are usually fou n d in chains o f various lengths, constitute an ex tremely small percentage o f the total num ber o f organisms in a given speci m en. T h e stained smear m ade from the same bacterial suspension (Fig. i, right) exhibits spirochetes with a marked degree o f distortion. T h e m orph ologic changes are m ost evident as regards the shape, size and spirals. T h e tendency o f the treponemes to uncoil their spirals in these dried film preparations and as sume a straight form is vividly portrayed. T here are present m any organisms that vary from almost straight form s to long wavy filaments exceeding in length the
Fig. 2.— D ark-field and stained sm ear preparation s o f Borrelia b u ccale and other oral organisms obtained from case o f m argin al gingivitis. ( X ¡455-)
the greater percentage o f individuals represented in such smears. T h e taper ing, pointed extremities and the fine terminal filaments are not apparent in such preparations. T h e conditions described can be better appreciated w hen a dark-field exam ina tion and a stained smear are m ade from the same sample o f organisms. Figure i shows tw o such preparations m ade from a pure actively grow ing culture o f one o f the smaller treponemes. T h e dark-field photom icrograph shows quite clearly the closely knit arrangement o f the fine symmetrically placed coils as well as the fine tapering extremities.
spirochetes in the control dark-field p h o tom icrograph. This change in length is apparently due to the u nfoldin g o f coils. Certain o f these forms cou ld be easily confused with the smaller types o f the oral Borrelia. Neither the tapered, pointed ends nor the terminal filaments are in evidence. T h e phenom enon o f altered structure o f the small treponemes in stained smears is not restricted to this group o f organisms, but can also be demonstrated in the larger oral spirochetes. Borrelia buccale and what is considered to be Borrelia vincenti, the two largest spiro chetal inhabitants o f the oral cavity, can
H a m p p — I d e n t if ic a t io n o f S p ir o c h e t e s
be recognized in the dark-field with a rather uniform degree o f accuracy. T he coils o f both organisms are larger, more open and extremely flexible as co m pared with the small, closely knit, sym metrically spaced spirals o f the oral treponemes. Figures 2, left, and 4, left, represent dark-field photom icrographs o f Borrelia buccale and Borrelia vincenti. T he form er organism was obtained from a case o f marginal gingivitis and the latter from a tonsillar lesion in a case o f V in cent’s angina. It is evident that B. buc cale is longer and wider, and the coils are proportionally larger and m ore widely spaced than those o f B. vincenti. In addition, B. buccale is doubly con toured, apparently ow in g to the extreme
F ig. 3.— D ark-field preparation o f Borrelia b u ccale from m ixed spirochetal c u ltu re ; third passage. T h e inoculum fo r this cultu re w as obtained from the same source as th at shown in Figu re 2, left. ( X > 4 5 5 -)
w idth o f the cell body, w hich reflects light o ff the opposite surface and is re solved separately.11 B. vincenti does not exhibit this double contoured appear ance, an im portant point in differentiat ing between the tw o form s. In co n trast, the w idth o f B. vincenti is from one-half to two-thirds that o f B. buccale, so that the form er appears as a long, thin, graceful spirochete. T h e extremi ties o f B. buccale, as a rule, present abruptly tapered, pointed ends; whereas B. vincenti has both extremities more
3 21
delicately tapered and pointed. T er m inal filaments are occasionally observed in both species. U nder low er m agnification ( X 6 4 5 ) , the cell bodies o f b oth B. buccale and B. vincenti reflect light in a characteristic manner, apparent as a yellowish tinge, while the smaller oral treponemes pre sent a brilliant white hue. This differ ence, although present, is not so p ro nounced under higher m agnification ( X 1455). Figures 2, right, and 4, right, are photom icrographs o f heat-fixed stained smears o f B. buccale and B. vincenti. These preparations were m ade from the same bacterial suspensions as Figures 2, left, and 4, left. H ere, as in the case o f the smaller oral treponemes, it is ap parent that the greatest am ount o f physi cal distortion is fou n d in relation to the shape, size and spacing o f the coils. T h e extreme length and the w ide coils char acteristic o f these species increase their tendency to assume a straight line when dried in air and fixed by heat. As seen, both photographs show spirochetes vary ing from almost straight forms to thin, wavy, ribbonlike filaments possessing several widely spaced irregular shallow spirals, and w hich are very unnatural when com pared with their mates shown in the control dark-field p h otom icro graphs. T h e shrinking and narrowing o f the cell bodies are m ost apparent in B. buccale. T h e extremities o f b oth or ganisms m ay exhibit the tapered, pointed ends, or they m ay be blunted, the shape depending on whether the smear prep arations were properly m ade. The terminal protoplasm ic filaments have never been observed in such prepara tions. In many cases o f oral infection, B. buccale is fou n d to be the predom inating spirochetal type by dark-field exam ina tion. W hen stained smears from such cases are prepared, the organisms in question m ay be confused with B. vin centi, particularly if a dark-field exam
322
T h e J o u r n a l o f t h e A m e r ic a n D e n t a l A s s o c ia t io n
ination is not m ade. This becomes evi dent w hen it is considered that the two organisms undergo distortion in a simi lar manner, the shrinkage being more severe in the case o f B. buccale. T here fore, a stained smear is misleading and cannot be used to establish the identity o f either organism. It is customary to find m orphologic descriptions in the literature6’ 7> 9’ 10 de picting B. vincenti as a delicate, loosely w ound spirochete with irregular coils. U nfortunately, there is n o indication as to whether such observations were m ade by dark-field or by stained smear examination. T h e irregularity o f the coils is often explained on the assump tion that the spirals are n ot fixed and the organism is extremely flexible.
due to a fixation-staining phenom enon, as previously described. O f the larger oral spirochetes, B. buccale has been reported isolated in pure culture on few occasions by technics d if ficult to reproduce.6’ 7’ 8 In our experi ments, this organism has been obtained in large numbers on numerous occasions in con ju n ction with the smaller treponemes and free from contam inating oral bacteria. Figure 3 represents a darkfield photom icrograph o f B. buccale in a m ixed spirochetal culture. T h e in oculum fo r this culture was obtained from the same source as the organisms portrayed in Figure 2, left. It is ap parent that the organisms from the cu l ture are shorter and narrower than those fou n d in the original gingival scrapings
F ig. 4.-— D ark-field and stained sm ear preparations of spirochetal form s th at conform w ith m orph ologic descriptions o f B orrelia vincenti. In the stained smear, the three form s alm ost p a rallel and in the center of th e field are the organism s in question. T h e rem ain ing spiral form s are B orrelia buccale. T h e m aterial was obtained from a tonsillar lesion in a case of V in c e n t’s angina. ( X *455-)
H ow ever, dark-field observation o f the V incent-type spirochete (F ig. 4, left) shows that the coils are not irregular, but are constructed uniform ly and widely spaced. W hen in m otion, the organism exhibits rapid flexion, looping, transla tion and rotation. T here is an apparent tendency to extension o f its spirals dur ing m otion, with reconstitution o f the spirals when it slows dow n or m ovem ent ceases. It is only in stained smears that m odified m orph ologic types are observed and, in all probability, the changes are
and, in addition, the spirals are p ro portionately smaller and m ore com pact. In b oth preparations, the spirochetes present double contoured form s with pointed ends. In the living state, they exhibited rapid flexion, looping, transla tion and rotation. It is often stated in the literature3’ 6’ 9>12 that the m ove ments o f this organism are serpentine. This type o f m otility was not apparent either in the organisms obtained directly from the m outh or in those obtained from m ixed spirochetal cultures. W hen
H a m p p — I d e n t if ic a t io n o f S p ir o c h e t e s
wet dark-field preparations began to dry so that the m ovements o f the spirochetes were restricted, and only then, were the serpentine movements observed, and we do not consider these m ovem ents charac teristic o f the species. C onclusive evidence has never been presented that B. vincenti has been o b tained in pure culture. K lein 9 asserts that he has obtained it. H ow ever, the only evidence submitted in support o f his assumption consists o f a single dark-field photograph o f three spirochetal forms o f which the m orph ologic details are obscure. N o attempt was m ade to pre sent a m orph ologic description o f his organisms in the text or with the illus tration.
323
pointed ends. (C om p a re Figure 4, left.) In the living state, it is extremely motile, exhibiting rotation, looping, flexion and translation, and coincides with descrip tions o f Borrelia vincenti presented in the literature.5’ 6*10’ 12 Figure 5, right, represents a stained smear m ade from the same suspension o f organisms as F ig ure 5, left. H ere, as in the case o f the other oral spirochetes previously de scribed, the distortion is m ost evident as regards the coils. These organisms vary from almost straight forms to wavy filaments possessing widely spaced irreg ular spirals. M an y o f the extremities are delicately pointed. Others are rounded and blunted. Numerous inter mediate forms are in evidence.
Fig. 5.— D ark-field and stained sm ear preparations of pu re culture, th ird passage, of large oral spirochetal form s w h ich conform w ith descriptions in literatu re for B orrelia vincenti.
During a series o f cultural experiments in this laboratory, a large oral spirochete was obtained on several occasions in primary cultures together with the smaller treponemes and B. buccale. F ig ure 5, left, is a photom icrograph o f a dark-field preparation o f one strain o f this large spirochete isolated in pure culture. It is m orphologically different from our pure strains o f smaller tre ponemes and the m ixed spirochetal cu l ture o f B. buccale. T h e dark-field illus tration shows clearly that it is a large organism possessing large, widely spaced symmetrically placed spirals. It is not double contoured and has delicately
SUMMARY
Differences in structures o f certain o f the oral spirochetes were observed when they were exam ined in the living state by dark-field illumination and in air-dried, heat-fixed stained smears by direct il lumination. T h e organisms used were from pure cultures o f the smaller oral treponemes, a large spirochetal form m orphologically similar to those de scribed in the literature as Borrelia vin centi and m ixed spirochetal cultures con taining Borrelia buccale. These pure strains were supplemented by organisms obtained from subgingival scrapings in cases o f oral infection.
324
T h e J o u r n a l o f t h e A m e r ic a n D e n t a l A s s o c ia t io n
O u r observations show that use o f air-dried, heat-fixed stained smears o f spirochetal form s is extremely unreliable, failing to indicate fine structural d if ferences between spirochetal species, and thus giving a false impression o f the organisms in question. Such prepara tion causes extreme distortion o f the smaller oral treponemes and shrinkage and distortion o f the oral Borrelia. These changes are sufficient to make it impose sible to recognize the organism referred to as B. vincenti, as it m ay be confused with B. buccale, or B. buccale m ay be mistaken for the pathogenic species. Such observations are equally applicable to the smaller oral treponemes the al tered m orph ologic forms o f w hich may be confused with certain o f the inter mediate form s o f the oral Borrelia. These oral spirochetes, when observed in the living state by dark-field illumina tion, present sufficient differences to be distinguished and identified. It cannot be overemphasized that as an aid in the diagnosis o f oral infection due to the presence o f spirochetes, the laboratory m ethod o f choice is observa tion o f living organisms by dark-field examination. Until this fact is recog nized, confusion will continue to exist in the identification o f the oral spirochetal types associated with such conditions. BIBLIOGRAPHY
I. Noguchi, Hideyo: Cultural Studies on Mouth Spirochaetae (Treponema Microden-
tium and Macrodentium). /. Exper. Med.., 1 5 :8 i, January 1912. 2. I d e m : Treponema Mucosum (New Spe cies), a Mucin Producing Spirochaeta from Pyorrhea Alveolaris, Grown in Pure Culture. /. Exper. M e d ., 16:194, August 1912. 3. Dobell, C.: Researches on Spirochaetes and Related Organisms. A rch. f. Protisten kunde, 2 6 :119, July 1912. 4. Mühlens, P.: Vergleichende Spirochä tenstudien. Ztschr. f. H yg., 57:405-416, 1907. 5. I d e m .: Andere, Sum Teil Als Pathogen Geltende Spirochäten. K olle a. Wasserman’ s H andb. pa th . M ikroorg., 7:921-950, 1913. 6. Smith, D. T .: Oral Spirochetes and Re lated Organisms in Fusö-Spirochetal Diseases. Baltimore: Williams and Wilkins Co., 1932. 7. Proske, H. D., and Sayers, R. R.: Pul monary Infection and Pneumoconiosis. I. Bacteriologic and Experimental Study. Pub. H ealth R e p ., 49:839, July 20, 1934. 8. Gins, H. A.: Untersuchungen über die Spirillen der Menschlichen Mundhöhle. Ztschr. f . H y g ., 124:460-479, 1942. Abstr. Gesellsch H y g ., 5 1 :2i 1, 1943. 9. Klein, H. S.: Oral Spirochetes, Their Occurrence in Diseases of Oral Cavity and a Simple Method of Pure Cultivation. A cta O dontol. Scandinav'., 5 : i - i 8, October 1943. 10. Thomson, J. G., and Thomson, D.: Some Researches on Spirochetes Occurring in Alimentary Tract of Man and Some of Lower Animals. Proc. R oy. Soc. M ed ., 7:47, July 1913-1914. ' 11. Rosebury, Theodor: Photomicrographs of Some of Parasitic and Pathogenic Spiro chetes and Spirilla as They Appear Under Dark-Field Microscope. /. Lab. & Clin. M ed ., 27:1470, August 1942. 12. Semple, D.; Price-Jones, L .: Report for Pathological War Office of Inquiry into Vincent’s Disease Occurring R oy. A rm y M . Corps., 3 3 : 28i ,
C., and Digby, Committee of Gingivitis and in Army. ] . October 1919.
i8
carditis. 1941.
T h e J o u r n a l o f t h e A m e r ic a n D e n t a l A s s o c ia t io n
J .A .M .A ., 117¡1870, November 29,
7 . K e e fe r , C. S., et al. : Penicillin in Treat ment of Infections. J .A .M .A ., 1 2 2 : 1 2 1 7 , Au gust 2 8 , 1 9 4 3 .
8. L o ew e , L eo, et al.: Combined Penicillin and Heparin Therapy of Subacute Bacterial Endocarditis. J .A .M .A ., 124:144, January 15, 1944. 9. D a w so n , M. H ., and H o bby , G ladys L.: Clinical Use of Penicillin. J .A .M .A ., 1 2 4 :6 i l , March 4, 1944. 10. D ubos, R. J.: Antimicrobial Agents of Biologic Origin. J .A .M .A ., 124:633, March 4, 1944.
1 1 . U l r i c h , J. L.: Blind Dental Abscess. J .A .M .A ., 6 5 :1619, 1916. 12. H artzell , T. B., and H enrici, A. T . : Study of Streptococci from Pyorrhea Alveolaris and from Apical Abscesses. J.A .M .A ., 64 :10 5 5 , 1915. 13. F raser, C. J.: Preliminary Report on Relation of Streptococcus Viridans to Peri apical Infection. Brit. D . J., 44:1350, De cember 1, 1923. 14. A ppleton , J. L. T .: Bacterial Infection, with Special Reference to Dental Practice. Ed 2. Philadelphia: Lea & Febiger, 1933. 15. R osenow , E. C.: Oral Sepsis in Rela tion to Focal Infection. J .A .D .A ., 14:1417, August 1927.
COM PARATIVE STUDY OF DARK-FIELD AND STAINED
SMEAR TECHNICS
FOR
IDEN
TIFIC A TIO N OF ORAL SPIROCHETES ON
THE
BASIS OF
M ORPHO
LOGIC CHARACTERISTICS E d w a r d G. H
am pp
* D.D.S.. M .S.. Bethesda. M d.
INTRODUCTION
U R knowledge of bacterial morph ology has been gained for the most part by systematic microscopic ob servation of pure cultures of organisms maintained in the laboratory on artificial culture media. In this respect, the stained smear and the hanging-drop preparation have been used to advantage in studies of most bacterial species; but such prepa rations are of relatively little value in morphologic studies of spirochetes. The dark-field microscope has been of great value in such work, representing the only simple method by which the details of spirochetal morphology may be eluci dated. In spite of knowledge of this technic, there persist a considerable num
O
*Fellow, Research Commission, American Dental Association; Passed Assistant Dental Surgeon (R ), National Institute of Health, United States Public Health Service. '
Jour. A .D .A ., V ol. 32, M arch 1, 1945
ber of workers who attempt to identify spirochetes by observation of fixed and stained smears. It is the purpose of this paper to compare the findings observed when pure cultures of spirochetes are studied microscopically, using fixed and stained smears observed with direct il lumination and wet preparations ob served with dark-field illumination, and to point out the fallacy of employing the former method as a means of establish ing the identity of the oral spirochetal species. The fine structural differences that exist among the various members of the group, which are the only means avail able for the differentiation of the vari ous species, may be so distorted or obscured when fixed and stained smears are used that identification of the spiral forms is difficult. This is especially true of the smaller oral treponemes whose
H a m p p — I d e n t if ic a t io n o f S p ir o c h e t e s
structure m ay be so altered in many instances that they may be confused with the intermediate forms o f the larger spirochetes o f the mouth. m a t e r ia l a n d m e t h o d s
T h e source o f the material in this study was subgingival scrapings from hum an cases o f gingivitis and V in cen t’ s stomatitis, and also from numerous pure cultures o f the smaller treponemes iso lated from oral infections. Pure cu l tures, when used as a source o f material, were examined within twenty-four hours after growth was initiated. T h e sample organisms were in all cases selected from the periphery o f the advancing edge o f growth so that young actively grow ing
crystal violet: i part o f a saturated solution o f the dye in 95 per cent a lco hol and 9 parts o f distilled water. Since m uch has been written con cern in g measurements o f the oral spiro chetes,1’ 2>3>4’ 5> 6111 n o attempt will be m ade to repeat this work, but such m or ph ologic variations as are m ade apparent by the tw o methods will be discussed. RESULTS AND COMMENT
Thirty-five pure cultures o f the smaller oral treponemes obtained from human cases o f oral infection were studied in detail. Numerous heat-fixed stained smears prepared from these cultures ex hibited organisms w ith from a m oderate to an extreme degree o f distortion, w hich
F ig. 1.-— D ark-field and stained sm ear preparation s obtained from pu re culture o f sm aller oral treponem es; fou rth passage. ( X I455-)
organisms w ould be obtained, since they are generally considered typical o f the species. W hether the material contain ing spirochetes was obtained directly from lesions in the m outh or from cul tures, the sample was suspended in sterile physiologic sodium chloride solu tion. Small amounts o f this bacterial suspension were then used to make heatfixed stained smears and, at the same time, wet preparations were m ade from the same sample. These served as co n trols fo r direct examination o f the living organisms under the dark-field m icro scope. T h e stain used fo r the dried films was
primarily affected the shape, size and spacing o f the coils. In many instances, the structure o f these organisms was so altered that they could have easily been confused with certain o f the interm edi ate forms o f the larger oral spirochetes. T h e probable explanation fo r such cellular changes m ay be based on sev eral facts. T h e act o f drying a smeared preparation in air will cause plasmolysis o f the spirochetal body.8 T h e extreme length o f the oral treponemes in co m parison with their w idth tends to cause the organisms to unfold their closely knit, symmetrically placed coils and as sume a straight position when dried in
320
T h e J o u r n a l o f t h e A m e r ic a n D e n t a l A s s o c ia t io n
air. This tendency is further accentuated by heating during fixation. T h e smaller treponemes have been fou n d to be m ore stain-resistant than other oral spirochetes. This necessitates prolonged staining and, in certain instances, m ay require steam in g o f the dye at some time during the procedure to obtain a suitable prepara tion. This further contributes to the alteration o f their structure. T h e endresult is w avy distorted filaments m od i fication o f the structure o f w hich m ay vary from a slight to an excessive de gree on com parison with the same or ganisms observed in the living state by dark-field examination. A lthough this altered structure is n ot fully evident in all the cells, it usually predom inates in
Several organisms are represented in w hich n o coils are in evidence. Such form s, w hich are usually fou n d in chains o f various lengths, constitute an ex tremely small percentage o f the total num ber o f organisms in a given speci m en. T h e stained smear m ade from the same bacterial suspension (Fig. i, right) exhibits spirochetes with a marked degree o f distortion. T h e m orph ologic changes are m ost evident as regards the shape, size and spirals. T h e tendency o f the treponemes to uncoil their spirals in these dried film preparations and as sume a straight form is vividly portrayed. T here are present m any organisms that vary from almost straight form s to long wavy filaments exceeding in length the
Fig. 2.— D ark-field and stained sm ear preparation s o f Borrelia b u ccale and other oral organisms obtained from case o f m argin al gingivitis. ( X ¡455-)
the greater percentage o f individuals represented in such smears. T h e taper ing, pointed extremities and the fine terminal filaments are not apparent in such preparations. T h e conditions described can be better appreciated w hen a dark-field exam ina tion and a stained smear are m ade from the same sample o f organisms. Figure i shows tw o such preparations m ade from a pure actively grow ing culture o f one o f the smaller treponemes. T h e dark-field photom icrograph shows quite clearly the closely knit arrangement o f the fine symmetrically placed coils as well as the fine tapering extremities.
spirochetes in the control dark-field p h o tom icrograph. This change in length is apparently due to the u nfoldin g o f coils. Certain o f these forms cou ld be easily confused with the smaller types o f the oral Borrelia. Neither the tapered, pointed ends nor the terminal filaments are in evidence. T h e phenom enon o f altered structure o f the small treponemes in stained smears is not restricted to this group o f organisms, but can also be demonstrated in the larger oral spirochetes. Borrelia buccale and what is considered to be Borrelia vincenti, the two largest spiro chetal inhabitants o f the oral cavity, can
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be recognized in the dark-field with a rather uniform degree o f accuracy. T he coils o f both organisms are larger, more open and extremely flexible as co m pared with the small, closely knit, sym metrically spaced spirals o f the oral treponemes. Figures 2, left, and 4, left, represent dark-field photom icrographs o f Borrelia buccale and Borrelia vincenti. T he form er organism was obtained from a case o f marginal gingivitis and the latter from a tonsillar lesion in a case o f V in cent’s angina. It is evident that B. buc cale is longer and wider, and the coils are proportionally larger and m ore widely spaced than those o f B. vincenti. In addition, B. buccale is doubly con toured, apparently ow in g to the extreme
F ig. 3.— D ark-field preparation o f Borrelia b u ccale from m ixed spirochetal c u ltu re ; third passage. T h e inoculum fo r this cultu re w as obtained from the same source as th at shown in Figu re 2, left. ( X > 4 5 5 -)
w idth o f the cell body, w hich reflects light o ff the opposite surface and is re solved separately.11 B. vincenti does not exhibit this double contoured appear ance, an im portant point in differentiat ing between the tw o form s. In co n trast, the w idth o f B. vincenti is from one-half to two-thirds that o f B. buccale, so that the form er appears as a long, thin, graceful spirochete. T h e extremi ties o f B. buccale, as a rule, present abruptly tapered, pointed ends; whereas B. vincenti has both extremities more
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delicately tapered and pointed. T er m inal filaments are occasionally observed in both species. U nder low er m agnification ( X 6 4 5 ) , the cell bodies o f b oth B. buccale and B. vincenti reflect light in a characteristic manner, apparent as a yellowish tinge, while the smaller oral treponemes pre sent a brilliant white hue. This differ ence, although present, is not so p ro nounced under higher m agnification ( X 1455). Figures 2, right, and 4, right, are photom icrographs o f heat-fixed stained smears o f B. buccale and B. vincenti. These preparations were m ade from the same bacterial suspensions as Figures 2, left, and 4, left. H ere, as in the case o f the smaller oral treponemes, it is ap parent that the greatest am ount o f physi cal distortion is fou n d in relation to the shape, size and spacing o f the coils. T h e extreme length and the w ide coils char acteristic o f these species increase their tendency to assume a straight line when dried in air and fixed by heat. As seen, both photographs show spirochetes vary ing from almost straight forms to thin, wavy, ribbonlike filaments possessing several widely spaced irregular shallow spirals, and w hich are very unnatural when com pared with their mates shown in the control dark-field p h otom icro graphs. T h e shrinking and narrowing o f the cell bodies are m ost apparent in B. buccale. T h e extremities o f b oth or ganisms m ay exhibit the tapered, pointed ends, or they m ay be blunted, the shape depending on whether the smear prep arations were properly m ade. The terminal protoplasm ic filaments have never been observed in such prepara tions. In many cases o f oral infection, B. buccale is fou n d to be the predom inating spirochetal type by dark-field exam ina tion. W hen stained smears from such cases are prepared, the organisms in question m ay be confused with B. vin centi, particularly if a dark-field exam
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ination is not m ade. This becomes evi dent w hen it is considered that the two organisms undergo distortion in a simi lar manner, the shrinkage being more severe in the case o f B. buccale. T here fore, a stained smear is misleading and cannot be used to establish the identity o f either organism. It is customary to find m orphologic descriptions in the literature6’ 7> 9’ 10 de picting B. vincenti as a delicate, loosely w ound spirochete with irregular coils. U nfortunately, there is n o indication as to whether such observations were m ade by dark-field or by stained smear examination. T h e irregularity o f the coils is often explained on the assump tion that the spirals are n ot fixed and the organism is extremely flexible.
due to a fixation-staining phenom enon, as previously described. O f the larger oral spirochetes, B. buccale has been reported isolated in pure culture on few occasions by technics d if ficult to reproduce.6’ 7’ 8 In our experi ments, this organism has been obtained in large numbers on numerous occasions in con ju n ction with the smaller treponemes and free from contam inating oral bacteria. Figure 3 represents a darkfield photom icrograph o f B. buccale in a m ixed spirochetal culture. T h e in oculum fo r this culture was obtained from the same source as the organisms portrayed in Figure 2, left. It is ap parent that the organisms from the cu l ture are shorter and narrower than those fou n d in the original gingival scrapings
F ig. 4.-— D ark-field and stained sm ear preparations of spirochetal form s th at conform w ith m orph ologic descriptions o f B orrelia vincenti. In the stained smear, the three form s alm ost p a rallel and in the center of th e field are the organism s in question. T h e rem ain ing spiral form s are B orrelia buccale. T h e m aterial was obtained from a tonsillar lesion in a case of V in c e n t’s angina. ( X *455-)
H ow ever, dark-field observation o f the V incent-type spirochete (F ig. 4, left) shows that the coils are not irregular, but are constructed uniform ly and widely spaced. W hen in m otion, the organism exhibits rapid flexion, looping, transla tion and rotation. T here is an apparent tendency to extension o f its spirals dur ing m otion, with reconstitution o f the spirals when it slows dow n or m ovem ent ceases. It is only in stained smears that m odified m orph ologic types are observed and, in all probability, the changes are
and, in addition, the spirals are p ro portionately smaller and m ore com pact. In b oth preparations, the spirochetes present double contoured form s with pointed ends. In the living state, they exhibited rapid flexion, looping, transla tion and rotation. It is often stated in the literature3’ 6’ 9>12 that the m ove ments o f this organism are serpentine. This type o f m otility was not apparent either in the organisms obtained directly from the m outh or in those obtained from m ixed spirochetal cultures. W hen
H a m p p — I d e n t if ic a t io n o f S p ir o c h e t e s
wet dark-field preparations began to dry so that the m ovements o f the spirochetes were restricted, and only then, were the serpentine movements observed, and we do not consider these m ovem ents charac teristic o f the species. C onclusive evidence has never been presented that B. vincenti has been o b tained in pure culture. K lein 9 asserts that he has obtained it. H ow ever, the only evidence submitted in support o f his assumption consists o f a single dark-field photograph o f three spirochetal forms o f which the m orph ologic details are obscure. N o attempt was m ade to pre sent a m orph ologic description o f his organisms in the text or with the illus tration.
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pointed ends. (C om p a re Figure 4, left.) In the living state, it is extremely motile, exhibiting rotation, looping, flexion and translation, and coincides with descrip tions o f Borrelia vincenti presented in the literature.5’ 6*10’ 12 Figure 5, right, represents a stained smear m ade from the same suspension o f organisms as F ig ure 5, left. H ere, as in the case o f the other oral spirochetes previously de scribed, the distortion is m ost evident as regards the coils. These organisms vary from almost straight forms to wavy filaments possessing widely spaced irreg ular spirals. M an y o f the extremities are delicately pointed. Others are rounded and blunted. Numerous inter mediate forms are in evidence.
Fig. 5.— D ark-field and stained sm ear preparations of pu re culture, th ird passage, of large oral spirochetal form s w h ich conform w ith descriptions in literatu re for B orrelia vincenti.
During a series o f cultural experiments in this laboratory, a large oral spirochete was obtained on several occasions in primary cultures together with the smaller treponemes and B. buccale. F ig ure 5, left, is a photom icrograph o f a dark-field preparation o f one strain o f this large spirochete isolated in pure culture. It is m orphologically different from our pure strains o f smaller tre ponemes and the m ixed spirochetal cu l ture o f B. buccale. T h e dark-field illus tration shows clearly that it is a large organism possessing large, widely spaced symmetrically placed spirals. It is not double contoured and has delicately
SUMMARY
Differences in structures o f certain o f the oral spirochetes were observed when they were exam ined in the living state by dark-field illumination and in air-dried, heat-fixed stained smears by direct il lumination. T h e organisms used were from pure cultures o f the smaller oral treponemes, a large spirochetal form m orphologically similar to those de scribed in the literature as Borrelia vin centi and m ixed spirochetal cultures con taining Borrelia buccale. These pure strains were supplemented by organisms obtained from subgingival scrapings in cases o f oral infection.
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O u r observations show that use o f air-dried, heat-fixed stained smears o f spirochetal form s is extremely unreliable, failing to indicate fine structural d if ferences between spirochetal species, and thus giving a false impression o f the organisms in question. Such prepara tion causes extreme distortion o f the smaller oral treponemes and shrinkage and distortion o f the oral Borrelia. These changes are sufficient to make it impose sible to recognize the organism referred to as B. vincenti, as it m ay be confused with B. buccale, or B. buccale m ay be mistaken for the pathogenic species. Such observations are equally applicable to the smaller oral treponemes the al tered m orph ologic forms o f w hich may be confused with certain o f the inter mediate form s o f the oral Borrelia. These oral spirochetes, when observed in the living state by dark-field illumina tion, present sufficient differences to be distinguished and identified. It cannot be overemphasized that as an aid in the diagnosis o f oral infection due to the presence o f spirochetes, the laboratory m ethod o f choice is observa tion o f living organisms by dark-field examination. Until this fact is recog nized, confusion will continue to exist in the identification o f the oral spirochetal types associated with such conditions. BIBLIOGRAPHY
I. Noguchi, Hideyo: Cultural Studies on Mouth Spirochaetae (Treponema Microden-
tium and Macrodentium). /. Exper. Med.., 1 5 :8 i, January 1912. 2. I d e m : Treponema Mucosum (New Spe cies), a Mucin Producing Spirochaeta from Pyorrhea Alveolaris, Grown in Pure Culture. /. Exper. M e d ., 16:194, August 1912. 3. Dobell, C.: Researches on Spirochaetes and Related Organisms. A rch. f. Protisten kunde, 2 6 :119, July 1912. 4. Mühlens, P.: Vergleichende Spirochä tenstudien. Ztschr. f. H yg., 57:405-416, 1907. 5. I d e m .: Andere, Sum Teil Als Pathogen Geltende Spirochäten. K olle a. Wasserman’ s H andb. pa th . M ikroorg., 7:921-950, 1913. 6. Smith, D. T .: Oral Spirochetes and Re lated Organisms in Fusö-Spirochetal Diseases. Baltimore: Williams and Wilkins Co., 1932. 7. Proske, H. D., and Sayers, R. R.: Pul monary Infection and Pneumoconiosis. I. Bacteriologic and Experimental Study. Pub. H ealth R e p ., 49:839, July 20, 1934. 8. Gins, H. A.: Untersuchungen über die Spirillen der Menschlichen Mundhöhle. Ztschr. f . H y g ., 124:460-479, 1942. Abstr. Gesellsch H y g ., 5 1 :2i 1, 1943. 9. Klein, H. S.: Oral Spirochetes, Their Occurrence in Diseases of Oral Cavity and a Simple Method of Pure Cultivation. A cta O dontol. Scandinav'., 5 : i - i 8, October 1943. 10. Thomson, J. G., and Thomson, D.: Some Researches on Spirochetes Occurring in Alimentary Tract of Man and Some of Lower Animals. Proc. R oy. Soc. M ed ., 7:47, July 1913-1914. ' 11. Rosebury, Theodor: Photomicrographs of Some of Parasitic and Pathogenic Spiro chetes and Spirilla as They Appear Under Dark-Field Microscope. /. Lab. & Clin. M ed ., 27:1470, August 1942. 12. Semple, D.; Price-Jones, L .: Report for Pathological War Office of Inquiry into Vincent’s Disease Occurring R oy. A rm y M . Corps., 3 3 : 28i ,
C., and Digby, Committee of Gingivitis and in Army. ] . October 1919.