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JouRNAL OF ENDODONTICS Printed in U,S.A.
COpyright ~'J 1987 by The Amer~,an Association of Endodo~tists VOL. 13, NO. 1, JANUARY 1987

Light and Electron Microscopic Studies of Root Canal

Flora and Periapical Lesions
P. N. Ramachandran Nair, BVSc,DVM

This study elaborates on (a) the structure of the root cally (1, 2, 4, 6-11). In view of the obvious contamina-
canal flora in periapically affected human teeth; (b) tion problem, only bacteria found within intact lesions
the relationship of flora to the dentinal wall of the with firm attachment to the root apex, particularly those
root canal; (c) morphological aspects of interaction found within phagocytic cells, were considered genuine
among diverse groups of bacteria; and (d) the dy- findings. With the exception of one investigation (9),
namic nature of inflammatory host response at the those studies (1, 6, 8, 10, 11) tend to suggest that only
periapex. Spontaneous human periapical lesions (30 a small fraction (5-15%) of periapical lesions, especially
granulomas and 1 radicular cyst) obtained by tooth those with acute inflammation may show the presence
extraction were processed for light and electron of bacteria within. Consequently, periapicai lesions
microscopy. All 31 teeth showed bacteria in their were considered to be caused not necessarily by mi-
root canals. The flora consisted of cocci, rods, fila- croorganisms alone but rather by other primary and
mentous organisms, and spirochetes. The rods independent cofactors such as decomposition products
often revealed Gram-negative walls. The bacterial of necrotic pulp tissue (12) or root canal fillings. Efforts
flora formed clusters of "self-aggregating" colonies have also been made to implicate immunopathological
of one distinct type or "coaggregating" communities mechanisms in the initiation of these lesions (13, 14).
of several types. Electron microscopically a con- Investigations using refined microbiological tech-
densed bacterial layer could be identified on the niques (15-19) resulted in repeated reporting of certain
dentinal wall of the root canal which when light groups of bacteria, particularly some obligate and fa-
microscopically visible, gave the palisade structure cultative anaerobes in combination with less fastidious
of bacterial plaques adhering to tooth surfaces. The Gram-positive organisms. Carefully designed patho-
endodontic flora was separated from the rest of the biological experiments using those organisms or their
lesion by a dense wall of neutrophils (PMN's) or by products (20-27) in animal models tend to suggest high
an "epithelial plug" at the apical foramen. Five spec- endodontopathic potential of the mixed root canal in-
imens revealed both intra- and extracellular bacteria fections and their probable involvement in the patho-
within the body of the lesions. The membrane-delim- genesis of periapical lesions. Nevertheless, modern
ited phagosomes containing bacteria showed inter- histopathological studies of intact periapical lesions
action between phagocytic cells and bacteria. The with firm attachment to the root apex are extremely
presence of the "immunocoating" on the surface of rare. Except for a couple of publications (28, 29), trans-
certain phagocytosed bacteria strongly suggests mission electron microscopic studies of the root canal
that they were not contaminants but actual tissue and periapical flora do not seem to exist. The purpose
invaders against which the host mounted a specific of this communication is to study the endodontic and
immune response at the periapex. periapical flora of diseased human teeth using corre-
lated light and transmission electron microscopy.

MATERIALS AND METHODS

The primary etiological agent of all periapical lesions
has long been considered to be bacterial. Nevertheless, The tissue specimens (31 lesions) formed part of a
attempts (1-4) to identify organisms which are signifi- collection of endodontically untreated and periapically
cant for the pathogenesis of periapical lesions have affected human teeth obtained by extraction. All 31
largely failed due to the enormous problem of bacterial teeth were profoundly carious. The pulps were nonvital
contamination or to inadequacy in microbial culture and the periapical regions were consistently radiolu-
techniques (5). In spite of this, samples of granulomas cent. Five teeth were symptomatic (pain) and were
gained after tooth extraction or removed by flap surgery clinically diagnosed as presenting acute periapical in-
have been studied both bacteriologically and histologi- flammation. The other teeth were asymptomatic and
29
 30 Nair Journal of Endodontics

were diagnosed as presenting chronic periapical gran- those tooth-adhering monobacterial aggregates, the
ulomas. Immediately after removal the teeth with the dentinal wall of the root canal, in isolated segments,
attached periapical lesions were fixed by immersion, for was covered by single (Fig. le) or multilayered (Fig. l f )
a period of 1 wk, in modified Karnovsky's (30) fixative, bacterial condensations which were visible only at the
consisting of 2% paraformaldehyde and 2.5% glutaral- electron microscopic level. Various types of bacteria
dehyde buffered in 0.02 M sodium cacodylate. There- could be observed in this condensed layer. The fila-
after, the lesions with the apical third of the affected mentous forms were often found to adhere perpendic-
root were severed and kept suspended in a decalcifying ular to the dentinal wall. The coccoid forms appeared
solution containing 0.25 M EDTA (Titriplex-III; Merck), to form cell strings orientated in the same direction.
4% g/utaraldehyde, and sucrose for a variable period Very rarely the condensed bacterial layer was thick
of 4 to 5 months. After decalcification the specimens enough to be seen with the light microscope, in which
were divided into 0.5- to 1-mm thick slices in the axial case it gave a pallisade-like structure (Fig. 4b, upper
plane of the root using a sharp razor blade and a Wild inset).
M-5 stereomicroscope. The endodontic flora was often seen against a dense
The specimen blocks were washed overnight in wall of PMN's (Fig. 1, a and b) and less frequently
0.185 M sodium cacodylate buffer (pH 7.4, 360 mos- against an ingrowth of epithelium from the body of the
mol), postfixed in 1.33% OsO4 buffered in 0.067 M s- periapical lesion into the entrance of the root canal. The
collidine (31) dehydrated in ascending grades of latter appeared to form an epithelial plug at the apical
ethanol, and embedded in Epon (32). Those tissue foramen (Fig. lc). Several apparently spherical clusters
blocks of all lesions in which the section planes passed and numerous single, scattered bacteria could be ob-
through the apical root canal were selected for this served within the epithelial plug and along the epithe-
investigation. They consisted of a total of 31 lesions, of lium dentin interface (Fig. 2). Morphologically, those
which 1 was a radicular cyst. From each selected block, mixed bacterial clusters consisted of cocci, rods, fila-
1- to 2-#m thick semithin ~=ctions were prepared using mentous bacteria (Fig. 2, large inset), and spirochetes
glass knives and a Reichert OM-U2 ultramicrotome. (Fig. 2, left lower inset). The intermicrobial space in
Sections were stained differentially in PAS and meth- those clusters appeared to be structureless. The epi-
ylene blue-Azur II (33) and photomicrographed using a thelial plug was heavily infiltrated by PMN's which were
Leitz-Orthoplan photomicroscope. Miniature pyramides seen scattered among the loose flora or appeared to
were made on nine selected blocks containing light surround the bacterial clusters. In one of the lesions,
microscopically visible collections of bacteria within root the endodontic flora was observed at the apical fora-
canals and/or the body of the lesion. Thin sections were men without being separated from the rest of the
cut with a LKB Ultrotome II, double contrasted using lesions by an epithelial plug or by a PMN wall (Fig. 3)
lead and uranyl salts (34, 35), and examined in a Philips The body of the periapical lesion was characterized by
201 transmission electron microscope. a collagen-rich connective tissue which was infiltrated
by a moderate number of mononuclear cells.
RESULTS Four granulomas revealed the presence of bacteria
within the body of the lesion, of which one was a clear
The root canals of all 31 lesions revealed the pres- case of actinomycosis and was described in detail
ence of a mixed bacterial flora. Although, in most cases elsewhere (29). The other three lesions showed the
the flora was restricted to the root canal, both intra- presence of distinct bacterial plaque adhering to the
and extracellular bacteria could be observed within the dentinal wall at the apical foramen (Fig. 4, a and b). In
body of four granulomas and the one radicular cyst. those cases, the floral front extended for varying depths
Teeth with the latter lesions were symptomatic. into the body of the periapical lesions causing limited
Structurally, the bulk of the root canal flora existed (Fig. 4, a and b) or extensive tissue necrosis and acute
as loose collections of a variety of morphologically PMN response. In the latter instance, the chronic gran-
distinct but taxonomically unidentifiable bacteria con- ulomatous tissue immediately around the tooth apex
sisting of cocci, rods, and filamentous forms. Most of appeared to be lysed and was occupied by a massive
the flora remained suspended in an aparently moist and aparently young apical plaque (Fig. 5). Character-
canal lumen (Fig. 1, a to c). Less frequently, dense istically, the flora consisted of numerous dividing cocci
aggregates of bacteria could be observed sticking to (Fig. 5, middle inset), rods, filamentous organisms (Fig.
the dentinal wall of the root canal or existing free among 5), and plenty of spirochetes (Fig. 5, upper inset). The
vast numbers of PMN's in the canal lumen (Fig. 1, a rods often showed Gram-negative cell walls and some
and b). Electron microscopically those dense aggre- of them revealed a third electron-dense outer layer (Fig.
gates were found to be clusters of bacterial cells of one 5, lower inset). In very rare instances, the flora con-
distinct morphological type (Fig. ld). The interbacterial tained isolated branching or hyphal-like structures (Fig.
spaces revealed an amorphous material, particularly 4, middle inset). The root dentin of some of the speci-
toward the center of the aggregates. Independent of mens revealed the presence of isolated clusters of
 Vol. 13, No. 1, January 1987 Root Canal Flora and Periapical Lesions 31

:-~' :~.'+,
9 -,,,~....
... .., :'+~ . : :

+' " "' ~. . . . D

, 9 ++

,-,i ~4. ' ~

~,r

. 9 e~

r ;

m + ' ~.+~++_'P~'~m_
.t,~.~ "~.~+,~+~'-~' ~ ; ' + ~ p l ~ ~=+.~ ~ . ~ + ~ .~ ~ ~ ~++~+ ~ ,~ ", . ,~ ~,~,.~+-,t+,:~
+,++ ,"

~-+ ~+~ ~.,

' ~
+ , +...-~, .
..+ + ., + ++,,, , ,
,,++
.'~;~-+
,++ + ,+ +,

FIG 1. The endodontic flora in the apical third of periapically affected human teeth. The flora appears to be blocked by a wall of neutrophils (NG
in a and b) or by an epithelial plug (EP in c) in the apical foramen. Note the dense aggregates of bacteria sticking to the dentin wall (AB in b) and
similar ones (SB in b) along with loose collections of bacteria (inset in C) remaining suspended in the root canal among neutrophils. A cluster of
an apparentfy monobacterial colony is magnified in d. Electron micrographs show bacterial condensation on the surface of the dentin wall,
forming thin (e)- or thick (/)-layered bacterial plaques. The rectangular demarcated portion in a and the circular one in c are magnified in b and
the inset in c, respectively. GR, granuloma; D, dentin, a, original magnification xf0; b, original magnification x400; c, original magnification x40;
d, original magnification • e, original magnification • f, original magnification • inset in c original magnification x400.
32 Nair Journal of Endodontics

FiG 2
 Vol. 13, No. 1, January 1987 Root Canal Flora and Periapical Lesions 33

bacteria in apparently disintegrating and widened den- Nevertheless, such studies provide information compli-
tinal tubles (Fig. 6). mentary to data obtained by bacteriological, biochemi-
One of the lesions happened to be a radicular cyst, cal, and pathobiological methods. Regarding the com-
which showed a distinct lumen lined by a stratified position of the root canal flora, only very general and
squamous epithelium of varying thickness (Fig. 7a). The limited conclusions can be drawn from this study. The
cyst cavity contained numerous erythrocytes, PMN's, root canal flora consisted of cocci, rods, filamentous
and other necrotic cells. Single and isolated aggregates forms, and spirochetes. Although the latter are known
of bacteria could be observed to remain suspended in to be important periodontal pathogens, only rarely they
the cyst lumen which apparently contained a fluid me- have been reported from infected root canals (15, 36).
dium. Light microscopically numerous neutrophils con- In a recent study using dark-field microscopy, Thilo et
tained long and filamentous organisms within them (Fig. al. (36) reported 6% of spirochetes among the root
7, right inset). Electron microscopically the neutrophils canal flora. Spirochetes seem to form a significant
revealed several morphologically distinct types of mem- component in our specimens. Similarly Gram-negative
brane-delimited phagosomes containing bacteria (Fig. rods also appear to be important. The electron-dense
7, b and c). In some of the phagosomes the host cell outer cell wall layer observed in some of the Gram-
membrane adhered to the bacterial cell wall, leaving no negative rods appear to be identical to such a layer
space between them (Fig. 7c, P1 and P2). In other described for certain species of Bacteroides under var-
instances, a clear and distinct space separated the ious names such as the "additional layer" (37), the
: bacterium from the phagosome membrane (Fig. 7c, P3). "external surface layer" or simply the "S-layer" (38), or
A third group of phagosomes contained bacteria show- the "outer membrane complex" (39). To my knowledge,
ing an electron-dense coating around them. This cell no mycotic species have been reported among the
wall coating appeared to fill partially (Fig. 7b, ,04) or infected pulp or the root canal flora. The observation of
completely (Fig. 7b, Ps) the space between the bacte- isolated, branching, or hyphal structures is suggestive
rium and phagosome-membrane. The fourth type of of mycotic presence in necrotic canals with periapical
phagosome contained bacteria without any surface involvement. They should, nevertheless, be considered
coating along with several membrane-bound, electron- as opportunistic invaders along with the more distinct
dense, cytoplasmic granula-like structures (Fig. 7b, P6). Actinomyces organisms encountered (29).
The entire cyst was encapsulated in a connective tissue The organization of the canal flora in certain definite
capsule that was firmly attached to the root apex (Fig. patterns and the relationship of the flora to the dentinal
7a, left inset). wall have to be interpreted in terms of the phenomenon
of microbial adherence (40). The bacterial clusters con-
DISCUSSION sisting of only one definite morphological type seems
The present study elaborates on the structure of the to be self-aggregating (41) organisms forming colonies.
root canal flora in periapically affected teeth, its rela- The loose amorphous material observed in those colo-
tionship to the dentinal wall of the root canal, morpho- nies may be the insoluble part of the extracellular matrix
logical aspects of interaction among diverse groups of of bacterial orgin. On the other hand, those apparently
bacteria, and the dynamic nature of inflammatory host spherical clusters consisting of several distinct types of
response at the periapex. Although a mixed bacterial bacteria seem to form "coaggregates" (42, 43) among
flora was observed in all 31 specimens under investi- themselves. In an ecological sense they appear to form
gation, it may be emphasized that a mere bacterial intricate microbial communities promoting a symbiotic
presence does not imply an etiological relationship to relationship. Although there does not seem to exist any
the development of the lesion. report on the formation of bacterial plaque on the
There are numerous modern studies on the bacteri- dentinal wall by the root canal flora, the condensed
ology of infected pulp and; root canals (15-19), but bacterial layer may be the earliest plaque-forming or-
structural descriptions of endodontic flora are still miss- ganisms similar to those described on human tooth
ing. Obviously taxonomical identification of the species surfaces (44, 45).
involved cannot be reached by morphological means: The interface between the bacterial front and the

FJG 2. The root canal flora near the apical foramen of a tooth with an apical epithelial plug. The transmission electron microscopic composite
shows the presence of several microbial clusters (double small arrow-heads) and numerous single bacteria existing between the root dentin (D)
and the epithelial plug (EP). NG, neutrophilic granulocytes. The circular demarcated cluster at the bottom is magnified in the large insert. Note
the presence of cocci, rods, filamentous organisms, and spirochetes (S in lower left inset) in the cluster. The periapical lesion(upper left inset) is
a Collagen.dense, resting granuloma (GR) containing epithelial strands (EP). The circular demarcated area in the large inset is magnified in the
left lower inset. Original magnification x2,400; insets: upper, original magnification x15; middle, original magnification x8,960, lower, original
magnification x 17,500.
34 Nair Journal of Endodonti(

periapical lesion deserves some attention. The flor

may be separated from the lesion by a dense PMN w~
or by an epithelial plug (5, 46) at the apical foramer
Only in a single specimen was the root canal flora foun
to exist at the apical foramen without being separate
from the body of the lesion by a structural barrier. Thi
case should be considered as a "resting granuloma" (5
in which the bacteria may remain more or less dormar
in the root canal and the body of the lesion shows
collagen-rich connective tissue with moderate monc
nuclear cell infiltration. The PMN wall found in mos
cases is explainable in terms of chemotactic respons~
of the PMN's to bacteria invading or their metabolic o
breakdown products egressing into the periapical area
Chronic periapical granulomas have long been showt
(47) to contain proliferating epithelial cells which, if
some instances, may contact the apical root surfac(
and may grow into the entrance of the root canal (47
48), forming a plug-like epithelial seal (5, 46) at th{
apical foramen. The interface between the apical epi
thelial plug and the root dentin reveals an ultrastructura
attachment device consisting of a basal lamina, hemi
desmosomes, and a cuticular structure (46). Althougr
the epithelial plug appears to block the advancing bac.
terial front against the periapical tissues, it cannot b~
counted as an effective, biological defense barrier ir
view of its infrequent and unpredictable development
Furthermore, the wide intercellular spaces allow bac-
teria to enter or their products to leak into the per/apex,
thereby maintaining the lesion at the periapex.
The observation of bacteria within the body of the
lesion is consistent with findings of previous research-
ers (1, 2, 4, 6-8, 10, 11). Except for the case of
periapical actinomycosis (29), all of the other three
lesions have to be considered as secondary abscesses
("Phoenix abscess") developing in response to bacteria
invading the body of the lesion from the root canal. This
happens when the delicate equilibrium existing at the
periapex, between the root canal flora and host defense
mechanisms, is disturbed either by a temporary decline
in the host resistance or by an increase in the collective
bacterial virulence through several possible factors (5,
23).
Another aspect to be considered is the presence of
bacteria within the radicular cyst. It may be argued as
a case of bacterial contamination during the process of
tooth extraction. However, it may be emphasized that
only well-encapsulated and unbroken lesions with firm
attachment to the root apex were selected for further
processing. Regarding the radicular cyst in question,

FIG 3. A collagen-rich and mononuclear cell-infiltrated periapical lesion

(resting granuloma, GR) showing the presence of bacteria (BA) at the
apical foramen. There is neither an epithelial plug nor a demarcating
walt of neutrophils. Inset, circular area outlined in the root canal.
Original magnification x80; inset, original magnification x400.
Fie 4. An apical plaque invading a resting granuloma. (The rectangular demarcated area in a is magnified in b.) The well-encapsulated granuloma
(GR in a) shows the bacterial front (arrowheads in b and lower inset) deep within the body of the lesion. Note the funnel-like area of tissue
necrosis immediately in front of the apical foramen (a and b) and the plaque-like bacterial condensation (BA in b and upper inset) along the root
dentin. This plaque is electron miroscopically shown in c. The middle inset shows a high magnification of a branching or hyphal-like structure
found among the plaque flora D, dentin, NG, neutrophilic granulocytes, a, original magnification x23; b, original magnification x l 0 0 ; c, original
magnification x2,680; insets: upper, original magnification x400; middle, original magnification x4,300; lower, original magnification x250.
36 Nair Journal of Endodontics

Fig 5
Vol. 13, No. 1, January 1987 Root Canal Flora and Periepical Lesions 37

F~e6. Presence of bacterial clusters in the root dentin. The area represents the root portion slightly coronal to the periapical area shown in Fig.
5. Note part of the apical plaque visible peripheral to the cementum (CD) and clusters of bacteria (BA) existing in apparently disintegrating
dentinal tubules. Onginal magnification x5,300; inset, original magnification x l 2,800.

microscopically there was no sign of breakage or con- with specific antiserum. Such a coating was missing on
tinuity of the cyst lumen to the exterior. Furthermore, control bacteria incubated with nonspecific sera. The
numerous PMN's revealed membrane-delimited pha- specificity of the immunocoating and its appearence in
gosomes containing bacteria. The morphology of the PMN phagosomes were described subsequently (52).
various phagosomes described are suggestive of sev- The electron-dense bacterial surface coating observed
eral aspects of interaction between bacteria and in some of the PMN phagosomes in this study is likely
PMN's. The "zipper mechanism" of phagocytosis (49) to be a product of a specific immune reaction that took
implies a close adherence of PMN plasma membrane place between the bacteria in question and its specific
to the bacterial cell wall during and immediately after homologous antibody. This is strong evidence against
engulfing. A definite spatial separation between the the possibility of the bacteria having reached the peri-
bacterial cell wall and the phagosome membrane begins apical area by contamination. On the contrary, the body
to appear after the onset of the respiratory burst (50). was challenged by the bacteria in question, through the
Some of the phagosomes encountered within PMN's root canal, for a sufficiently long period to mount a
in the cyst lumen appear to fall into those two cate- specific immune response at the periapex.
gories. The membrane-bound, granula-like bodies ob-
served along with bacteria within some other phago- CONCLUSIONS
somes appear to be lysosomal granules having fused
and released into the phagosome vacuole. The elec- This study shows that the root canals of all peri-
tron-dense surface coating of bacteria observed within apically affected teeth contain bacteria. The endodontic
several phagosomes deserves particular attention. This flora consists of a mixture of cocci, rods, filamentous
coating appear to be identical to what has been de- forms, and spirochetes. Only a small fraction of the
scribed as the immunocoating (51). While attempting to periapical lesions reveals bacteria within the body of
develop a suitable electron microscopic technique to the lesions. Such lesions are invariably acute and symp-
localize bacterial surface antigens using unlabeled sera, tomatic. These observations seem to reflect recent
those authors (51) observed a thick electron-dense findings based on modem bacteriological and patho-
layer on the surface of the bacteria that were treated biological investigations.

FIG 5. A massive periapical plaque associated with an acute lesion. Note the mixed .nature of the flora. Numerous dividing cocci (DC, middle
inset), rods (lower inset), filamentous bacteria, and spirochetes (S, upper inset) can be seen. Rods often reveal a Gram-negative cell wall (double
arrowhead, lower inset) some of them showing a third outer layer (OL). The circular areas 1, 2, and 3 are magnified in the middle, upper and
lower insets, respectively. D, dentin; C, cementum; NG, neutrophils. Original magnification x2,680; insets: upper, original magnification x19,200;
middle, original magnification x 11,200; lower, original magnification x36,400.
 38 Nair Journal of Endodontics

FiG 7. Presence of bacteria in a radicular cyst. Note the distinct epithelial lining (EP, a) of the cyst lumen (LU, a) and a cluster of neutrophils (NG
right inset) showing phagocytosed bacteria. (The upper inset in a shows an overview of the well-encapsulated cyst (CY).) Several types o
membrane-delimited phagosomes (P1 to P6) containing bacteria are electron microscopically shown in b and c. Note the close adherence o
bacteria and the phagosome-membrane in P~ and ,~ while a distinct and clear space is visible between them in ,~ An electron-dense coating o
varying thickness can be distinguished on the bacterial surface in P4 and P~. Note the bacterium in ,~ which is devoid of such a coating, but the
phagosome contains several membrane-delimited granu[a-like structures. D, dentin; NU, nucleus, a, original magnification x l 0 0 ; insets: [eft
odginal magnification x l 0 ; right, original magnification x850. b, original magnification x12,800, insets: upper, original magnification x8,900
lower, original magnification x l 7,500. c, lower inset, original magnification x8,900; upper inset, original magnification x l 7,500.
 Vol. 13, No. 1, January 1987 Root Canal Flora and Periapical Lesions 39
I thank Dr. E. Schmid, Surgical Polyclinic, Dental Institute, University of oral bacteria isolated from infected root canal after varied times of closure.
Zurich, Professor P. Brunner, Social Dental Clinic, Canton of Z0nch, for Scand J Dent Res 1982;90:134-44.
collecting extracted teeth with periapical lesions, Mrs. K. Rossinsky for excellent 26. Dahl(m G, Fabricius L, Heyden G, Holm SE, Moiler AJR. Apical perio-
technical assistance, Mrs. R Kroni for secretarial and editorial help, and dontitis induced by selected bacterial strains in root canals of immunized and
professor H. IS. Schroeder for critical reading of the manuscript. nonimmunized monkeys. Scand J Dent Res 1982;90:207-16.
27. Dahl~rl G, Fabricius L, Holm SE, Moiler AJR. Circulating antibodies after
Dr. Nair is affiliated with the Department of Oral Structural Biology, Dental experimental chronic infection in the root canal of teeth in monkeys. Scand J
institute, University of Zurich, Zurich, Switzerland. Dent Res 1982;90:338-44.
28. Lin L, Laegeland K. Light and electron microscopic study of teeth with
carious pulp exposures. Oral Surg 1981 ;51:292-316.
29. Nair PNR, Schrneder HE. Periapical actinomycosis. J Endodon
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