7

Socransky reported that periodontosis lesions contained

Studies of the Microbiology of significantly more gram-negative anaerobic rods than
normal sites within the same patient. Control sites
Periodontosis* harbored microorganisms similar to those found in
supragingival plaque of normal individuals. However, in
the periodontal pocket areas, gram-negative anaerobic
by
rods were dramatically increased in proportions compris­
MICHAEL G . N E W M A N | ing 40 to 78% of the total cultivable microbiota. The
majority of these organisms could not be identified by
S l G M U N D S . SOCRANSKY
current classification schemes and were divided into five
EUGENE D . SAVITT groups based on morphologic and physiologic character­
istics.
DEMITRIA A . PROPAS
Strains representing three of the five groups of gram-
ANN CRAWFORD negative anaerobic rods were implanted in germ-free
8
rats as monocontaminants by Irving et al., and New­
7
man and Socransky. In each case implantation of
T H E PURPOSE OF these studies was to investigate the
the organisms led to extensive alveolar bone destruction
microbiota in the apical portion of lesions of human
accompanied by a marked osteoclastic response without
periodontosis. The evidence that bacterial plaque is the
microscopically visable plaque formation or root caries.
major etiologic agent in human periodontal diseases has
1 4 These studies suggested that specific gram-negative
been documented in recent reviews. " In addition a
anaerobic rods may participate in the pathogenesis of
number of studies have examined the predominant
periodontosis.
cultivable microbiota of the normal gingival sulcus and
5,6 The present investigation confirms and extends obser­
the periodontal pocket. These reports suggest that
vations on the nature of the microbiota associated with
differences may exist in the microbiota associated with
normal and pathologic sites. No studies were available periodontosis lesions. In addition, the nature of the
concerning the microbiota in the periodontosis lesion. gram-negative anaerobic rods present on molars and
Thus the present investigation was undertaken to deter­ incisors of younger siblings of periodontosis patients as
mine the nature of the microbiota in the depths of the well as "normal" adolescents was sought. Finally, the
periodontosis pocket as a prerequisite to the understand­ pathogenic potential of representative strains of two of
ing of the role which microorganisms might play in this the major groups of isolated organisms was tested in
unusual clinical condition. gnotobiotic rats. The data presented represents cum-
mulative information from earlier studies and the present
Studies of the predominant cultivable microbiota of
investigation.
any oral site present a number of problems which if not
appreciated may lead to information which is difficult to M E T H O D S AND MATERIALS
interpret. Such problems include difficulties in diagnos­
Patient Selection—Periodontosis. A total of 34 lesion
ing clinical disease status, sample taking, cultivation of
sites and seven clinically normal sites were studied in 20
the many diverse organisms and identification of the
patients diagnosed as having periodontosis according to
organisms once they have been isolated. For example, 9 7
criteria of Baer and, Newman and Socransky. The
pooled samples examined in earlier studies probably
patients were between the ages of 14 to 20 years and
obscured differences in bacterial composition between
demonstrated radiographic evidence of greater than 50%
various sites by combining bacteria from periodontally
bone loss on at least one or more of the first permanent
involved areas with organisms from normal sites. In
molar teeth and/or incisors. There was little evidence of
addition the inability to cultivate oxygen sensitive orga­
clinical inflammation, as measured by a score of 1 or less
nisms and to classify many organisms resident in plaque 10
using the Gingival Index, and no history of pain or
led to further confusion. abscesses in the affected areas. There was no history of
One of the most intriguing questions in periodontal any contributory systemic disease, systemic antibiotic
research is whether specific agents cause specific forms of therapy or dental manipulation in the experimental sites
periodontal disease. In an earlier study of 14 patients in the previous 6 months. An example of such a patient is
diagnosed as having periodontosis, Newman and shown in Figure 1. In seven of the patients, at least three
sites were examined—two diseased sites and one control
Harvard School of Dental Medicine and Forsyth Dental Center, or "normal" site. Normal sites were those which did not
Boston, Mass. 02115.
This project was supported by Grants DE02847 and DE03488. demonstrate any clinical or radiographic evidence of
* Awarded first place in the Balint Orban Memorial Program at the bone loss. The bacterial samples from the normal sites
1975 meeting of the American Academy of Periodontology in Min­ were taken in exactly the same way as the diseased sites.
neapolis, Minnesota.
t Presently at U C L A School of Dentistry, Los Angeles, Calif. In the remaining 13 patients, at least one diseased site
90024. was sampled.

373
 J. Periodontol.
374 Newman, Socransky, Savitt, Propas, Crawford July, 1976

FIGURE 1. Nineteen year old patient with periodontosis. Note spacing of anterior teeth. Radiographs of this patient reveals severe
localized alveolar bone destruction around first molars and incisors.

Nonperiodontosis Patients. Two groups of five pa­ rial samples from every patient were taken from the base
tients were studied. Five individuals were preadolescent or most apical location of each site. A syringe and
children who were siblings of periodontosis patients cannula were modified to permit continuous flushing
mentioned above. These children were from two families with oxygen-free gas (Fig. 2). The plunger end of the
in which two out of four and two out of five siblings had syringe was altered to permit the passage of a long
periodontosis. stainless-steel wire soldered to a barbed broach through
The other five patients were clinically "healthy" the syringe barrel and needle. The barbed broach was
adolescents who served as "age-matched" controls. In all wound with sterile calcium alginate fibers. In use, the tip
10 individuals, there was no evidence of bone loss, no of the needle was "capped" with aluminum foil. The
history of systemic disease, and no history of antibiotic capped needle was inserted into the base of the pocket (or
therapy or scaling within the previous 6 months. Two sulcus) (Fig. 3). The aluminum foil cap prevented micro­
samples were taken from the first molars and/or incisors organisms from entering the cannula as it was inserted
of each patient since these teeth are most likely to lose into the pocket. The barbed broach was then passed out
supporting structures in periodontosis patients. of the needle, the cap was dislodged and the broach was
used to scrape the bottom of the pocket (or sulcus) and
Bacterial Sampling for Cultural Studies adjacent tooth surface. The calcium alginate fibers
The sample-taking method described below has been trapped the loosened microorganisms. The barbed
7
used for sampling periodontosis, rapidly-advancing peri­ broach was withdrawn back into the needle and the entire
11 12
odontitis in adults, periapical abscesses and normal apparatus was removed (The aluminum foil cap was
13
individuals. This device permits reasonably precise later removed by means of a scaler). The barbed broach
localization of the sample site and removal of samples was introduced into a prereduced anaerobically-sterilized
with limited contamination from adjacent areas. Bacte­ medium containing 1% sodium metaphosphate in one-
 Volume 47
Number 7 Periodontosis Microbiology 375
quarter strength Ringers solution which was continu­
ously gassed with an oxygen-free gas mixture. The sam­
ple was dispersed, subsequently diluted, plated and proc­
essed as described below. The sample depths were deter­
mined by comparing millimeter markings on the needle
barrel and the barbed broach while the apparatus was in
place. A second recording of sample depth was made af­
ter the sample was taken using a calibrated periodontal
probe. In this way, localization of the sample at the most
apical extent of each site could be verified.

Cultural Techniques
The microbiological techniques including conditions of
cultivation, purification, and identification of isolates
7
have been described by Newman and Socransky, Man-
14 15 16
ganiello, Manganiello, et al., Socransky, et al.,
17 18
Darwish, Hyppa and Socransky and Savitt, et a l .
FIGURE 3. Diagrammatic representation of sample-taking de­
Pathogenic Potential vice in a deep periodontal pocket. Magnified view depicts ap­
proximate relationship of fiber-wound barbed broach to tooth
The pathogenic potential of strains N-21 and N-27 of
surface.
gram-negative anaerobic rod groups II and III respec­
tively were tested in germ-free rats using methods
8
described by Irving et al. and Socransky, et a l . The 19
RESULTS
strains chosen for study, supplemented previously re­ Total Cultivable Flora
7 8
ported data ' so that representatives of all five groups of
"periodontosis gram-negative anaerobic rods" were stud­ The predominant cultivable microbiota of periodonto­
ied. sis lesions and control sites in some of the same
periodontosis patients, normal sites in noninvolved sib­
lings and in normal adolescents are shown in Table 1.
S A M P L E T A K I N G DEVICE
The data are expressed as the mean percent of the total
cultivable flora according to standard classification
groups.
The microbiota of the normal sites in all patients
consisted mainly of gram-positive organisms. In some of
the samples these organisms were shown to include
strains of Streptococcus sanguis and Streptococcus mitis,
Staphylococcus epidermidis and large numbers of gram-
positive filamentous rods which-resembled Actinomyces
viscosus, and Propionibacterium acnes. In contrast, the
microbiota from diseased sites had an increased propor­
tion of gram-negative anaerobic rods coupled with a
proportionate decrease in the gram-positive facultative
cocci and rods. Though variability occurred from site to
site in the normal flora as well as the pathologic flora, the
pattern (trend) of distribution of gram-positive faculta­
tive and gram-negative anaerobic organisms was surpris­
ingly consistent. It should be pointed out that the
reported number of gram-negative anaerobes was proba­
bly lower than the actual number since these organisms
20 2 1
are more sensitive to the sonic oscillation procedures -
than the gram-positive flora.
The gram-negative anaerobic rods in the periodontosis
lesions made up a mean of 55% of the cultivated
microbiota. Ten percent of this flora included strains of
Bacteroides corrodens, Campylobacter sputorum, Sele-
nomonas sputigena, Fusobacterium nucleatum, and Bac­
FIGURE 2. Construction of sample-taking device, magnified teroides melaninogenicus. The majority of gram-negative
view depicts aluminum foil cap in place. anaerobic rod isolates (45% of total organisms culti-
 J. Periodontol.
376 Newman, Socransky, Savitt, Propas, Crawford July, 1976

TABLE 1. Predominant Cultivable Microbiota from 20 Peiodontosis and 10 Nonperiodontosis Patients

{Mean Percent)

Periodontosis Non Periodontosis

Lesion Control Siblings Adolescents

Total Gram P o s i t i v e 37 78 41 63

Total Gram Negative 63 22 59 37

Gram Negative Anaerobic Rods 55* 10 45 24

Selenomonas, C. sputorum
F. nucleatum, B. melaninogenicus 10 8 31 13

"Five Periodontosis Groups" 45 2 14 11

Spirochetes Few Few Few Few

Patients Examined 20 7 5 5

Number of Sites Sampled 34 7 10 10

Sample Depth (mm) 8(±2) 2(±.5) 1.5(±.5) 2(±.5)

•Standard e r r o r of mean = ±15%

vated) from the diseased sites were not identifiable on the TABLE 2. Frequency of Detection of
22 25
basis of current classification schemes. " The microbi­ Gram-Negative Anaerobic Rods in Periodontosis
Lesions
ota from these sites consisted primarily of gram-negative
7
anaerobic rods described by Newman and Socransky as Percent Lesions Number of Groups Detected
the "five periodontosis groups." These organisms were
13 one group
placed into groups based on morphologic and physiologic
19 two groups
criteria. Group I consisted of anaerobic saccharolytic
vibrios. Group II organisms included the "Capnocyto- 31 three groups

phaga" species discussed below, Group III isolates were 37 four groups

tiny gram-negative anaerobic rods (0.3 x 0.8 p). Group 0 five groups
IV organisms were gram-negative saccharolytic Bac-
teroides-like organisms and Group V consisted of TABLE 3. Occurrence of Gram-Negative Anaerobic Rods in
gram-negative anaerobic surface translocating mi­ Periodontosis Lesions
croorganisms with differing morphological and bio­ c
Percent of Lesions Mean ' when Percent Group
chemical characteristics from the "Capnocytophaga" Group Isolated Isolated Predominated
(Group II) species.
Group I 50 7 13
In contrast to the predominance of the five periodonto­
II 88 17 38
sis groups in the lesions, the unclassified gram-negative
anaerobic rods were found to make up 14% of the total III 63 7 25

microbial flora in the normal sites in the noninvolved IV 50 12 19

siblings and 11 % of the flora in patients without peri­
V 44 8 6
odontosis.
Low numbers of small spirochetes were detected
periodontosis lesions. The most common organisms de­
microscopically and culturally from lesions and normal
tected were of Groups II and IV. Both the frequency of
sites. In the lesions Treponema denticola and Treponema
26
isolation and the mean percentage of the organisms were
macrodentium were identified. These spirochetes com­
higher than other groups averaging 17 and 12% of the to­
prised a small percentage of the total flora.
tal cultivable microorganisms resident at the base of the
The frequency of detection of gram-negative anaerobic sampled pocket. Organisms similar to those detected in
rods in the periodontosis lesions is shown in Table 2. It periodontosis lesions also could be isolated from nonperi­
could be demonstrated that 1 or more of gram-negative odontosis patients but in low numbers. In the nonin­
anaerobic rod groups were present in all 20 periodontosis volved siblings "Capnocytophaga", Group II, averaged
patients studied. In none of the patients could all five 5% of the total cultivable flora, and in the adolescents
groups be detected. The most common situation was less than 3% of the total cultivable flora. In the control
three or four of the five major types being present in sites in periodontosis patients, Group II organisms were
individual patients. In approximately 13% of the sites less than 1 % of the isolates.
studied, only one group of gram-negative anaerobic rods
was detected, usually Group III and IV organisms or the Pathogenic Potential
"Capnocytophaga" (Group II) species. Table 3 shows The strains of Groups II and III initiated a form of
the occurrence of gram-negative anaerobic rods in the periodontal destruction similar to that observed in previ-
Volume 47
Number 7 Periodontosis Microbiology 377
7,8
ous studies. The changes occurring in rats monoin- The distribution of microorganisms seen in periodon­
fected with these organisms differed remarkably from tosis siblings is similar to previously reported data for the
30
changes seen previously using gram-positive organisms same aged children with the exception of thefindingof
as monocontaminants. Infection by the two strains tested low numbers of "Capnocytophaga" strains. This discrep­
resulted in minimal plaque formation, undetectable root ancy is most likely due to the different cultural tech­
surface caries, and extensive alveolar bone destruction. niques employed in the two studies.
The finding of large numbers of gram-negative
DISCUSSION
anaerobic rods such as Bacteroides melaninogenicus
The finding of large numbers of specific types of in the preadolescent siblings, also agrees with the pre­
gram-negative anaerobic rods in periodontosis lesions viously reported results. In one 6-year-old patient in
and their pathogenic potential in animals, suggest the our study, B. melaninogenicus accounted for 38% of the
possibility that these organisms may contribute to the total flora in one sample located adjacent to a recently
27
pathology seen in periodontosis. Recently, Tanzer has erupted first molar.
reported finding high numbers of gram-negative anaero­
bic rods in other periodontosis patients. Crawford, et Plaque Morphology
11
al. demonstrated the presence of large numbers of Of major importance in understanding the coloniza­
gram-negative anaerobic rods in patients diagnosed as tion of microorganisms in oral ecologic sites, has been
having rapidly-advancing periodontitis. These patients, the morphologic description of plaque and plaque
who were older than 30 years of age, demonstrated more development. 31

than 50% bone loss on individual teeth. Sites in which Light and electron microscopic observation of ex­
rapid destruction had taken place (greater than 2 mm of tracted teeth and adjacent tissues from some of the same
bone loss) in the previous year were determined by serial patients from which microbiologic samples were ob­
radiographs. Anaerobic sampling and cultural techniques tained revealed a minimum amount of attached bacterial
similar to those used in the periodontosis studies were plaque on the tooth surface near the apical portions of
used. The microorganisms isolated in this study were 32 4 0
the periodontosis pockets. ' (Fig. 4). When the plaque
extensively characterized and were shown to consist was attached to the tooth surface, it was usually located
primarily of asaccharolytic gram-negative anaerobic close to the cemento-enamel junction. The plaque in the
rods differing from the saccharolytic organisms consist­ apical portion of the pocket appeared to be unattached to
ently found in periodontosis. These organisms comprised the tooth surface. In addition, fewer morphologic forms
from 48 to 92% of the total cultivable flora. were seen when compared to the microbiota associated
Although studies to date have been limited, there is a with gingivitis or periodontitis. In comparison, plaque
suggestion that the microbiota at the apical portion of associated with these clinical conditions is complex,
both periodontosis and advanced periodontitis lesions heterogeneous, and at least a portion appears to be firmly
was made up predominantly of gram-negative anaerobic attached to the tooth surface. Organisms in the advanc­
rods. The periodontosis microbiota is characterized by ing front of the periodontosis lesions had cell wall struc-
saccharolytic microorganisms of five distinct groups. The
advanced periodontitis microbiota was characterized by
the presence of large numbers of asaccharolytic microor­
ganisms including Fusobacterium nucleatum, Bacter-
oides melaninogenicus, Eikenella corrodens, Bacteroides
corrodens, Bacteroides capillosus and anaerobic vibrios.
The finding of large numbers of "Capnocytophaga"
organisms in periodontosis lesions and their subsequent
identification in nonpathologic sites in nonperiodontosis
patients promoted an intensive taxonomic study of these
18,28 29
organisms by several investigators. ' A total of 66
isolates from periodontosis lesions, advanced periodonti­
tis lesions and supragingival dental plaque were studied.
The results of the investigation demonstrated that the
organisms were significantly different from existing
genera and thus a new genus was proposed and called
"Capnocytophaga" These organisms required C 0 for 2

growth, produced acetate and propionate as major acid FIGURE 4. Artists conception of periodontosis pocket with
approximate proportion of attached {diagonal lines) vs. unat­
end products, most were sensitive to Actinomycin D , and
tached plaque (dots). Histologic sections of plaque demon­
were saccharolytic. In addition, the fusiform-shaped non- strate the sparsely colonized tooth surface. The unattached
flagellated microorganisms have the ability to "surface plaque is surrounded by polymorphonuclear leukocytes from
translocate" or "glide" over agar surfaces. the adjacent soft tissue. (Courtesy Dr. Max Listgarten).
 J. Periodontol.
378 Newman, Socransky, Savitt, Propas, Crawford July, 1976

tures consistent with those described for gram-negative trast to the results of 14 previously tested gram-negative
organisms as determined by electron microscopy. ' 32 4 0
isolates which failed to show pathogenic potential. These
included one or more strains of Veillonella parvula,
Host-Bacterial Interactions—Immunologic Veillonella alcalescens, Fusobacterium nucleatum, Cam­
Considerations pylobacter sputorum, Escherichia coli, Selenomonas
Until recently there have been few systematic reports sputigena, Bacteroides melaninogenicus, Bacteroides or­
directly concerned with the immunologic aspects of alis, Treponema macrodentium, and Treponema
periodontosis. In one report, cell mediated and humoral denticola. 36

immunologic investigations were carried out by Lehner, The histopathologic changes occurring in rats monoin-
33
et al., in 23 patients diagnosed as having periodontosis. fected with gram-positive organisms were shown to be
Periodontosis patients had significantly increased con­ distinctly different from changes induced by the
centrations of serum IgG, IgM, and IgA compared to 37 38
gram-negative organisms. ' Strains of Actinomyces
matched controls. The level of IgM increased to a naeslundii, Actinomyces viscosus and Streptococcus
relatively greater extent than that of IgG and IgA. It was mutans when implanted as monocontaminants in gnoto­
postulated that the increase in IgM may reflect a biotic rats formed massive amounts of plaque, caused
response to gram-negative organisms. Whether the im­ root caries, and led to extensive resorption of alveolar
munoglobulin response was induced by bacteria "caus­ bone. The disappearance of alveolar bone was associated
ing" the disease or by microorganisms inhabiting the with a decrease in number of osteoblasts; and osteoclasts
lesion sites subsequent to bone loss was not clear. In the were not noticeably increased in number in comparison
study, lymphocytes from periodontosis patients failed to to germ-free controls. In contrast, infection with
respond (transform) to plaque and selected gram-nega­ gram-negative organisms resulted in minimal plaque
tive bacteria in spite of intact macrophage migration formation and no root caries, but extensive loss of
inhibition. These and other findings led the authors to alveolar bone which was accompanied by a marked
postulate a selective cell mediated immunodeficiency 8
osteoclastic response. ' 3 9

which would result in an abnormal defense reaction to Recent evidence has suggested that gram-negative
specific bacteria. anaerobic rods may play a significant role in the rapidly
34
In another immunologic study, Kaslick et al. demon­ destructive periodontal diseases in man. The circumstan­
strated that patients with periodontosis had a lower tial evidence of the location of these organisms at the site
incidence of human leukocyte antigen, H L - A , present
2 of rapid periodontal breakdown, their pathogenicity in
on the surface of lymphocytes using a microdroplet animal model systems, and their potential to initiate
lymphocyte cytotoxicity test to identify H L - A antigens. osteoclastic destruction suggest that this group of mi­
These authors suggested that H L - A antigen may be a
2 croorganisms may be of major significance.
means to identify susceptible individuals. The concept of bacterial specificity suggests that
Future immunologic studies will undoubtedly elucidate periodontal disease may be a group of diseases with
important aspects of the disease process. The recognition different etiologies and clinical courses but with similar
of a specific resident microbiota to a specific periodontal clinical symptomatology. With the recognition of a
disease process will allow future investigators to more diverse group of etiologic agents, more accurate clinical
accurately assess the response of the host to potentially and laboratory diagnosis could be made. Our inabilities
pathogenic bacteria and their products. to separate such disease entities at the present time
reflects a lack of clinical diagnostic acumen, as well as an
Pathogenic Potential
insufficient basic understanding of periodontal disease
Additional information regarding microbial specificity pathogenesis. Accurate identification of organisms is a
in periodontal disease may be gained from animal model prerequisite to future studies.
systems. It should be stressed that only certain microor­
ganisms can cause disease in a particular animal model. ACKNOWLEDGEMENTS
Representative isolates from each of the five predomi­ Appreciation is expressed to: R. Kaslick, T. West, A .
nant cultivable gram-negative anaerobic rod groups Chasens, E. Ledbetter, S. Holt, S. Sapiro, J. Stolman, R.
found in the advancing front of periodontosis lesions Lobene, R. Pollack, R. Barbanell, L . Butler, C. Lai, S.
were pathogenic in germ-free rats. In a recent study by Darwish, and S. L. Newman whose cooperation helped make
35 these studies possible.
Crawford and Socransky, two isolates from the numer­
ically dominant asaccharolytic gram-negative rods from REFERENCES
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