Intemationai Endodontic Journal (1985) 18, 35-tO
The antibacterial action of sodium hypochlorite and EDTA in
60 cases of endodontic therapy
A. B Y S T R O M & G. S U N D Q V I S T Departments of Endodontus and Oral Microbiology, University
of Umed, Sweden
Summary. In this study the antibacterial effect of
irrigating infected root canals with 0.5 and 5 per
cent sodium hypochlorite solutions was evaluated
clinically. The results indicated that there was no
difference between the antihacterial effect of these
two solutions. The combined use of EDTA and
5 per cent sodium hypochiorite solution was more
efficient than the use of sodium hypochiorite
solutions alone.
An important observation was that bacteria
surviving instrumentation and irrigation rapidly
increased in number in the period between appoint-
ments when no intracanal medicament was used.
Introduction
Endodontic treatment includes the cleansing
of pulp tissue and bacteria from infected root
canals. This is performed mechanically by files
and supported by irrigation witb antibacterial
and tissue dissoKing solutiotis. Sodium hypo-
chiorite Js the most widely used component of
these irrigating solutiotis. Sodium hypochlorite
has a good antimicrobia! effect in vitro (Shih
etal. 1970, Bioomfield & Miles 1979, Foleyrt
al. 1983), a good tissue dissolving capacity
{The 1979, Hatid et al. 1978, Koskinen et al.
1980, Mower & Wesselink 1982) at»d a low
irritant effect on tissues (The et al. 1980,
Lamersrta/. 1980).
The cleansing effect of irrigation with
sodium hypochlorite solution has recently
been studied by scannitig electron microscopy.
Some of these studies indicate that irrigation
with sodium hypochlorite solutions does not
dean oi^anic matter and debris from the canal
efficiently (Baker et al. 1975, Bolanos &
Correspondence: A. Bystrfim, Department of
Endodontics, University of Umei, S-901 87 Umea,
Sweden.
Jensen 1980), while other studies show that
irrigation with sodium hypochlorite solutions is
highly effective in this respect (McComb &
Smith 1975, McComb etal. 1976, Goldman et
al. 1981, 1982). The difference can most likely
be explained by the various amounts and con-
centrations of sodium hypochlorite that were
used. In the studies reporting good cleansing
effect concentrations of 5 per cent or higher
were used, while a 1 per cent concentration
was used in the other studies.
During the instjuimentation of the root canal
a smeared iayer of mainly inorgamc matter is
formed on the canal walls. It has been con-
sidered important to prevent the formation of
this layer (Goldberg & Abramovich 1977).
Sodium hypochlorite acts on organic matter
but has very little effect on this layer (Baker
et al. 1975, McComb & Smith 1975, McComb
etd. 1976, Goldman rta^. 1981,1982, Bolanos
& Jensen 1980). The smeared layer may,
however, be removed by the chelating agent
ethylene diamine tetra-acetic acid (EDTA)
and solutions containing EDTA have been
recommended for irrigation (McComb &
Smith 1975, McComb et al. 1976, Goldberg
& Abramovich 1977, Goldberg & Spielberg
1982). Irrigation with both sodium hypo-
chlorite and EDTA solutions has been reaim-
mended to remove the smeared layer and
organic matter in the canal (Goldman et al.
1982).
The scanning electron microscopic studies
have, however, given very sparse infonnation
concerning the antibacterial efficiency of irri-
gation with these solutions. In the present
study the clinical efficiency of sodium hypo-
chlorite and EDTA as components of irrigating
solutions was evaluated by bacteriological
methods.
35
36 A. BystrUm IS G. Sundqvist
Material and methods
Clinical material
Sixty single-rooted teeth with necrotic pulps,
intact pulp chamber walls and radiographic
evidence of periapical bone destruction were
studied. In 20 root canals 0.5 per cent sodium
hypochlorite solution (pH 9) was used for irri-
gation, and in another 20 canals 5 per cent
sodium hypochlorite solution (pH 9) was used,
and in a further 20 canals both a S per cent
sodium bypochlorite solution and a 15 per
cent EDTA solution (pH 8) were used for irri-
gation. The results from 15 ofthe teeth treated
with 0.5 per cent sodium hypochlorite sol-
ution have already been published (Bystrom
& Sundqvist 1983). Radiographic examination
was carried out using the long-cone technique
(Oralix 65, Philips) with Kodak ultra-speed
film (24x36 mm) in a filmbolder (Eggen
1974).
Procedure to obtain bacterial samples fiom the root
canals
The procedure to obtain samples from the root
canals was the same as previously reported
(Bystrom & Sundqvist 1981, 1983). An asep-
tic technique was used throughout treatment.
Before the puip chambers were opened control
samples were taken to test sterility. The root
canals were enlarged and samples from the
canals were taken with charcoaled paper points
(Moller i%6). The sampling fluid was sucked
up into the paper points and these were trans-
ferred to a tube with 5 ml anaerobic peptone-
yeast extract-glucose (PYG) broth (Holdeman
et al. 1977). Precautions were taken to
avoid oxygen contamination of the PYG-
broth (Fulghum 1971). The root canals were
thoroughly instrumented for 15 minutes and
during this treatment repeatedly irrigated with
sodium hypochiorite solution; the total volume
was 6 ml. The inigwit was introduced into the
canal by a syringe with a 23 gauge needle
and removed by an ejector. When the root
canals were treated with both sodium hypo-
chlorite and EDTA solutions, the treatment
was initiated by instrumentation and irrigation
with sodium hypochlorite solution for 10
minutes. Thereafter, an EDTA solution was
applied to the canals and repeatedly changed
for 10 minutes. Finally the canals were irri-
gated with sodium hypochlorite solution for 5
minutes. To inactivate remnants of sodium
hypochlorite, the canal was finally rinsed with
a 5 per cent sodium thiosutphate solution
(Moller 1966). After this treatment the access
cavity was sealed with zinc oxide-eugenol
cement. No dressing was used in the canals.
At the second appointment a sample was
taken from the canal before the canal was
instrumented and irrigated. At the third
appointment a sample was taken before the
root canal was dressed with calcium hydroxide
paste (Calxyl'). The interval between the
appointments was 2—4 days.
Microbiological examination ofthe specimens
The PYG-broth tube with the specimen was
introduced into an anaerobic box (Sundqvist
1976) and treated in accordance with the
method in earlier studies (Bj'strom &
Sundqvist 1981, 1983). The tube, which con-
tained glass beads, was agitated in a mechan-
ical mixer until the paper points disintegrated
and 10-fold serial dilutions to 10' were made
in dilution blanks (Holdeman « al. 1977).
Aliquots of 0.5, 0.2, and 0.1 ml from the PYG
broth and atiquots of 0.1 ml from each of
the dilutions were inoculated onto duplicate
blood agar plates (Holdeman et al. 1977), one
mitis saiivarius agar plate (Difco 0298-01)
and one Rogosa SL medium plate (Difco
0480-01). One set of plates was incubated for
10 days in the box in an atmosphere of 5 per
cent COj, 10 per cent Hj in nitrogen at 37°C
and the other set of blood agar plates
aerobically for 48 hours at 37°C.
Identification tif bacterial strains
Samples taken at the first and second appoint-
ment were analysed solely for number of
bacterial cells and no identification was made
of the isolated bacteria. Bacterial strains iso-
lated at the third appointment were identified
(Bystrom & Sundqvist 1981, 1983).
Results
Bacteria were found in all initial specimens
from the root canals. The median value of the
number of bacterial cells prraent in the initial
•Scanii Dental AB, Knivsta, Sweden.

APPOINTMENT
Fig. 1. Recovery of bacteria from samples taken at
the beginning of each of three appointments. The
dark part of the bars represent the number of root
canals from which bacteria were recovered; light
parts the number of canals with no recovery of bac-
teria at the appointment. 0.5 and 5.0: canals irri-
gated with 0.5 per cent and 5 per cent sodium
hypochlorite solution, respectively. E: canals irri-
gated with 5 per cent sodium hypochlorite and
EDTA solutions.
specimens from canals before treatment with
0.5 per cent sodium hypochlorite solution was
1.6x10' (range 8 x l'O^-2.5 x ]0»). Corres-
ponding figure for teeth treated with 5 per
cent sodium hypochlorite solution was 3 x 1 0 '
(range 6 X IO'-2 x 10') and for canals treated
with 5 per cent sodium hvpochlorite solution
and EDTA 3 x 1 0 ' (range 'l X 10'-1.6 X 10').
After the first appointment ejcacerbations
occurred in 12 teeth, four from each group.
At the second appointment bacteria were
recovered from 12,10 and 11 ofthe root canals
in the respective groups (Fig. 1). At the third
appointment bacteria were recovered from
eight, six and three of the root canals in the
respective groups (Fig. 1). The median number
of baaerial ceils present at the third appoint-
ment was 2X 10' (range 8 X W-l.Sx !0*) in
specimens from root canals treated with 0.5
per cent sodium hypochlorite, ! x l C (range
1.4 X10*-2 X 10') for canals treated with 5 per
cent sodium hypochlraite, and 8 X 10' (range
1 X 10*-2 X 10*) for canals treated with 5 per
cent sodium hypochlorite and EDTA. Bacteria
were never recovered from specimens obtained
during the operative procedure of preparing
the entrance to the prfp chambers.
Antibacterial actton of NaOCl and EDTA 37
The 44 bacterial strains that were recovered
at the third appointment are presetited in
Table I. Eighty per cent of the strains were
anaerobic; most could be classified to the
species level. However, a group of non-motile,
non-saccharolytic, small. Gram-negative, non-
pigmenting rods which produced a small
amount of butyTic acid could only be identified
as Fusobacterium species, and other strains
whicb produced minor amounts of acetic and
succinic acid as Bacteroides species. Also atuer-
obic strains producing lactic acid could only be
identified as Lactohacillus species. There was
no indication that any specific bacteria were
resistant to the treatment.
Discussion
Tbe mechanical instrumentation of root canals
has been considered one of the most important
phases in endodontic treatment (Grossman
1981, Ingle et al. 1976, SchUder 1976). In a
previous study (Bystrom & Sundqvist 1981)
mechanical instrumentation and irrigation with
saline significantly reduced the number of
bacteria in the root canal but in half the cases
bacteria remained in the canals after four
treatments and it was concluded tbat a sup-
porting action of a disinfectant is necessary
for successful extermination of living bacteria
from root canals.
Antibacterial solutions have been used in
treatment for irrigation of canals and as dress-
ings in canals between appointments. In the
present study the antibacterial effect of irri-
gation with 0.5 per cent and 5 per cent sodium
hypochiorite solutions was evaluated clinically.
The results indicate that there was no differ-
ence between the antibacterial effect of these
two solutions. These results are in accordance
with those of Cvek et al. (1976) who could not
demonstrate any significant difference in anti-
bacterial effect between 0.5 per cent and 5 per
cent sodium hypochlorite solutions in a clinical
study.
Hypochlorite reacts with organic debris in
the root canal and in that way facilitates clean-
ing; however, this reaction inactivates tbe
hypocblorite and reduces its antibacterial
capacity. Therefore, fresh hypochlorite sol-
ution should be applied frequently into the
root canal. The results in thfe study indirated.
 A Bystrdm iS G. Sund/pisi

Tabte I. Bacterial strains persisting at the third appointment in eight root canals treated with
0.5 per cent NaOQ, six treated with 5 per cent NaOQ and three root canals treated with 5
per cent NaOQ and EDTA

0.5 per cent 5 per cent 5 per cent

Bacterial species N a O a & EDTA

Streptocoeem miileri
Streptococcus mutans
Streptococcus sanguis
Streptococcus intermedius
Streptococcus morkllorum

Peptostreptococcus aaaerohius
Peptostreptococcus micros

Arachnia propionica
Euhacterium alactotyticum
Eubacterium brachy
Eubacterium lentum
Euhacterium timidum

Lactohacillus sp.'
Lactobacillus sp. group 1>

Fusohacterium sp.
Pusobacterium nucleatum

Bacteroides sp.
Bacteroides gingivalis
Bacteroides intermedius
Bacteroides oralis

Capnocytopkaga ochracea
Wolmdla recta

'These anaerobic strains do not fit into recognized species. Group 1 corresponds to strains
classified by numerical taxonomy (Sundqvist 1976).

however, that in spite of frequent application
5 per cent sodium hypochiorite solution did
not exterminate ail bacteria frotn mfected root
canals. The sodium hypochloHte solution may
not have penetrated Into confined areas of the
root canal (Senia rtai 1971, Bakerrta/. 1975,
McComb et al. 1976, Rosenfeld et al, 1978,
Rubin <?fl/. 1979).
When the cleaning and debriding properties
of irrigating solutions have been evaluated
in scanning electron microscopy it has been
found that instrumentation produced a smeared
layer on the dentinai wails, which may com-
pletely obturate the dentinat tubules (Baker et
al 1975, McComb et al 1976, Lester & Boyde
1977, Rubin « al 1979). This layer may con-
tain bacteria and also prevent the penetration
of the antibacterial solutions into infected
dentinal tubules (Chimside 1958, Shovelton
1964). By including EDTA in the irrigating
solution the formaticm of the smeared layer
may be prevented but the clinical significance
of this has not been proved (McComb et al.
1976, Goldman rtai 1981,1982, Goldberg &
Abramowich 1977, Bolanos &Jettsen, 1980).
The present study indicated that the combined
use of EDTA and 5 per cent sodium hypo-
chlodte solutions was mwe efficient than the
use of sodium hypochlcnite solutions done, but
none the less three out of 20 erf the treated

canals contained living bacteria at the third
appointment (Fig. 1). Recent results indicate
that decreasing the surface tension of irri-
gadng solutions by adding a detergent (Abou-
Rass & Patonai 1982, Cunningham et al.
1982a, or the use of ultrasound may improve
the efficiency of treatment. The use of ultra-
sound has been shown to improve the cleaning
of the canal by removing the smeared layer,
and by increasing the tissue-dissolving and
antimicrohial effects of sodium hypochlorite
(Cunningham et al 1982b, c, Cameron 1983,
Moorer & Wesselink 1982).
An important observation in this and earlier
studies (Bystrom & Sundqvist 1981, 1983)
was that bacteria surviving instrumentadon
and irrigation rapidly increased in number in
the empty root canals in the pyeriod between
appointments. Similar results have been
obtained in other studies in which no intra-
canal medicament was used between appoint-
ments (Stewart rta/. 1969, Bence etal. 1973).
Therefore, the efficiency of antimicrobial
agents as inter-treatment dressings should be
evaluated.
References
ABOU-R*SS, M . & P*TONM, F.J. (1982) The effects
of decreasing surface tension on theflowof irri-
gating solutions in narrow root canals. Oral
Surgery. Oral Medicine and Oral Pathology. 53,
524-526.
BAKER, N.A., ELEAZER, P.D., AVERBACH, R . E . &
SELTZER, S . (1975) Scanning electron micro-
scopic study of the efficacy of various irrigating
si:i\tAon&. JoumtU of Endedontics. 1, 127-135.
BENCE, R . , MADONIA, J.V., WEINE, F . S . & SMULSON,
M.H. (1973) A microbiologic evaluation of endo-
dontic instrumentation in puipless teeth. Oral
Surgery, Oral Medicine and Oral Pathology. 35,
676-683.
BLOOMFIELD, S . F . & MILES, G.A. (1979) The anti-
bacterial properties of sodiuin dichloroisocyan-
urate and sodium hypochlorite formulations.
Journal of Applied Bacteriology, 46, 65-73.
EkiLANos, O.R. & JENSEN, J.R. (1980) Scanning
electron microscope comparisons of the efficacy
of various methods of root canal preparation.
Journal of Endodontics, 6, 815-822.
BYSTROM, A. & SUNDQVIST, G . (1981) Bacteriologic
evaluation of the efficacy of mechanical root canal
instrumentation in endodontic therapy. Scandin-
avian Journal of Denial Research, 89, 321-328.
Antibacterial action ofNaOCland EDTA 39
BYSTROM, A. & SUNDQVIST, G . (1983) Bacteriologic
evaluation of the effect of 0.5 per cent sodium
hypochlorite in endodondc therapy. Oral Surgery,
Oral Medicine and Oral Pathology, 55, 307-312.
CAMERON, J.A. (1983) The use of ultrasonics in the
removal of the smear layer: a scanning electron
microscope study. Journal of Endodontics. 9,
289-292.
CHiRNsniE, l.M. (1958) The bacteridogical status of
dentine around infected pulp canals. A'np
Zealand Dental Journal, 54, 173-183.
CUNNINGHAM, W.T., COLE, J . S . & BAUEKJIANT, A.Y.
(1982a) Effect of alcohol on the spreading abilit)'
of sodium hypochlorite endodontic irrigant. Oral
Surgery, Oral Medicine and Oral Pathology, 54,
333-335.
CUNNINGHAM, W.T., MAIITIN, H . & FORREST, W . R .
(1982b) Evaluation of root canal debridement by
the endosonic ultrasonic synergistic system. Oral
Surgery, Oral Maiicinr and Oral Pathology, S3,
401W4
CUNNINGHAM, W T . , MARTIN, H . , PELLEU, G . B . &
STOOPS, D . E . (1982C) A comparison of anti-
microbial effectiveness of endosonic and hand
root cana! therapy. Oral Surgery. Oral Medicine
and Oral Pathology. 54, 238-241.
CVEK, M . , NOIID, C - E . & HOLLENDER, L . (1976)
Antimicrobial effect of root canai debridement in
teeth with immature root. A clinical and micro-
bioiogic study. Odontoiogisk Revy, 27, 1—10.
EcGEN, S. (1974) Rontgenografiske tannnialinger i
dagUg praksis. Tandlakartidningen, 66, 10-12.
FoLEV, D.B., WEINE, F.S., HAOEN, J.C. &
DEOBMUUO, j.J. (1983) Effectiveness of selected
irrigants in the elimisation of Bacteroides melani-
nogenicus from the root canal system: an in vitro
study. Journal ofEndodontics, 9, 236-241.
FULGHUM, R.S. (1971) Mobile anaerobe laboratory.
Applied MicrMology. 21, 769-770.
GOLDBERG, F . & ABRAMOVICH, A. (1977) Analysis of
the effect of EDTAC on the dentinai walls of the
root canal. Journal ofEndodontics, 3, 101-105.
GoLt>BERG, F. & SpiELffiRG, C. (1982) The effect of
EDTAC and the variation of its working time
analj'zed with scanning electron microscopy.
Oral Surgery. Oral Medicine and Oral Pathology.
53, 74-77.'
GOLDMAN, L.B., GOLDMAN, M . , KRONMAN, J . H . &
LIN, P.S. (1981) The efficacy of several irrigating
solutions for endodondcs: a scanning electron
microscopic study. Oral Surgery. Oral Medidne
and Oral Pathology, 52, 197-204.
GOLDMAN, M . , GOLDMAN, L.B., CAVALEIU, R., BOGIS,
J. & LIN, P.S. (1982) The efficacy of several
endodontic irrigating solutions: a scanning
electron microscopic study: part 2. Journal of
Endodontics, 8, 487-492.
40 A. BystrSm & G. Sundqvist
GKOSSMAN, L.I. (198!) Endodmtic Practice. 10th
edn, p. 200. Lea & Febiger, PhfladelphU.
HAND, R.E., SHOTH, M . L . & HAWBSON, J.W. (1978)
Analysis of the effect of dilution on the necrotic
tissue dissolution property of sodium hypochlor-
ite. Joamal sfEndodonHa, 4, 60-64.
HoLDEMAN, L.V., a i o , E.P. & MooKE, W.E.C.
(1977) Anaerobe Laboratory .Manual, 4th edn,
VPI AnaenAe Laboratorj, Virginia Polytechnic
Institute and State University, Blacksburg, Va.
INGLE, J.I., BEVEBIDGE, E.E., LUEBKE, R.G., WALTON,
R.E. & ZiDELL, J.D. (1976) In Endodontics (eds
J. I. Ingle & E. E. Beveridge), 2nd edn, pp.
101-215. Lea & Febiger, Philadelphia.
KOSKINEN, K . P . , SlENVALL, H . & UnTO, V-J.
(1980). Dissolution of bovine pulp tissue by
endodontic solutions. Scandinavian Journal of
Dental Rtsmrch, 88, 406-411.
LAMEHS, A . C , VAN MuLtxM, P.J. & SIMON, M .
(1980) Tissue reactions to sodium hypochlorite
and iodine potassium iodide under clinical con-
ditions in monkey teeth. Journal of Endedontics.
6, 788-792.
LKIER, K . S . & BoYDE, A. (1977) Scanning electron
microscopy of instrumented, irrigated and filled
root canals. British Dental Jounml, 143, 359-367.
McCoMB, D. & SkUTH, D.C. (1975) A preliminary
scanning electron microscopic study of root
canals after endodontic procedures. Journal of
EndodoHtics, I, 238-242.
McCoMB, D., SuflTH, D.C. & BEAGHE, G . S . (1976)
The results <^in vivo endodontic chemomechani-
cal mstrumentation—A scanning electron micro-
scopic study. Journal of the British Endodontic
Society, 9, 11-18.
MooBM, W.R. & WESSELINK, P.R. (1982) Factors
promoting the tissue dissolving capability of
sodium hypochlorite. International Endodontic
Journal, 15, 187-1%.
MoLLER, A.J.R. (1966) Microbiological examination
of root canals and ptriafncal tissues of human teeth.
TTiesis, AkidemifSrkget, Gothenburg.
D, E.F., JAMES, G . A . & BURCH, B . S .
(1978) Vitai pulp tissue response to sodium
hypochlorite./(iBm«/o/£m<a(ioB*i«, 4, 140-146.
RuwN, L.M., SKOBE, Z . , KRAKOW, A.A. & GHON, P.
(1979) The effect of instrumentation and flushing
of freshly extracted teeth in endodontic therapy:
i scanning electron microscope study. Journal of
Endodontics. 5, 328-335.
ScHiLDui, H. (1976) Casal d^bridement and disin-
fection. In Pathmiys of the Pulp (eds S. Cohen
and R. C. Bums)^ pp. 111-133. C. V. Mosby
Company, St Louis.
SEMA, E.S., MMISHALL, F . J . & ROSEN, S. (1971)
The solvetst action of sodium hypochlorite on
pulp tissue of extracted teeth. Oral Surgery, Oral
Medicine and Oral Pathology. 31, 96-103.
SMH, M . , MARSHALL, F.J. & ROSEN, S . (1970) The
bactericidal efficiency of sodium hy^iochlorite
as an endodontic irrigant. Oral Surgery, Oral
Medicine and Oral Pathology. 29, 6i3-619.
SHOVELTON, D . S . (1964) The presence and distri-
bution of microorganisms within non-vital teeth.
BrUish Dental Journal, 117, 101-107.
STEWART, G.G., KAPSIMALIS, P. & RAPPAPORT, H .
(1969) EDTA and urea peroxide for root canal
preparation. Journal of the American Dental
Association, 78,335-338.
SuFTOQUST, G. (1976) Bacteriological studies of
necrotic dental pulps. Ume4 University Odonto-
logical Dissertation No. 7, Universit)' of L'mea,
Sweden.
THE, S O . (1979) The solvent action of sodium
hypochlorite on fixed and unfixed necrotic tissue.
Oral Surgery, Oral Medicine and Oral Pathology.
47, 558-561.
THE, S.D., MALTHA, J.C. Sc PLASSCHAERT, A . J . M .
(1980) Reactions of guinea pig subcutaneous
connective tissue following exposure to sodium
hvpochlorite. Oral Surgery, Oral Medicine and
Oral Pathology, 49, 46(M66.