A number of studies have suggested that apical
enlargement may reduce the microbial flora compared
with instrumentation to smaller file sizes. These studies,
some of which are listed in Table 3, have been
performed under different conditions with divergent
results. On the basis of selected findings, some authors
have advocated that it should then be possible to
complete endodontic treatment in one visit if cleaning
to a large apical size completely eradicates bacteria.
With significant root canal enlargement, it is not
surprising that it might result in a reduction in the
bacterial flora, but it happens at the expense of tooth
structure. Can this method of instrumentation totally
and predictably eliminate bacteria, or is it simply a
further reduction of the overall bacterial load? It should
be appreciated that whilst bacterial reduction is
undoubtedly desirable, the goal of endodontic treat
ment is bacterial eradication. Whenever bacterial cells
survive, there remains a risk that they may multiply and
reach numbers that have the capacity to maintain
periapical inflammation.
It is known that after instrumentation there are very
few remaining cells, which may number less than 10
and special procedures and precautions are required to
recover the remaining bacterial cells. Only a few culture
studies have adopted the measures necessary to recover
these cells. As an example, the steps taken in one of
these studies
for recovery of small numbers of cells
after instrumentation is shown in Table 4. In contrast,
a study cited as showing that increased apical
enlargement results in bacterial elimination claimed
that 89 to 100 per cent of canals were rendered bacteria
free when molars were instrumented to a size 60 and
canines/premolars to a size 80, respectively. The
bacteriological methods for sampling after
instrumentation and culture in that study are shown in
Table 4.
When a sensitive technique is used
the chances are optimized for bacterial recovery.
For example, where three samples are taken and no growth is
seen with one
sample the others can be used as backup and check of
the first sample. An ‘enrichment’ medium, fluid
thioglycolate medium (FTM), was used so that if
colonies did not appear on the plates, new plates were
inoculated from the other PYG broth and the FTM. If
none are positive for bacteria, one can be more assured
of the veracity of a negative result. Even the application
of paper points must be done with utmost care to
maximize recovery of fluid from the root canal. Figure
7 shows two paper points from the same canal. One is
negative and the other is positive for bacteria, because
the left one is thinner and reached further apically in
the canal to capture bacteria. The light microscopic
view of a sectioned tooth root apex shown in Fig 5
illustrates where bacteria may be located and how they
can escape recovery if a paper point does not reach the
full canal length.
Dilution of a sample is another critical step in
bacterial cultivation. The mean number of cells in an
untreated case may be as high as 10 to 10
cfu/ml and
in order to quantify cells and distinguish species from
one another, serial dilution is necessary to obtain about
50–200cfu per agar plate. However, when small
numbers of cells and species are anticipated in a
sample, such as after antimicrobial instrumentation or
filling, the method of dilution is markedly different.
Under these conditions, minimal dilution and large
aliquots of the sample inoculated onto the plate are
more likely to recover a few cells than a small aliquot
and an automated spiral plate dilution device (Table 4).
Other factors may also influence recovery of cells.
After instrumentation, the surviving bacteria are in a
fragile state and are vulnerable to handling and oxygen
exposure. Thus, handling the sample in an anaerobic
box and growth on media for at least 10d enhances the
chances for cell survival. Identification of recovered
species helps ensure sample accuracy and protects
against contamination. Thus, recovery of species not
normally identified in the root canal, e.g.,
Staphylococcus epidermidis , would imply a contaminant
and this provides a check of sample integrity. Similarly,
tracing a species from sample to sample through a
single clinical case helps ensure that a species isolated
after treatment was present in the canal from the
beginning of treatment. If a species is identified in a
post-treatment sample that has not been isolated in
pretreatment samples, it implies contamination. Simple
bacterial counts without species identification cannot
provide the same level of information.
Without a deeper insight of bacteriological
procedures, these differences may appear slight yet the
consequences of a less sensitive method for cultivation
and culturing are that it is unlikely that small numbers
of cells will be identified in a sample. This is likely to
account for the perceived differences in recovery of
bacteria from cleaned root canals and limits the
conclusions that can be drawn from such material. That
complete eradication of infection cannot be achieved by
apical enlargement is confirmed by immunohistological
and microscopic analysis of canals instrumented in
lower molars,
which revealed that microbes are often
located as biofilms in inaccessible areas of the canal
system such as isthmuses or accessory canals (Fig 6).
Thus, on the basis of currently available information
there is insufficient scientific support for the idea that it
is possible to eliminate infection by apical enlargement
of the canal space.