Biological indicators for /certified fixed orthodontic courses by Indian dental academy

BIOLOGICAL INDICATORS
FOR
STERLIZATION EFFICACY
A DISCUSSION
INDIAN DENTAL ACADEMY
Leader in continuing dental education
www.indiandentalacademy.com


INTRODUCTION:
• A recent study found that orthodontists have the highest
incidence of hepatitis B among dental professionals.
• Saliva is about half as infectious as blood.
• So, sterilization monitoring improves the assurance that
the instruments have been adequately sterilized.
• Biological monitoring fulfills the valid requirements for
sterilization procedures given by national health
authorities and it is reliable, convenient , and self
contained and should be routinely used in dental office.

• A number of studies
have been conducted
to determine the
efficacy of the
Biological Indicators
and to determine
sterilization efficacy in
different dental offices.

STUDIES:
1.

The ability of Biological Indicators to detect sterilization
failure. J.Dent.Res.(1990).

2.

Evaluation of Biological Monitors in Harvey
Chemiclave.J.Dent Res.(1980)

3.

Effectiveness of Dental Office instrument sterlization
procedures.JADA (1991).

4.

Proper monitoring of sterilization procedures used in oral
surgery.Int j. of oral Surg.(1983).

5.

Characterization of a rapid Biological Indicator
utilizing B.Stearothermophilus spore associated
alpha glucosidase enzyme.J of App. Micro(1998).

The Ability Of Biological Indicators To
Detect Sterilization
Failure:C.H.Miller,M.A.Sheldrake(1990)
• AIM OF THE STUDY:
To compare immediate and delayed
incubation on the ability of BI’s to
detect sterilization failure.

METHOD:
• The BI’s were exposed to previously determined
sublethal condition in steam or chemical vapour
sterilizer.
• Portion of each brand were incubated (56 0C for 7
Days) immediately, after storage at 270C for 7
days, and in one case after mailing (3-day delay)
to the laboratory.
• Non exposed control BI’s were used and all
showed growth after immediate and delayed
incubation.

RESULT:
•

Eighteen of 18 strips,60 of 60 strips and 26 of 26 strips yielded
growth of B. Stearothermophilus after immediate, 7-day delay and 3
day mailing delayed incubation.

•

Three brands of BI strips and one brand each of BI vented vial and
glass ampule were used in chemical vapour sterlizer.

•

After sublethal exposure for 1,1,1,3 and 7 min,respectively,at 271 0F
all 20 of each BI showed growth of B.Stearothermophilus after
immediate incubation.

•

All 20 of two strip brands and of ampule showed growth after 7-day
delay in incubation.

DISCUSSION:
•

These result show that appropriate BI’s can detect sterilization failure
after immediate and delayed incubation.

Evaluation of Biological Spore Monitors
in Harvey Chemiclave:B.Matis et al.
(1980)
AIM OF THE STUDY:
•
•

To determine permeability of protective covering of
BI’s in chemical vapor sterilization.
To determine how much sterilization bags limits vapor
penetration.

METHOD:
•
•

Monitors were enclosed in polypropylene vials with
filter paper caps and glassine paper.
Sterilizer bags were sealed and placed inside one of
another with innermost bag containing side by side
BI’s.

RESULT
• There was decrease in permeability of 67% with filter
paper covered containers & 90% with glassine covered
containers compared to controls.
• BI’s inside the filter paper-covered containers were
negative for growth up to eight layers and glassineenclosed up to four layers of sterilizer bags, after
completion of normal 20-min cycle.

DISCUSSION
• It is recommended that the user of chemical vapor
sterilizers should avoid layering packages of
instruments.
• Where it is necessary, the time of cycle must be
adjusted.
• Filter-paper covered spore strips are the most accurate
and time efficient monitors of sterilization.

EFFECTIVENESS OF DENTAL
OFFICE INSTRUMENT
STERILIZATION PROCEDURES :
Richard J. Hastreiter(1991)
AIM: This study evaluates the effectiveness of
dental instrument sterilization procedures in
Minnesota dental offices.


METHOD
• A random sample of 900 dentists was selected from 2,808
dentists in active practice. The sample size was chosen to
provide a sufficient response rate to test 400 dental office
sterilizers.
• Each selected dentist was assigned an identification
number and sent a study participation form. The form
contained: the dentist’s printed name, address and
telephone number; space for entering the results of the
microbiological culturing of up to three BI series; and
questions regarding dental office instrument sterilization
methods and use of BI’s to monitor sterilization
performance.

•

Study participation forms were returned by 55 percent (497/900) of
sampled dentists.

•

Each of these dentists was mailed the first BI test series which
contained sterilization testing procedure instructions and an
addressed return mailing envelope containing four BIs. Each BI
consisted of a spore strip enclosed in a glassine envelope
containing both B. stearothermophilus and B. subtilis spores.

•

Three of the four spore strips in each BI series served as test BIs
that were each run through a different sterilizer cycle with a
moderate to heavy load of dental instruments. Specific instructions
were given to operate the sterilizer in a manner normally used by
the office and to place each BI in the most difficult area of the
chamber to sterilize, such as in the center of a load of bagged or
wrapped instruments. Dentists were requested to provide
information about sterilizer operating conditions for each tested
sterilizer cycle (for example, for steam autoclaves the temperature,
pressure and time of the cycle).

• The fourth spore strip in a BI series served as a
study control BI that was used to determine the
effect of mail transit, handling and storage on
the viability of BI spores. The control BI was not
processed through a sterilizer cycle but was
returned with the other three test BIs for
culturing.
• On receipt, each BI was placed in sterile Amsco
spordi culture medium and incubated for seven
days at 560 C for B. stearothermophilus and for
seven days at 370 C for B. subtilis.

• Dentists whose three test BIs cultured negative (indicating
adequate sterilization) and whose control BI cultured positive
(indicating no lethal damage to spores as a result of mail
transit, storage or handling) were not sent any additional BIs.
• If a control BI cultured negative (indicating lethal damage to
spores as a result of mail transit, storage or handling), the
dentist was sent another BI series of three test and one
control BIs to perform the sterilizer testing procedure again.
• Dentists whose sterilizer produced one or more BIs that
cultured positive (indicating inadequate sterilization) were
called with the finding, counseled on methods of improving
sterilization performance, and sent another BI test series of
three test and one control BIs to perform the sterilizer testing
procedure for a second time.

RESULT
• In the first BI series, 18 percent (74/406) of all sterilizers
failed, 24 percent (18/74) failed the second BI series and
18 percent (3/17) failed the third BI series.
• Sterilizer specific failure rates for the first BI series were:
steam autoclaves, 16 percent; chemical vapor sterilizers,
16 percent; dry heat ovens, 43 percent; and ethylene
oxide sterilizers, no failures.
•

Dry heat ovens failed to
sterilize significantly more
often than steam autoclaves
or chemical vapor sterilizers.

• There was no significant difference in
sterilization failure rates among steam
autoclaves, chemical vapor sterilizers and dry
heat ovens with either the second or third BI
series.
• After the third BI series, only three of the
original 406 sterilizers had failed to kill the
spores in all three test BIs in a single BI
series.

CAUSE
• 87 percent of sterilization failures were the
result of operator error.
• The balance of failures was primarily caused
by faulty equipment maintenance, resulting in
equipment defects such as inadequate
sterilizer door seals.

Discussion
• Biological indicators are useful in monitoring
sterilization performance only when sterilization
procedures are consistently performed in a
competent manner by well-trained staff using
adequately maintained equipment.
• Four elements are essential to assuring proper
instrument sterilization:
- Quality sterilization equipment and maintenance;
- Correct sterilization equipment operation;
- Comprehensive operator training;
- Use of BIs to monitor the effectiveness of
sterilization procedures.

Proper monitoring of sterilization
procedures used in oral surgery:
N.Skaug(1982)
AIM:
• To survey the sterilization procedures used by oral surgeons
and,
• To monitor efficacy of the procedure used by Biological
Indicators.
MATERIAL& METHOD:
• All oral surgeons full or part-time basis in the institution or
private practice were included in Norway.
• Steam autoclaves were tested twice using 4 BI’s units for
each sterilization cycle.
• Dry heat sterilizer and gas autoclaves were tested once with 6
BI’s units.

• The system includes the Attest No. 1242 BI’s containing
spores of B.stearothermophilus & Attest Biological
Incubator for steam sterilizer
• For dry heat sterilizer and gas autoclaves B.Subtilis
spores were used.
• BI’s were placed at different locations inside the sterilizer
containing instruments.
• Immediately after sterilization, the indicators were send
by mail to laboratory.


RESULTS
•
•
•
•

35 of the 36 oral surgeons responded.
22 steam autoclaves and 4 dry heat sterilizer were tested.
In 17 of the 22 steam autoclaves BI’s were killed.
Where inadequate sterilization occurred, more BI’s
survived the sterilization cycle in a medium load than in
heavy load of instruments.
• In one autoclave BI unit got deformed
due to excessive heat.
• 2 out of 4 dry-heat sterilizer inactivated
all spores.
• B.Subtilis spores survived in gas sterilization.

DISCUSSION
• 5 out of 22 steam autoclaves and 2 out of 4 dry
heat sterilizer & all of gas sterilizer tested did not
kill the spores, and therefore did not fulfill the
standards of sterilization procedures.
• The standards states that probability of having
an inadequate sterilization result should not be
greater than 10-6 .This means that sterilization
procedure will be acceptable if 1 out of 10 6
sterilization procedure performed is inadequate
or if 1 out of 106 objects sterilized is not sterile.

BIOLOGICAL INDICATORS FOR STEAM
STERILIZATION: CHARACTERIZATION
OF A RAPID BIOLOGICAL INDICATOR
UTILIZING B. STEAROTHERMOPHILUS
SPORE-ASSOCIATED ALPHAGLUCOSIDASE ENZYME :
H.ALBERT ET AL.(1998)


GENERATIONS OF BI’s:
• The original format of biological indicators was
inoculated paper strips inside envelopes, which were
transferred to sterile culture medium following
processing, and incubated for 7 d . Sterilization failure
was measured by turbidity of the growth medium.
• Second generation biological indicators are selfcontained systems which comprise the spore strip and
growth medium required for recovery in a primary pack
ready for use. These biological indicators rely on a pH
indicator to measure production of acid metabolites in
the growth medium by outgrowing spores and replicating
cells.
These systems remove the problem of contamination
during manipulation encountered with the first generation
indicators, but 24-168 h readout time is still required for
detection of surviving spores .

•

Recently, the third generation biological indicator, Attest TM 1291
Rapid Readout Biological Indicator , was developed for use in
132 °C flash sterilization in view of the need for rapid results in
emergency sterilization procedures used in the operating room.
These third generation biological indicators have a dual
readout system. The rapid portion detects active sporeassociated alpha glucosidase enzyme surviving the
sterilization process within 1-3 h.
It is consistently detectable in viable spores but is destroyed by
steam sterilization just after spore kill. The survival of the sporeassociated alpha-glucosidase enzyme following exposure to steam
sterilization correlates well with spore survival.
The second readout system comprises a pH indicator which
detects acid metabolites produced by outgrowing spores and
replicating cells, and gives confirmation of the rapid result
within 24-168 h.

AIM:
This study was undertaken to characterize the alpha-glucosidase enzyme
utilized as a rapid monitor of sterilization.
MATERIALS & METHODS:
• Bacterial strains and growth conditions:
Bacillus stearothermophilus was provided as spore strips by 3M, St Paul,
MN, USA, derived from ATCC 7953 culture.
For culture maintenance, a spore strip was grown in soy broth overnight in
a shaking incubator. This stock vegetative cell culture was stored in 1 ml
aliquots in liquid nitrogen. For the primary inoculum, 1 ml of the stock
vegetative cell culture was used.
• Preparation of crude spore extract:
A culture medium was prepared to extract spores which was stored at 40C.
• Alpha-glucosidase assay:
Alpha-glucosidase was determined by spectrophotometric measurement
and fluorimetric detection.
• Properties of the alpha-glucosidase:
1. Molecular weight was determined.
2. The effect of pH on alpha -glucosidase activity was determined.
3. The effect of temperature on alpha -glucosidase activity was determined.
• Electron microscopy :
Ultra thin sections werewww.indiandentalacademy.com
prepared to see the spores in electron
microscope.

RESULT
• The optimum pH for enzyme stability was approximately 7·35.
The activity of the enzyme increased rapidly from 20 °C,
reaching an optimum activity at approximately 63 °C in the crude
extracts The alpha-glucosidase activity dropped sharply at
temperatures between 63 °C and 77 °C. The alpha-glucosidase
was stable up to approximately 65 °C, above which its stability
rapidly decreased .
• This high thermo stability of the enzyme in liquid extract reflects
the expected increase in stability of the enzyme in anhydrous
form, and its use as a monitor of steam sterilization.
• Correlation was found between alpha -glucosidase activity
(measured by fluorescence after 1 h incubation) and 24 h growth
readout of AttestTM 1291 Rapid Readout Biological Indicators
following exposure to 132 °C gravity sterilization.

DISCUSSION
• The alpha-glucosidase proved to be a
useful predictor of spore survival as it is
consistently present in viable spores and
vegetative cells, and it survives just longer
than the spore following moist heat
sterilization.
• It is the fastest method to determine
sterilization efficacy for high speed pressure
steam sterilizer as the enzyme becomes
fluorescent yellow within 60 minutes as the
spores are killed.

SUMMARY
•

The advantage of BI monitors over indicators like
bags,strips,tapes with chemical formulation which
changes colour with given temperature is that they
only assure instruments have been exposed to
sterilization cycle ,they do not verify that
sterilization have occurred. BI indicators provide
the only real method of verifying the effectiveness
of sterilization procedure.

•

Four elements are essential to assure proper
instrument sterilization:
Quality sterilization equipment and maintenance;
Correct sterilization equipment operation;
Comprehensive operator training;
Use of BIs to monitor the effectiveness of
sterilization procedures.

1.
2.
3.
4.


Thank you
For more details please visit
www.indiandentalacademy.co
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Biological indicators for /certified fixed orthodontic courses by Indian dental academy

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