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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.


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


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


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


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


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.


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.


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.


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.


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 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.


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.

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