From Volume 40, Issue 8, October 2013 | Pages 630-636
A literature search revealed six laboratory-based microbial challenge studies which support the opinion that oil contamination does not prevent successful sterilization of the lumen of dental handpieces. However, a detailed appraisal of these papers revealed lack of detail in the cleaning methods used prior to sterilization and none of the papers considered the risks of prion contamination.
A literature search was performed with the assistance of librarians at the BDA Information Centre using OVID search facility on Medline, EMBASE, CINALH and The Cochrane Library.
On the suggestion of the reviewer, the BDA Information Centre provided a list of publications on the subject of the history of sterilization from the Index of Dental Literature from 1896 to 1949. This produced key papers from the Lancet and the authors, Perkins and Fallon.
A12 BDA advice sheet produced guidance on infection control from the devolved countries forming the UK.
Two search parameters are listed in Table 1. The selected papers produced 21 references. The reference lists of each of the 21 papers were consulted and further papers selected. The references of the references were examined.
| Search Strategy 1: |
An internet search of PubMed and Google Scholar was performed using the keywords ‘sterilization’, ‘decontamination’, ‘dental’, ‘handpiece’, ‘lumen’, ‘prion’, ‘internal’, ‘disinfection’. This led to a number of useful lectures published online.
A search of the University of Birmingham library catalogue was performed which led to many papers from The British Library. Search engines attached to Dental Update and British Dental Journal websites were searched for relevant papers.
Manufacturers of dental handpieces were contacted and websites consulted for all the manufacturers listed below. Email requests were made to each company for relevant literature on the subject of decontamination of dental handpieces. The companies contacted were Kavo Dental, Sirona Dental, Bienair, W+H Dental, NSK Dental Ltd and Eschmann Equipment. A request was made to manufacturers of endospore preparations where it was deemed necessary.
Information was obtained from the HTM 01-05 Infection Control document published in 2009 and a list of references supplied to support this document supplied by the Department of Health was consulted.
The following criteria were developed against which the quality of the paper was judged:
The papers are presented in Table 2 in the form of final outcome in answer to the question posed.
| YES | NO | REJECTED PAPERS |
|---|---|---|
| Hegna et al, 19781 |
Nolte & Arnim, 19552 |
Five papers fitted all the criteria set out above and claimed that a sterile lumen was possible. A sixth paper by Silverstone and Hill11 also claimed a sterile lumen, but with higher temperatures resembling dry heat sterilization. A detailed appraisal of these papers is presented below.
Hegna et al claimed successful sterilization of samples of cotton cloth impregnated with Geobacillus stearothermophilus spore preparation in 50% serum. Samples were inoculated from Oxoid spore strips with a viable count of 1x105 organisms grown in a jar of blood agar at 56°C for 10 days. Samples were sprayed with handpiece oil, dried and autoclaved at 134°C for 8 minutes in an Emmer 430I laboratory autoclave, and then incubated in an agar-free dithionite thioglycollate (HS-T) broth for 7 days at 56°C. No growth was observed, indicating that the organisms did not survive the process despite a coating of oil. Organisms that were dried for 5 hours and 24 hours also did not survive.
Oxoid, the manufacturer, confirmed a ‘spore kill time’ of 15 minutes and ‘spore survival time’ of 5 minutes, for the organisms at 121°C. A D-value was not quoted in the paper or obtainable from the manufacturer. The lack of a D-value makes it difficult to make comparisons since spore resistance differs widely among spore strains. The nature of the recovery medium is critical to the promotion of growth and, since the manufacturer's recommended recovery medium was not used, there is the potential for false negatives. This raises doubt as to the validity of the result.
In a separate experiment, five new handpieces were lubricated and autoclaved. The handpieces were connected to a motor and run, thus inoculating them with Geobacillus stearothermophilus spores. Handpieces were once more lubricated and autoclaved, then disassembled and immersed in broth for 15 days. The results again showed no growth. The author acknowledged that oil prevents the contact of steam to the organisms but concluded that moisture around the oil heated up to 134°C would be sufficient to kill the organisms. In fact, direct contact of saturated steam is needed to cause protein coagulation by the release of latent energy.
Other experiments involving used contaminated handpieces, which had not been used for up to a week, indicated that these could not be sterilized. The author concluded that material dried on for longer periods of time created an effective barrier, preventing sterilization of a variety of other organisms which would normally have far less resistance to steam sterilization. The paper did not explicitly state that the results obtained from handpieces were used as positive controls. A negative control was not mentioned in the method. The autoclave used was a gravity displacement type, which is normally less efficient than a vacuum autoclave.1
Edwardsson et al compared the effectiveness of sterilization of handpieces which had been lubricated with oil with and without an antimicrobial agent to that of non-lubricated handpieces. Multiple series of 10 handpieces were first disassembled and autoclaved at 127°C for 20 minutes. Nine of them were inoculated with saliva-spore suspension of Geobacillus stearothermophilus with a viable count of 106 organisms. Inoculations were performed in a standardized way in an attempt to seed organisms into parts of the handpiece considered most difficult to sterilize.
The count was estimated after incubation of the organisms on brain heart infusion agar after 10 days. The handpieces were immediately assembled and autoclaved in a downward displacement autoclave at various prescribed temperatures and times. Negative and positive controls were used. Temperature was monitored with the use of two thermo-electric elements, one placed inside a handpiece and the other elsewhere in the autoclave chamber. The temperature measured inside the lumen was used to calculate the D value of the organisms in two of the cycles. After the sterilization phase, the handpieces were again disassembled, and various components separated into three test tubes each containing a different type of broth. Test tubes containing the broth and components were shaken for 15 minutes and then transferred to new tubes containing the same broth. The reason for different broths was to minimize the risk of metal ions from the handpieces being released into the broth and causing a bacteriostatic effect. The different broths were also tested independently to confirm similar performance in spore germination. Thus for each sterilization cycle there were six tubes which were then subsequently incubated and examined for growth. The broth was also checked for possible contaminating organisms, such as Staphylococcus aureus. Results indicated that growth occurred from those handpieces which were not lubricated and those lubricated with oil containing no antimicrobial agent, when sterilized at 120–124°C for 20 minutes and 134–136°C for 10 minutes. No growth occurred for handpieces that were lubricated with oil containing 5% isopropanol and formaldehyde sterilized at 134°C for 10 minutes. The author concluded that the lumen could not be sterilized as the presence of conventional oil impeded access for steam3 and sterility could only be achieved with the use of oil containing an antimicrobial agent. Furthermore, the lumen could not be sterilized in the absence of oil, although the expectation was that the opposite result should occur. Many series of handpieces were sterilized and, although the loading configuration was described in some detail, minor differences in loading configuration could have affected the outcome. This is particularly the case for gravity cycle steam sterilizers. An aseptic technique is vitally important during assembly of the handpiece components, which could have led to unwanted contamination and led to false positive results. Despite the promise shown in this paper, concerns about toxicity do limit the use of formaldehyde in this way.3
Barnes et al investigated the ability of steam sterilization to kill Geobacillus stearothermophilus spores from re-usable dental prophylaxis angles. The items tested were inoculated with spores sourced from spore preparations combined with 40% ethanol, which underwent serial dilution, to produce numerous samples with viable counts 1.3 x 106 determined by the ‘most probable number’ (MPN) method. The D value was not stated explicitly but it was claimed that assay sensitivity was determined by quality control measures from the manufacturer. Inoculated items were autoclaved using an Eagle Series 2021 gravity displacement type sterilizer. Multiple series of prophylaxis angles were tested in this way, designed to compare assembled versus disassembled items with the internal portions exposed during sterilization; lubricated versus non-lubricated items; and those inoculated with pure spore preparations versus spore preparations mixed 1:1 with bovine serum albumin. Experimental procedures were carried out aseptically with a laminar flow hood and all items were tested at 121 °C for 20 minutes at 2 bar pressure. Results indicated negative growth for all tests, indicating successful sterilization which was unaffected by state of assembly of the test item, presence of oil, and presence of serum and all combinations of these factors. Negative and positive controls responded as expected. The resistance of organisms in liquid suspension is not known so it was not possible to determine the sterility assurance level of the test items.5
Silverstone and Hill investigated the use of heated handpiece oil as an alternative to autoclaving for the sterilization of handpieces. The technique was assessed in light of knowledge gained from earlier studies claiming the destruction of spores using hot oil at no detriment to handpiece performance. Handpieces were inoculated in the same manner as that described by Barnes and immersed in preheated turbine oil at temperatures of 150°C and 160°C. Handpieces were taken out at set time intervals, allowed to drain for 10 minutes, dried with paper towels and incubated on tryptase soy broth for 7 days at 56°C. As time intervals increased, reducing numbers of spores were demonstrated. Inoculum concentrations were evaluated by dilution and counting on nutrient agar plates. Results indicated that all handpieces immersed in oil at 160°C for 45 minutes showed no growth, this being the only successful regime demonstrated. A significant disadvantage of this technique was the unwanted seepage of oil draining from upright handpieces for weeks after the experiment.11 Modern handpieces are specified to operate up to a maximum of 135°C. Although this technique has no modern practical relevance, it may be useful in less developed countries where autoclaving is not possible.
Andersen et al examined the effect of steam sterilization on handpiece turbines contaminated with spores and compared the effectiveness of four different non-vacuum and one vacuum bench top steam sterilizers. Twelve used handpieces formed the sample population. Handpieces were first cleaned and lubricated with handpiece oil. The method of the initial cleaning was not specified in the paper. Spores were inoculated on cotton carriers creating viable counts of 3x 105 with a D value of 1–2 minutes at 121°C. Turbine chambers were opened and inoculated by placing one cotton carrier on the turbine wheel and the chamber then closed. The handpieces were sealed in bags and autoclaved for 20 minutes at 121°C. After the cycle, cotton carriers were transferred to broth, incubated and examined for growth. Results indicated that varying amounts of growth were observed from carriers that had been autoclaved in all non-vacuum autoclaves. Carriers sterilized in the vacuum autoclave all showed negative growth, indicating successful sterilization. Negative and positive controls performed as expected. Unlike other papers, no drying of samples was performed and additional soil was not added to simulate the clinical situation. Bagging of items, though considered appropriate for vacuum cycles, should not be done for items processed in a non-vacuum autoclave, except for solid instruments, a fact that was acknowledged in the paper. The reason given for bagging was to present the same challenge to the sterilization process to compare autoclaves. However, the risk of this approach is the possible introduction of false positive results for non-vacuum cycles. This problem would have been minimized if the author had performed additional cycles of items in the non-vacuum autoclaves without bagging.
Larsen et al evaluated the ability of a device called the Sterimax 3 (Akeda Dental) to decontaminate handpieces. This device was designed to combine the processes of lubrication, cleaning and steam sterilization.7 Two organisms were used, Streptococcus salivarius and Geobacillus stearothermophilus. Handpieces were inoculated with Geobacillus spores on cotton carriers inoculated with 3 x 105 organisms. The cotton carriers were placed aseptically inside the turbine chamber. No D value was specified in the paper and a search for the manufacturer was unsuccessful (SSI KJ1, Denmark).
In addition, handpieces were inoculated with Streptococcus salivarius in the air and water channels of the handpiece and in the turbine chamber. Handpieces underwent two different cycles (Table 3). It is notable that oil lubrication was part of the cycle on two occasions.
| Set up 1 | Lubricate | Flush with warm water at 50°C for 60 sec | Dry | Sterilize at 121°C 15psi for 6min | Lubricate | Dry for 60 sec | Total Time 20 min |
| Set up 2 | Lubricate | Flush with warm water at 70°C for 120 sec | Dry | Sterilize at 121°C 15psi for 20 min | Lubricate | Dry for 90 sec | Total time 30 min |
Handpieces were connected to a dental unit and samples for inoculation of S. salivarius were collected by flushing. The water supply of the dental unit underwent regular chemical disinfection. Both organisms were collected for culture aseptically, incubated using appropriate media and examined for growth.
The author reported the effects of flushing alone and in combination with a vacuum cycle for both organisms in all internal sites of inoculation.7
Results are summarized in Tables 4 and 5.
| Organism | Site of inoculation | Flushing only | After autoclaving |
|---|---|---|---|
| S. salivarius | Water channel | Growth (3x log reduction) | Growth (4x log reduction) |
| Air channel | Growth (3x log reduction) | Growth (4x log reduction) | |
| Turbine chamber | Growth (no change) | No growth (5x log reduction) | |
| Geobacillus | Turbine chamber | - | Growth |
| Organism | Site of inoculation | Flushing only | After autoclaving |
|---|---|---|---|
| S. salivarius | Water channel | Limited growth (5–6 log reduction) | No growth |
| Air channel | Limited growth (5–6 log reduction) | No growth | |
| Turbine chamber | Growth (little change) | No growth | |
| Geobacillus | Turbine chamber | – | No growth |
The cleaning phase had a significant 3x log reduction effect on Streptococcus salivarius in water and air channels, further improved by autoclaving. Flushing alone with water at 50°C did not affect the organisms in the turbine chamber. A sterilization time of 6 minutes failed to kill endospores.
Flushing with warmer water at 70°C resulted in an improved 5–6 log reduction to Streptococcus salivarius and, once again, had little effect on organisms in the turbine chamber. Autoclaving was successful in the elimination of all organisms.
With reference to the tables above, the author concluded that Set up 2 was successful as the bioburden of Streptococcus salivarius had been reduced more effectively and showed that a combination of pre-cleaning and sterilization was necessary to achieve an effective process.
The author also tested a further six handpieces under Set up 2, which had been used clinically. Viable counts of 6.2 x 106 were estimated and the sterilization process was not successful owing to the high initial bioburden. The cleaning phase did not include detergents or disinfection, which may have partly accounted for failure to sterilize handpieces used clinically. In this situation, it would be reasonable to expect serum/saliva to increase the challenge to sterilization further.7
Two of the papers did not examine dental handpieces. One assessed orthodontic bands16 and the other dental syringes.14 These form further evidence of the ability to sterilize a lumen. Many listed papers were a collection of editorials, opinion and marketing. One paper was not suited to this review as it tested hydrogen peroxide as a decontamination method.6 Other papers employed test culture methods for a wide variety of micro-organisms and demonstrated sterile lumens for Bacillus globigli17Streptococcus mutans, Staphylococcus aureus,1,15,19Streptococcus salivarius7 and duck hepatitis B virus.14 All these organisms have far less resistance to steam sterilization than Geobacillus and therefore the results of these studies are less meaningful.
A huge variation in sample sizes determined from the number of tests carried out was shown in these studies. One paper suffered from a small sample size and was rejected from consideration.8 The results from another paper demonstrated sterile lumens in dental syringes but a single failure in one test was not considered significant.14
No paper justified the number of subjects used for its sample size and the power of the study could not be evaluated. The most popular sample size was 30 handpieces.
An important aspect of quality was the use of controls in these papers. Both positive and negative controls should be incorporated into the ideal study design and the general principles governing their use are illustrated in the Table 6.
| Device | Bacterial contamination | Cleaning/sterilizing protocol | Results expected for non-sterile lumen | Results expected for sterile lumen |
|---|---|---|---|---|
| Test handpiece | Inoculated | Autoclaved | Contaminated | Not contaminated |
| Positive control | Inoculated | Not autoclaved | Contaminated | Contaminated |
| Negative control | Not inoculated | Autoclaved | Not contaminated | Not contaminated |
The positive control ensured that the decontamination result could only be explained by the decontamination protocol, whereas the negative control ensured that contamination could not occur by any means other than the intended bacterial inoculation. All papers employed controls but varied in the number of controls used.
There have been calls from the profession for a water-based lubricant but it seems that the majority of manufacturers will continue to insist on oil-based lubricants.
The presence of an oil-based lubricant has been studied in a comparison of the effect of steam sterilization on oil-lubricated and non-lubricated spore strips by Halleck et al.20 Spore strips prepared by inoculating paper strips with spores in ethanol, and then coated in lubricating oil, was shown to be a suitable method for use in sterilization studies for large numbers of samples. A reliable method of recovery of oil-coated spores was achieved by incubation and agitation on a rotary shaker for a minimum of 48 hours at 200 rpm followed by a further five days in a stationary state at 55°C. Where no growth was observed, subcultures were prepared and incubated for a further seven days. This procedure aimed to dilute any inhibitory oil residue that may have been present to allow growth of viable spores. Results indicated shorter sterilization times for non-oil-coated spores than for oil-coated spores. The oil coating increased the time required to kill the spores but did not prevent sterilization. The most effective method was shown to be a pre-vacuum cycle at 132°C.20 Rutala et al were able to show successful sterilization of surgical instruments that had been coated with hydraulic oil, again with a pre-vacuum cycle of 132°C for 4 minutes. A variety of surgical instruments, including lumened devices, were inoculated with Geobacillus spore suspension with a viable count of 2 x 105 and incubated on tryptase soy broth. Handpieces were immersed in broth, incubated and examined for growth. No growth was observed, indicating a successful process.21
The lack of detail in most of these papers with regard to cleaning processes prior to sterilization is a concern.
Much of the work on spores was carried out on seeded colonies. It has become increasingly recognized that contaminating organisms are more likely to be found in a biofilm, which is more resistant to sterilization. It may be that these studies demonstrating a sterile lumen underestimate the risk and are producing false positive results.
Small differences in microbiological technique, such as incubation temperature and recovery medium, can make a significant difference to the outcome.
Studies by Andersen and Rutala provide evidence for the benefit of vacuum autoclaves, supporting the principles of sterilization that evacuation of air is necessary for efficient steam penetration of the lumen.9,21
Spore suspensions in liquid suspension do not have the same D value as those supplied by manufacturers on biological indicators, so the sterility assurance level cannot be accurately determined.
Steam sterilization is an energetic process. The conversion of liquid vapour at 100°C to liquid water at 100°C releases considerable energy in the form of latent heat in the confined space of a lumened device. It may be that this energy is sufficient to agitate the surface of the liquid oil film for the steam to cause enough disruption to organisms within the oil. Proteinaceous material is more solid and adheres tenaciously to stainless steel, so may act as a more efficient shield for micro-organisms against this energy (in the opinion of the author).
Considering the body of literature above, it can be concluded, from the use of microbiological techniques, that a sterile lumen is possible and that oil does not prevent sterilization.
