Microwaves in the laboratory: Effective decontamination

Microwaves in the laboratory: Effective decontamination

Border, Barbara G

OBJECTIVE: We hypothesize that microwave irradiation of certain contaminated materials typically used in a clinical laboratory or a home healthcare setting could produce efficient and effective sterilization when compared to standard autoclave methods.

DESIGN: A standard household carousel microwave oven unit used at the High setting was employed to expose certain materials that had been contaminated with either bacteria or yeast to microwaves for specific intervals of time. Following each time interval, materials were checked for effectiveness of decontamination using standard culture techniques and colony counting. Additionally, powdered media was prepared and microwave irradiated for specific times. The media was then poured into plates and checked for microbial contamination; another set of plates was examined to determine the ability of the irradiated media to support bacterial growth.

SETTING: This study was carried out at Texas Tech University Health Sciences Center in Lubbock TX.

MAIN OUTCOME MEASURE: Standard culture and colony counting techniques were used to determine the efficacy of microwave sterilization.

RESULTS: The study indicated that microwave irradiation provided effective and efficient sterilization of all materials tested. Of the bacteria studied, only E coli survived beyond 30 seconds of microwave exposure. Yeast did not survive beyond 15 seconds of microwave exposure. Swabs and gauze contaminated with bacteria or yeast were completely sterilized after 30 seconds. After three minutes in the microwave oven, powdered, prepared media was free of contamination while able to support growth when inoculated with S. aureus.

CONCLUSION: We conclude that a household carousel microwave oven unit can provide fast, effective sterilization of certain contaminated materials typically used in a clinical laboratory, student laboratory, or home healthcare setting.

ABBREVIATIONS: CLS = clinical laboratory science; POL = physician office laboratory; TSA = trypticase soy agar; TSB = trypticase soy broth.

INDEX TERMS: bacteria; decontamination; media; microwaves; sterilization; yeast.

Clin Lab Sci 1999;12(3):156

Sterilization, defined as any physical or chemical process that destroys all forms of life, is used especially to destroy microorganisms, spores, and viruses. Typically, sterilization occurs through the application of a chemical agent or by heat, either wet steam under pressure at 121 deg C at 15 psi for at least 15 minutes, or by dry heat at 160 to 180 deg C for two hours. Autoclave sterilization typically involves superheated steam under pressure, and was introduced by Dennis Papin who invented the steam digester, a prototype of the autoclave that is still used in modern kitchens as the pressure cooker.’ Present day methods of sterilization include steam autoclaving insippation, filter membrane sterilization, peracetic acid, gas plasma, and ethylene dioxide gas. The most commonly used methods for sterilizing large materials are steam autoclaving and gas sterilization. Both require large pieces of equipment, space, knowledgeable operative personnel, special system hookups, i.e., steam, water, or gas, and constant surveillance. None of these methods address the issue of small-volume users such as physician office laboratories, dental laboratories, student laboratories, or homebound patients and caregivers.

With this in mind, the efficacy of the microwave oven for decontamination purposes was examined. In a microwave oven, electricity passes through a magnetron tube to produce microwaves, electromagnetic radiation with a wavelength of about 10 centimeters. These microwaves are channeled to a rotating fan that distributes them into an oven cavity containing some substance, usually food. The water in this substance absorbs the microwave radiation, creating high internal energy in the water molecules and producing extremely elevated internal temperatures. The mechanism through which microwaves produce their killing action is unclear, although it is thought that in addition to the production of heat within an organism, certain changes unrelated to heat production occur in intracellular molecules causing observable changes in cell morphology and eventual cell disintegration.2 In previous studies, contaminated medical materials have been exposed to microwave sterilization with mostly positive results. These materials include polyethylene catheters contaminated with Proteus sp., acrylic resin dentures, scalpel blades, dental instruments, contact lenses, and injection ampoules.2-7 The aim of this study was to determine whether microwaves produced by a typical home microwave unit could effectively sterilize contaminated media, gauze, and swabs as well as to demonstrate that microwave irradiation provides a method of sterilization that is faster, more efficient, less expensive, and as effective as standard autoclaving for certain materials.

MATERIALS AND METHODS

Microwave oven

The oven used for microwave decontamination was a Sharp Carousel model R4620 with 0.9 cubic feet of space and inner cavity dimensions of 13.25 inches (width) X 9.25 inches (height) X 14 inches (depth). Published output power for this particular model of microwave is 650 watts at a water load of 2000 mL.8 A stopwatch was used for consistent timing of exposure cycles. The temperature of the microwave oven was initially measured at each time interval by placing a laboratory thermometer into room temperature media contained in a 125 mL flask in the center of the microwave and irradiating on the high setting.

Autoclave

A Sybron Castle Steam Sterilizer was used to provide autoclave steam sterilization of materials. For steam sterilization, a setting of 121 deg C and 30 psi for a period of 15 minutes in accordance with our laboratory standards was used. Completion and success of the sterilization cycle was determined by the use of Baxter Biohazard Autoclave Tape. This procedure served as the negative control technique compared with microwave irradiation.

Bacteria and Yeast

Stock cultures of E coli, S. aureus, S. epidermidis, P aeruginosa and C albicans were obtained from Chrisope Technologies, Lake Charles LA and stored at -70 ‘C.

METHODS

Microwave irradiation of contaminated media and materials On day one, bacterial (E. coli, S. aureus, S. epidermidis, and P aeruginosa) and yeast (C albicans) isolates were removed from the -70 deg C freezer, allowed to come to room temperature in approximately 10 minutes, inoculated to a turbidity of the 0. 5 McFarland standard corresponding to 10^sup 8^ bacteria per mL into 10 mL of trypticase soy broth (TSB) and incubated for 24 hours at 37 ‘C. The following day, 4 mLs of each of the five inoculated TSBs were added separately to five flasks of 196 mLs of non-sterile trypticase soy agar (TSA; 1:50 dilution) for a total of five flasks, mixed well to ensure adequate distribution, and kept warm on hot plates.9

In the first set of experiments, 25 mL of each solution was poured into separate 125 mL flasks, sealed with sterile cotton stoppers, placed into the microwave oven, and microwaved for 5 seconds on the high setting. Following irradiation, the inoculated TSA was poured into sterile Petri dishes under a bacteriological hood. Plates remained at room temperature for one hour to allow hardening of the media. This procedure was repeated using clean 125 mL flasks but increasing the irradiation time by 5 second intervals up to 15 seconds, then by 15 second intervals up to 90 seconds. The final irradiation lasted 120 seconds. During the 90 and 120 second irradiations, approximately half of the solution boiled away. A negative control consisted of inoculated media that was not irradiated and poured into Petri dishes. The positive control consisted of media that had been inoculated, autoclaved by standard steam autoclave methods, and poured into Petri dishes. All plates were incubated for 24 hours at 37 deg C.

For contamination of other materials, dilutions were made of inoculated TSBs made in the initial portion of this experiment by mixing one part inoculated broth with two parts fresh broth in sterile 16 x 100 tubes. Sterile swabs were submerged in the diluted broth, allowed to remain in the broth for a period of two minutes, then reamed against the wall of the glass test tube. Sterile two-inch gauze squares were submerged in the same broth dilution, allowed to remain in the broth for two minutes, then removed, and reamed using sterile forceps. Swabs and gauze squares were separately placed into autoclave bags and sealed, placed into the microwave oven, and irradiated for 15 seconds. Again, microwave exposure time was increased by 15 second intervals up to 90 seconds with a final sterilization time of 120 seconds. Upon removal from the oven, the gauze squares and swabs were rubbed onto a sterile TSA plate for examination of growth. The positive control consisted of rubbing a TSA plate with an inoculated swab and a gauze square that were not exposed to microwaves or steam autoclaving. In negative control experiments, an inoculated swab and gauze square were placed in autoclave bags and sterilized by standard steam autoclave methods, then inoculated onto sterile TSA plates. All plates were incubated at 37 deg C for 24 hours.

ASSESSMENT OF STERILIZATION

Following incubation, all plates were examined for growth by two independent investigators. A quad growth grid, a grid divided into four equal quadrants, placed under each Petri dish was utilized for colony quantification, and the number of bacterial colonies per grid section determined the effectiveness of sterilization. Growth was rated as follows: up to five colonies per quadrant = +1; six to ten colonies per quad = +2; eleven to fifteen colonies per quad = +3; sixteen to twenty colonies per quad = A; greater than twenty colonies per quad = +5. Complete sterilization was considered when no growth was observed on the TSA plates; this was rated as 0. Each plate of organisms was compared to the appropriate negative and positive control. Organism growth vs. time of irradiation is shown in Tables 1, 3, and 4 to illustrate the effectiveness of microwave sterilization.

Microwave irradiation of media

To determine whether moderate amounts of powdered media could be prepared and sterilized by microwave exposure, a 500 mL flask of distilled water and 20 grams of trypticase soy agar, (soybean casein digest agar; BBL, a division of Becton Dickinson and Co., Cockeysville MD), was mixed thoroughly. No organisms were added to the media. The flask was heated for one minute on a hot plate to ensure the powder was dissolved, then sealed with a cotton stopper and microwave irradiated at one minute intervals for a total time of five minutes. Following each exposure interval, 5 mL of the media was plated into two sterile Petri plates. One plate was labeled for sterility testing and the other plate was labeled to determine if the media would support the growth of S. aureus. Plates remained at room temperature for one hour to allow hardening of the media and then incubated at 37 ‘C. Plates were observed at 24 and 48 hours. If the plates demonstrated sterility (no growth present) at 48 hours, those plates labeled for growth were inoculated with S. aureus, re-incubated, and observed at 24 hours for growth.

A quad growth grid was placed under each Petri dish and the number of bacterial colonies per grid section determined (Table 1).

Microwave oven temperature measurement

To measure the temperature in the oven following an allotted period of time, a Celsius thermometer was placed in a 125 mL flask containing 25 mL of TSA in the center of the oven. The relationship between the time of exposure and the heat generated within the microwave oven is demonstrated in Table 2.

RESULTS

Following microwave irradiation of contaminated TSA, contaminated swabs or contaminated gauze and subsequent plating and incubation, bacterial or yeast colonies were counted to determine the amount of growth present. The number of bacteria was assessed by two investigators and growth was ranked as +5, indicating greater than 20 colonies per quarter plate; +4, indicating 16 to 20 colonies per quarter plate; +3, 11 to 15 colonies; +2, 6 to 10 colonies; +1, 3 to 5 colonies; and 0 indicating no growth. Any obviously contaminated plates were discarded and the entire experiment repeated for that particular microorganism. This counting procedure was followed for both microwave irradiated materials and steam autoclaved materials.

Following the protocol outlined in the previous section, it was observed that a maximum time of 60 seconds of microwave irradiation was required for all bacteria tested in this study to exhibit no growth. In the agar experiments, the most fastidious bacterial organism that took the longest to destroy was E coli and the least fastidious were the staphylococci. S. epidermidis and S. aureus were no longer viable after 30 seconds of irradiation, while E coli remained viable until 60 seconds of exposure time (Table 1). Candida albicans did not survive beyond 15 seconds of microwave irradiation. With no exposure to microwaves, the positive control, all organisms tested exhibited heavy lawn (+5) growth when plated and incubated. In negative control experiments involving the standard steam autoclaving protocol, no growth of any organisms was observed after plating and incubation.

In those experiments involving contaminated swabs and gauze, two organisms, E coli and P aeruginosa, remained viable after 45 seconds of exposure to microwaves(Tables 3 and 4). However, following 60 seconds of microwave irradiation these organisms exhibited no growth after culture.

Regarding sterilization of moderate amounts of prepared media, Petri plates were observed, and colonies were counted to determine the effectiveness of microwave sterilization. The same scale for quantifying bacterial growth was utilized to determine colony counts. It appeared that a maximum irradiation time of three minutes was required to exhibit no growth. Media solutions irradiated at one and two minutes intervals exhibited +I growth at both 24 and 48 hours. With no exposure to irradiation, the media solution exhibited + I growth at 24 hours and +2 growth at 48 hours. On those plates prepared to determine the ability of the media to support growth, all plates examined at and after three minutes of irradiation demonstrated +5 growth of S. aureus.

DISCUSSION

Microbiological sterilization is an all-or-none process: nothing living, including bacterial spores, must remain on a surface or in a liquid after it has been sterilized. There are a variety of traditional sterilization methods available that achieve this including steam, dry heat, gas, chemical, and ionizing radiation. These methods are approved for the sterilization of critical materials such as those that enter sterile tissues or enter the vascular system. Semicritical materials such as those that contact mucous membranes or skin that is not intact are disinfected, as opposed to sterilized, by exposure to glutaraldehyde or chlorine dioxide, while noncritical materials such as those that contact unbroken skin are disinfected by exposure to bleach, isopropyl or ethyl alcohol, or phenol. Typical methods of sterilization are time-consuming and require special and costly equipment. However, it is essential for the sterility of particular items to be attained and maintained in certain environments such as hospitals or laboratories to provide a safe and quality controlled work environment. Small laboratories have the same needs. In home healthcare situations where bacterial contamination of wound dressings or other materials might be present, it is important to remove the possibility of infection for both caregivers and patients.

Traditionally, different materials are sterilized by different methods. Glassware and metallic medical and dental instruments, for example, are typically dry heat sterilized, while liquids and other materials that can be penetrated by moist heat are steam autoclaved. Various gases such as ethylene oxide, hydrogen peroxide gas, and gas plasma for sterilization purposes are used in hospital, pharmaceutical, and manufacturing settings for certain instruments such as gastrointestinal endoscopes and other plastic materials. Ionizing radiation, such as gamma rays, is commonly used to sterilize disposable supplies, while non-ionizing radiation such as ultraviolet light is commonly used to disinfect surfaces.

One method of sterilization that has received some attention is microwave radiation sterilization. This form of electromagnetic radiation has been used for sterilization of dental instruments, plastic catheters, contact lenses, and other medical materials. Some of these studies demonstrate the efficacy of microwave sterilization while others obtain mixed results. One study examined a suspension of S. aureus dried within the lumen of a plastic tube and microwaved for ten minutes. The tubing was subsequently rinsed with sterile broth and plated. In this experiment, no bacteria were destroyed.10 Other studies, however, state that microwave irradiation is very effective in destroying bacteria and yeast dried onto scalpel blades and coverslips…… Microwave irradiation for a duration of six minutes was effective in complete sterilization of polyethylene catheters with Proteus sp. drawn into the lumen of the catheter.3 There was no change in the physical appearance of the polyethylene as long as a heat sink (300 mL of water in a flask) was used during the irradiation period. A study assessing a large decontamination unit that utilizes microwave-generated heat to disinfect clinical waste demonstrates the ability of microwaves to produce reliable microbiological decontamination. 13

The data collected in the present study demonstrate the microwave oven’s sterilizing capabilities relative to contaminated media and other contaminated materials that might be used in a hospital, laboratory, or home setting. Microwaves provide a safe, efficient method of decontamination of small loads of contaminated materials. The procedure is rapid, and equipment cost is minimal. Of course, other organisms might require different irradiation times, and heavily contaminated materials might require longer time of exposure to microwave irradiation. These possibilities need further study. The present results provide background for a more elaborate investigation into the utilization of a microwave unit as a replacement or an addition to a steam autoclave in a variety of clinical/research settings and needs. A microwave oven, dedicated to sterilization, could be an addition to a nurses station for prevention of accidental contamination of personnel when transferring unsterile materials. ln addition, a home microwave unit could be used to sterilize contaminated dressings and other materials such as swabs used by patients that are house-bound. A further use of the microwave oven would be in a student laboratory requiring sterilization of powdered media or instruments for bacteriology experiments. All of these settings could incorporate a microwave oven into their situation.

ACKNOWLEDGMENT

We would like to acknowledge Mr. Raymond Beduhn for his original idea and question regarding microwave sterilization. We also wish to acknowledge the effort put forth by several CLS students at TTUHSC involved in the initial stages of this project: Marcus Knight, Shaun Matthews, Justin Haynes, and Brian Pendelton.

REFERENCES

1. Encyclopedia Britannica ’98 CD-ROM, multi-media ed. Search terms: autoclave; microwaves; sterilization. Chicago; Encyclopaedia Britannica, Inc: 1998.

2. Rosaspina S, Salvatorelli G, Anzanel D. The bactericidal effect of microwaves on Mycobacterium bovis dried on scalpel blades. J Hosp Infect 1994;26(l):45-50.

3. Griffith D, Naceyj, Robinson R, Delahunt B. Microwave sterilization of polyethylene catheters for intermittent self-catheterization. Aust NZ J Surg 1993;63(3):203-4.

Thomas Cj, Webb BC. Microwaving of acrylic resin dentures. Eur i Prosthodontics and Restorative Dent 1995;3(4):179-82.

5. Rohrer MD, Bulard RA. Microwave sterilization. j Amer Dent Assoc 1985; 110: 194.

6. Starr MB. A critical appraisal of contact lens disinfection. New York State J Med 1990;90(1):12-6.

7. Sasaki K, Honda W, Shimizu K, and others. Microwave continuous sterdization of injection ampoules. PDA J Pharm Sci and Tech 1996; 50(3):172-9.

8. Sharp(R) Carousel Microwave Oven Operation Manual, Model R-4620, Sharp Electronics Corporation, New Jersey USA. 800-237-4277

9. Balows A, Hausler W Jr, Hermann K, and others, editors. Manual of Clinical Microbiology. 5th ed. Washington DC: American Society of Microbiology, 1991.

10. Hengen P. Methods and reagents. Emergency sterilization using microwaves. Trends in Biochem Sci 1997;22(2):68-9.

11. Rosaspina. S, Anzanel D, Salvatorelli G. Microwave sterilization of enterobacteria. Microbios 1993;76(309):263-70.

12. Rosaspina S, Salvatorelli G, Anzanel D, Bovolenta R. Effect of microwave radiation on Candida albicans. Microbios 1994;78(314):55-9.

13. Hoffman PN, Hanley MJ. Assessment of a microwave-based clinical waste decontamination unit. J App] Bacteriol 1994;77:607-12.

Barbara G Border PhD is an Assistant Professor at Texas Tech University Health Sciences Center, Lubbock TX

Lori Rice-Spearman MS is an Associate Professor at Texas Tech University Health Sciences Center, Lubbock TX.

Address for correspondence. Barbara Border PhD, Department of Diagnostic and Primary Care, TTUHSC, 3601 4th Street, Lubbock TX 79430. (806) 743-3248, (806) 743-3249 (fax).

alhbb@ttuhsc.edu

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