0017-9078/91 $3.00 + .OO 0 1991 Health Physics Society Pergamon Press plc

Heollh Physics Vol. 61,No. I (July),pp. 137-142, 1991 Printed in the U.S.A.



Margaret C. Stinson, *,+ Barbara L. Green, Charles J. Marquardt* and Alan M. Ducatman* *Environmental Medical Service, Massachusetts Institute of Technology, 20B-238, Cambridge, MA 02 139 and *Costar Corporation, One Alewife Center, Cambridge, MA 02 140 Abstract-A model system was developed previously for disposal of solid laboratory waste that is both radioactive and heat sensitive, e.g., HIV. A double polypropylene bag with charcoal vent filter and absorbent was designed to meet requirements for both steam sterilization and disposal as solid radioactive waste. Earlier work demonstrated the effective containment of radioactive gases by the filter and inactivation of organisms as heat sensitive as HIV. We sought to broaden the application of this model to ensure inactivation of microorganisms that are more heat resistant than HIV. The efficacy of steam sterilization using water or solutions of iodophor, hypochlorite, or hydrogen peroxide was studied under constant temperature and time conditions. The systems were monitored with internal probes, physical, chemical, and biological indicators. Biological indicators documented inactivation when bags containing hydrogen peroxide (3%)were autoclaved for 60 min at 121OC. Synergistic activity between hydrogen peroxide and autoclave conditions significantly reduced processing time.

of 12 1 "C is reached. The autoclave tape changes color at 121°C. The waste bag is then placed in an autoclavable tub and autoclaved for 30 min. After the autoclave cycle is complete, the waste bag is removed. The Diack control and the autoclave tape are then visually inspected to ensure that the autoclave was working properly, achieving a temperature of 121"C. Waste disposal requirements introduce three constraints into this system. First, the charcoal filter functions to adsorb any volatile radioactive materials and necessarily restricts air flow into the bag. In doing so, steam penetration is limited. The bag functions as a closed system with concurrent increased heat transfer requirements. Second, it is necessary to absorb any liquid so that all waste is dry, solid waste. The absorbent acts as a heat sink and increases the heat transfer requirements of the bag. Third, containment is within two polypropylene bags (rather than a single bag) to assure safe radiation practices. This also increases the heat transfer requirements. Our goal was to devise a model that overcomes these disadvantages while maintaining the integrity of the system. In particular, two system requirements were varied in an attempt to achieve this goal. These were: the type and volume of liquid added to the waste system and the

INTRODUCTION INFECTIOUS radioactive waste is generated in medical and biological research facilities. Ideally, the infectious nature of this waste is inactivated on-site to minimize health risks associated with its handling and transport. A method (Stinson et al. 1990) has been developed previously for treating and disposing of heat-sensitive infectious radioactive waste, such as HIV. This study increases the utility of the earlier model to ensure inactivation of the conventional steam sterilization indicator, Bacillus stearothermophilus. Once optimum sterilization conditions were identified, radioactive material was used to confirm the integrity of the system. In the previously developed model system, dry solid waste is placed in double polypropylene bags; 300 g (one pint) of absorbent is added to the bags. The solid waste is then accumulated in the bags. A minimal amount of water is added to the waste bag. The charcoal filter drying tube is secured at the opening of the bag by autoclavable rubber bands or autoclave tape. A Diack control$is taped to the outside of the bags. Both the Diack control and autoclave tape are temperature sensitive. The Diack control pellet melts and changes color when a temperature

Environmental Health, 80 E. Concord St., T-3C, Boston, MA 02 1 182394, Phone: (617) 638-4620. Diack, Inc., Beulah, MI.

(Manuscript received 15 October 1990;revised manuscript received 23 January 1991, accepted 1 I February 1991 ) Corresponding author and address for reprint requests: Margaret C. Stinson, Boston University School of Public Health, Department of 137


Health Physics

determination of the minimal amount of absorbent needed to ensure that the system be considered "dry solid waste." The amount and type of liquid added to the system was first analyzed. The Centers for Disease Control and biohazard bag manufacturers'' recommend that bags be kept open and water be added prior to steam processing (CDC 1980). The premise is that water placed among the waste will generate its own steam and replace residual air that might interfere with the steam sterilization process (Rutala et al. 1982). Additionally, the thermal conductivity coefficient of water is much higher than air, facilitating heat transfer via convection through the steam that has been generated within the bags. This is important for penetrating any dry air pockets found in areas within the autoclave bags. In our model, the radioactive hazard requires that the bag must be closed and air-flow restricted through a charcoal vent. We sought to overcome the disadvantage this closed system introduces by using a liquid that enhances the effectivenessof the steam sterilization process. Once a suitable solution was discovered, we were able to determine the minimal amount of absorbent required by the system. Processing temperature plays a vital role in steam sterilization. Ideally, the sterilization temperature is reached quickly and maintained throughout the process. The constraints described increase the time required to heat the system, subsequently reducing the amount of time the waste is exposed to the desired temperature (121°C). The theoretical relationship between sterilization time and temperature is a logarithmic function (Nickerson and Sinskey 1972) expressed as:




where t is the sterilization time at temperature r; F, is the sterilization time at 121"C (250°F); and z is the increase in temperature required to increase the kill rate by a factor of 10. For B. stearothermophilus, F, = 11.8 min and z = 11.3"C (20.5"F) (Lee et al. 1979). This equation demonstrates the important effect that even small temperature changes may have on achieving the predicted process parameters. It is known that a saturated steam processing time of 1 1.8 min at 121 "C is required to achieve a 6D reduction (99.9999% kill) of B. stearothermophilus (Whitbourne et al. 1976). (The D value is the thermal death time and is a measure of the time needed to reduce the spore population by one log when exposed to 121 f0.5"C.) By reducing the processing temperature by only 1"C to 120°C, the calculated D value

July 1991, Volume 6 1, Number 1

increases from 11.8 min to 14.8 min. Time difference may determine whether B. stearothermophilus inactivation is successful. Our technical goal was to vary the described process parameters so that adequate processing time and temperature could be assured. MATERIALS AND METHODS A gravity displacement autoclave' operating at 121"C was used throughout the experiment. Two biological monitoring systems that contained spores of B. steurothermophilus, spore strips' and Kilit ampules, * * were used to monitor the sterilization of the bag contents. Spore strips (Raven lot 315041) with an assayed population of 1.7 1 x I o colony-forming units per strip were used in the first experiment to test the single-bag system. The D-value for these spores was 1.85 min. The corresponding minimum kill time was calculated to be 9.68 min. The time for survival was calculated to be 20.78 min. Spore strips (North American Science Associate lot S2970 1 ) with assayed population of 1.4 X lo6 colonyforming units per strip were used for all remaining experiments. The D-value for these spores was 1.5 min. The corresponding minimum kill time was calculated to be 9.22 min. A period of 18.22 min at 121"C was calculated for a survival. All spore strips were incubated for a 7-d period at 56°C in Trypticase Soy Broth and monitored for growth. Nonautoclaved spore strips were used as controls throughout the experiment. One control was tested with each autoclave run. Kilit ampules contained a spore suspension in a culture medium with a pH indicator. Ampules were incubated at 56°C and examined for the appearance of turbidity and a color change, indicating a failure of the sterilization process. If no change in color occurred within 72 h, it was held for 7 d and a final observation made. Nonautoclaved ampules were used as controls throughout the experiment. One control ampule was tested with each autoclave run. A standardized waste load weighed approximately 500 g with added liquid and absorbent. Volume of liquid and amount of absorbent varied with each experiment. All waste bags were placed in a stainless steel container for autoclaving. The standardized waste load consisted of: 20 pipette tips, 10 disposable petri dishes, four pairs of latex gloves, 10 test tubes (glass), and absorbent plasticbacked bench paper (304.8 cm X 9 1.4 cm). Initially, time, moisture, and absorbent requirements were determined for a single-bag charcoal filter system with no waste contained in the bag. For each condition

'American Sterilizer Co., Erie, PA.


II Baxter


Bio-Check Biohazardous Bags, Baxter Healthcare Corporation, McGaw Park, IL.

'North American Science Associates, Inc., Northwood, OH and Raven Biological Laboratories, Omaha, NE. * * BBL Microbiology Systems, Cockeysville, MD.

Autoclave inactivation of infectious waste 0 M. C. STINSONet al.


Table 1. Time, moisture, and absorbent requirements for inactivation of B. stearothermophilus spore strips in an empty, single-bag charcoal filter system.

Table 2. Time and moisture requirements for inactivation of B. stearothermophilus spore strips in an empty, double-bag charcoal filter system.

Time (min)

Water (mL)


Inactivation ratio"


Time (min)

Water (mL)

Inactivation ratio

Inactivation (70)


30 30 30 30 30 30 30

5 5 5 5

0 90 150 300 0 90 150

4:4 1 :4 0:4 04 4:4 04 0:4


30 30 45 45 60 60

5 10 5

1:lO 3:lO 1010

10 30

10 5 10

1O:lO 1010 1O:lO

10 10 10

(%I 25 0 0 100

100 100

0 0

a Inactivation ratio is the number of spores strips (or ampules) inactivated:total number of spores (or ampules) tested.

tested, two spore strips were placed within a single 30.5 X 6 1.O cm ( 12 X 24 in.) polypropylene bag.tt A charcoal vent filter was placed at the opening of the bag and the bag sterilized for 30 min (Table 1 ). Time and moisture requirements were then determined for a double-bag charcoal-filter system with no waste contained in the bags, For each set of variables, three bags were tested containing a total of 10 spore strips (Table 2). Distilled water and disinfectants with known sporicidal activity were investigated for sterilization efficiency. These included: an iodophor, 1% Wescodyne,$$ a hypochlorite, Clorox bleach, $$ and hydrogen peroxide ( 3% solution). The standardized waste load was placed in polypropylene bags with spore strips distributed throughout. Spore strip positions were chosen to represent adverse conditions for steam penetration. These were wrapped inside approximately 9 1.4 cm2 ( 3 ft2)of bench paper, at the bottom of the interior waste bag, inside a test tube, inside a petri dish, and inside two latex gloves that were contained within approximately 9 1.4 cm2( 3 ft2)of bench paper. Volumes of 5, 10, 15, 25, and 50 mL of water, Clorox, and Wescodyne were tested for a 60-min cycle; 25 and 50 mL of hydrogen peroxide were tested. Additionally, the effectiveness of hydrogen peroxide solution (25 and 50 mL) was tested using Kilit ampules in standardized waste loads. Once the effectiveness of hydrogen peroxide had been established, the addition of absorbent, Speedi-Dri,1111 was studied, Spore strips and ampules were placed in the wasteload between a layer of bench paper. The dimensions of this paper were 152.4 X 91.4 cm ( 5 X 3 ft.). Each bag contained three spore strips and ampules. The volume of hydrogen peroxide remained constant at 50 mL while the weight of absorbent added varied from 0 to 300 g. To compare the effectivenessof the peroxide to water, several runs were conducted using 50 mL of distilled water and 150 g of absorbent. Clavies, Bel-Art Products, Pequannoc, NJ. **WestChemical Products Inc., Princeton, NJ. Clorox Company, Oakland, CA. Clint Sales, Beverly, MA.

100 100

Time and temperature profiles were recorded for three independent hydrogen peroxide and water trial runs. An Omega thermocouple probe" was placed within waste loads and the temperature recorded at 2-min intervals throughout the 60-min cycle. The most effective system was then tested using 35Slabeled cysteine, a radioactive material that is known to be volatile when heated (Stinson et al. 1990). The goal of this test was to confirm the integrity of the system when it contains volatilized radioactive material. The radiation containment test system included the standardized waste load, 50 mL of hydrogen peroxide, 150 g of absorbent, and 1.4 X lo8 Bq (67 pCi) of "S-labeled cysteine, processed for a 60-min sterilization cycle. Radiation contamination was monitored in the autoclave using techniques previously described (Stinson et al. 1990). RESULTS Tables 1 and 2 merely confirm expectations that double polypropylene bags would increase the time required for inactivation of bacterial spores. The double bagging is required for radiation protection purposes. Double bagging increased the time requirements for 100% kill by 15 min when 5 mL of water was added to the system. Increased volume of water in the bags improved the inactivation rate. All bags remained intact with no observable leaks. Growth was observed in all controls. The absorbent, Speedi-Dri, was effective in the absorption of any liquid condensation droplets that formed as the bag cooled to room temperature. While effective in its absorption capacity, Speedi-Dri acts as a heat sink more time is required to heat the mass of the absorbent and the additional waste in the bag. Table 1 demonstrates the adverse effect of increasing the volume of absorbent on percent inactivation of spores under constant autoclave conditions. Adding waste to the system changed the requirements substantially. Figure 1 shows that only 40% inactivation was obtained when adding up to 50 mL of water to the system. Neither bleach nor Wescodyne enhanced steam inactivation of B. stearothermophilus (Fig. 1). The highest


Omega HH-70 Pocket Size Thermometer, Omega Engineering, Inc., Stamford, CT.



Health Physics nn

50 mL of added liquid plus any additional moisture from waste or steam entering the bag through the charcoal filter. When 50 mL of hydrogen peroxide was added to the double-bag charcoal filtration system containing 1 50 grams of absorbent and a simulated waste load, 56 out of 58 (97%)of the biological indicators were inactivated. When the same volume of water was added to the bags, only nine out of 28 (32%) of the indicators were inactivated. Figure 2 indicates that model systems containing hydrogen peroxide solution rather than water achieve consistently accelerated temperature profiles until the plateau of 12 1 "C is obtained.







Water Wescodyne

e n a




July 199I , Volume 6 1 , Number 1

Clorox Hydrogen peroxide






Volume (ml)

Fig. 1. Effectiveness of varying volumes of water, Wescodyne, Clorox, and hydrogen peroxide in the inactivation of bacterial spores in a double-bag charcoal filtration system.

percent inactivation occurred when 50 mL of solution was added to the system. This resulted in 2 1% activation using bleach and 25% inactivation using Wescodyne. The addition of hydrogen peroxide solution to the bag improved results significantly ( Fig. 1 ) . When 50 mL of peroxide was added to the system, 49 out of 50 spore strips (98%)were inactivated. Paper products in the waste load turned brown. Visible condensation droplets were formed on the inside of the bag. Using 25 mL of solution provided mixed results; nine out of 20 spore strips (45%) were inactivated. Similar results were obtained when Kilit ampules were used rather than spore strips. When 50 mL of peroxide was added to the system, 16 out of 20 ampules (80%) were inactivated. Fifty percent inactivation was obtained in the ampules when 25 mL of peroxide solution was added to the system. Table 3 documents the dramatic effect of the weight of the absorbent on the efficiency of inactivation of the biological indicators. Since the absorbent acts as a heat sink, the amount added was minimized. One-hundred fifty grams was found to be more than adequate to absorb

Autoclaving is one generally accepted approach to sterilizing potentially hazardous microbiological waste. It is considered to be the most reliable and easily controlled procedure for on-site decontamination (Richardson and Huffaker 1980) and is widely practiced. The processing time for the sterilization of this waste depends on the load characteristics and containment. Load characteristics include the volume and type of liquid added and amount of absorbent added to the waste. Our method for containing radioactivity during sterilization introduces many constraints into the system. These include the barrier properties of the polypropylene bag and charcoal filter, double-bagging, and the addition of absorbent. Sealed polypropylene bags prevent steam penetration. Steam entry is limited to the orifice of the charcoal filter. Using an essentially sealed bag is not ideal. It requires the addition of a liquid to what is considered "dry, solid waste."






? Y

e z







Table 3. Effect of absorbent and hydrogen peroxide on the inactivation of B. stearothermophilus indicators used in a doublebag charcoal filtration system. Type of solution (50 mL) Hydrogen peroxide Hydrogen peroxide Hydrogen peroxide Hydrogen peroxide Distilled H20

Weight of absorbent Inactivation Inactivation (g) ratio (W)

0 90 I50 300 150

4050 6:12 5658 2:6 928


90 20 50 2 25 97 f 18 33 f 27 32+_ 12









Time (minutes)

Fig. 2. Temperature profiles in loaded bags containing 50 mL of hydrogen peroxide (3%)or water during a 60-min autoclave cycle at 121°C (250°F).

Autoclave inactivation of infectious waste 0 M. C. STINSONet al.

Containing the waste within two polypropylene bags affected both heat and steam transfer. The additional bag increased the amount of time required for heat to transfer from the autoclave environment and established equilibrium conditions with the interior of the bag. Certain physical characteristics of a waste load may interfere with steam penetration and the attainment of desired thermal conditions. Examples of waste having such adverse effects would be disposable petri dishes that encapsulate pockets of air and culture media, impeding steam penetration as dishes melt together during the autoclave process. Similarly, melted latex gloves may form enclosed air pockets within the gloves. The advantage of using spore strips and ampules as biological indicators is the ability to monitor the uniformity of the steam sterilization process. Indicators may be placed in areas with limited steam penetration to enable the monitoring of areas where temperatures may be lower and steam penetration poorer, resulting in inadequate processing. In practice, every waste load will vary in contents or configuration. In these experiments, the standardized waste load was chosen to represent typical solid radioactive waste from the laboratory. To limit experimental variables, the same randomly distributed materials were used for each load, and spore strips were distributed in the most difficult-to-penetrate areas, such as inside petri dishes, within latex gloves, or wrapped in bench paper. Placement of indicators in this manner was intended to simulate a worst-case condition. Safe radiation practices require the use of a tub as further protection against autoclave radioactivity contamination. Previously, a polypropylene tub was used with the model system (Stinson et al. 1990). Investigators have shown that heat transfer is better in a stainless steel tub than in one constructed of polypropylene (Gillespie and Gibbons 1975; Lauer et al. 1982; Rutala et al. 1982). Stainless steel tubs were used throughout this experiment and are recommended for those who intend to use this system. Earlier experiments showed survival of B. stearothermophilus spores after a steam sterilization time of 60 min using water as a solution. We sought to obtain inactivation of B. stearothermophilus in our system using various antibacterial solutions and a steam sterilization time of 60 min. In search of improved performance, these solutions were tested independently and jointly with varying amounts of absorbent. Hydrogen peroxide has been recognized widely as a bactericide. It has many applications including aseptic packaging and the sterilization of equipment (Turner 1983). We found that the hydrogen peroxide solution demonstrated a much higher efficiency of B. stearothermophilus inactivation than other agents with known sporicidal activity, such as bleach or Wescodyne. Additionally, biological indicators were inactivated more efficiently when hydrogen peroxide was added to the mixed waste bags than when an equal volume of water was added. In the model system studied, there was 100%inac-


tivation of spores when waste bags containing 50 mL hydrogen peroxide (3%) were autoclaved for 60 min at 121°C. After 150 g absorbent was added to the model system, 98% inactivation of the bacterial spores was maintained. The 2%not inactivated were located in areas with limited steam penetration, such as inside rubber gloves and inside petri dishes. Why does hydrogen peroxide work while water doesn't? We believe that there is a synergistic activity between hydrogen peroxide and the autoclave conditions that significantly reduced steam processing time. Specifically, the decomposition of hydrogen peroxide to water and oxygen is an exothermic reaction, The following hypothesis is proposed: Heat is released during the reaction. This heat facilitates generation of heat inside the bag (Fig. 2 ) . The decomposition of hydrogen peroxide may be represented as: 2H202 + 2H20


0 2

Heat of formation: -44.8 kcal mole-' Molecular weight: 34 g mole-' Boiling point: 150°C Using this information to calculate the heat released in this exothermic first-order reaction, we have: AH


Hprd- , X



50 mL of H 2 0 with 3% H202 = 1.5 mL of H202 (1.5 mL H202)(1.44 g mL-') (44.8 kcal X 34 g-') = 2.8 kcal of heat released to the system. One calorie is required to raise the temperature of 1 g of water 1 "C. The 2.8 kcal of heat released and distributed throughout the waste represents sufficient heat to raise the temperature within the bag by approximately lO"C, as seen in Fig. 2. There are several other possible explanations for the increased effectiveness of peroxide. The sporicidal action of peroxide has been shown to increase at higher temperatures (Turner 1983). If hydrogen peroxide were volatilized without decomposing to water, direct contact with the antibacterial peroxide solution would provide efficient inactivation of spores. Other suggestionsinclude hydroxyl radical formation and an exothermic reaction produced by the hydrogen peroxide reacting with the absorbent. Further studies would be necessary to determine the exact nature of its increased effectiveness. The pH of the system was determined before and after the autoclave process. No change was observed, and the pH was deemed not to be a critical factor in the sterilization process. Radioactivity containment of the system has been previously established (Stinson et al. 1990). These results were validated using the modified model system containing hydrogen peroxide solution and a heat volatile radionuclide, 35S.Our results confirm the radiation containment properties of the system. Future studies should be


Health Physics

performed to retest the modified containment system using various radionuclides.

July 1991, Volume 6 1, Number 1 Acknowledgments-We thank Mitch Galanek and Frank Masse for their support, and the MIT Biohazard Assessment Office for use of their facilities and assistance in microbiological assessment.

REFERENCES Centers for Disease Control. Disposal of solid wastes from hospitals. Atlanta, GA: Bacterial Diseases Division, Bureau of Epidemiology, Center for Disease Control; 1980. Gillespie, E. H.; Gibbons, S. A. Autoclaves and their dangers and safety in laboratories. J. Hyg. 75:475-487; 1975. Lauer, J. L.; Battles, D. R.;Vesley, D. Decontaminating infectious laboratory waste by autoclaving. Appl. Environ. Microbiol. 44:690-694; 1982. Lee, C.; Montville, T. J.; Sinskey, A. J. Comparison ofthe efficacy of steam sterilization indicators. Appl. Environ. Microbiol. 37:1113-1117; 1979. Nickerson, J. T.; Sinskey, A. J. Microbiology of foods and food processing. New York American Elsevier Publishing Co.; 1972: 44. Richardson, J. N.; Huffaker, R. H. Biological safety in the clinical laboratory. In: Balows, A.; Hausler, W. J.; Truant, J. P., eds.

Manual of clinical microbiology. 3rd ed. Washington, D.C.: American Society for Microbiology; 1980960-964. Rutala, W. A.; Steigel, M. M.; Sarubbi, F. A., Jr. Decontamination of laboratory microbiological waste by steam sterilization. Appl. Environ. Microbiol. 43:1311-1316; 1982. Stinson, M. C.; Galanek, M. S.; Ducatman, A. M.; Masse, F. X.; Kuritzkes, D. R. Model for inactivation and disposal of infectious human immunodeficiency virus and radioactive waste in a BL3 facility. Appl. Environ. Microbiol. 56:264268; 1990. Turner, F. J. Hydrogen peroxide and other oxidant disinfectants. In: Block, S. S., ed. Disinfection, sterilization and preservation. 3rd ed. Philadelphia: Lea and Febiger; 1983: 240-250. Whitbourne, J. E.; Ferris, B. K.; Morien, L. L. Dynamics of steam sterilization. Dev. Ind. Microbiol. 18:353-363; 1976.

Autoclave inactivation of infectious radioactive laboratory waste contained within a charcoal filtration system.

A model system was developed previously for disposal of solid laboratory waste that is both radioactive and heat sensitive, e.g., HIV. A double polypr...
201KB Sizes 0 Downloads 0 Views