Validating Autoclave Cycles for Carcass Disposal in Animal Biosafety Level 2/3 Containment Laboratories

Methods

Water Tests

A steam sterilizing autoclave (model PSS5-B-MSDD, Primus Sterilizer, Omaha, NE) was validated in this study. The cycle parameters (cycle A, Table 1) were first tested using 21-qt, 22-gauge stainless steel pans (20 in width × 12 in length × 6 in height; Central Restaurant Products, Indianapolis, IN) filled with cold tap water to within 2 inches of the top of the pan. The pan lids were placed upside down on each pan; this placement allowed a piece of string to be looped around the pan lid. The string was used to suspend a BI halfway into the pan (Figure 1). Four pans were placed in the autoclave, stacked 2 on 2, and the cycle was run.

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Table 1.

Autoclave Parameters Tested.

Cycle Name	Experiment	Sterilizing Temperature, °C	Pulse Pressure, psi	Number of Prevacuum Pulses	Sterilizing Time, min
Cycle A	Water pan tests and ferret test 1	123.1	20	3	240
Cycle B	Ferret tests 2 and 3	125	15	5	240

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Figure 1.

Configuration of water pans in the autoclave. Pans were stacked one on top of the other with the biological indicators suspended halfway into the water.

Often carcasses are frozen for multiple reasons prior to autoclaving. To mimic the conditions of frozen carcasses, the pans were filled with cold tap water and placed in a –80°C chest freezer until frozen. The pans were removed from the freezer, and a drill was used to core a 1-inch diameter hole, approximately 6 inches deep, into the center of the ice. The hole was then filled with cold tap water and the BI suspended inside the hole as described for the water tests. The pans were configured inside the autoclave as indicated in Figure 1, and cycle A was used for the initial water test.

Ferret Carcass Test 1

Sixteen ferret carcasses, each weighing approximately 1.5 to 2 kg, were used to validate autoclave cycle A. BIs were labeled and implanted into the carcasses (Figure 2). To test different bag-load capacities, 1, 2, or 4 carcasses were placed inside 2 autoclave bags. The bags were placed inside the steel pans, to a total of 4 bagged carcasses per pan (Figure 3). The 4 pans were placed inside the autoclave (Figure 4), and the cycle was run (cycle A, Table 1).

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Figure 2.

A 1- to 2-inch incision was made between the ribs on either side of the thoracic vertebrae, approximately 2 to 3 inches below the neck area (opposite the chest/abdominal cavity area). Scalpels were used to cut into the flesh and through to the chest cavity, taking care to minimize the width of the incision hole. To implant the biological indicators (BIs) inside the abdominal cavity of the carcass, a flexible wire write-on metal tag (VWR, Radnor, PA) was wrapped around the BI (A), then the BI was fed through one of the incisions made on the back of the carcass and fixed into place by twisting the wire around the spine through the second incision hole (B).

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Figure 3.

Placement of ferret carcasses inside pans. Each pan contained 4 carcasses with either (A) a single carcass per bag, (B) 2 carcasses per bag, or (C) 4 carcasses per bag. Each carcass was implanted with a biological indicator (BI). An additional BI was placed between the 2 middle carcasses in the 4-per-bag configuration.

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Figure 4.

Configuration of pans within the autoclave. The 4-per-bag carcass pans were placed one on top of the other at the front of the autoclave. The 2-per-bag carcass pan was placed on top of the single-bagged carcass pan at the back of the autoclave. The picture on the right shows how much space the pans use within the autoclave.

Once the cycle was completed, the pans were removed from the autoclave and allowed to cool for 4 hours. The BIs were removed from the carcasses and cleaned with 70% isopropanol, dirty tags were removed and replaced with fresh labeled tape, and BIs were incubated for 48 hours at 55°C.

Ferret Carcass Tests 2 and 3

For test 2, 8 carcasses were prepared as indicated above. Four carcasses were placed inside a double bag and placed inside a steel pan. Two BIs were placed between the carcasses (Figure 5). On the basis of the results of the first carcass test, an augmented cycle (cycle B, Table 1) was established.

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Figure 5.

Configuration of carcasses in autoclave for second ferret test. (A) Four carcasses were placed in a double-bag with 2 biological indicators placed between the carcasses. (B) Two pans, each containing 4 carcasses, were stacked and placed inside the autoclave.

For test 3, a 6-inch section of a 25-mL disposable serological pipette (with a diameter slightly wider than the BIs) was placed inside the chest cavity of each carcass to maintain a spot to place the BI after freezing. Eight carcasses were first frozen by placing in a –80°C freezer overnight (a minimum of 8 hours). The carcasses were removed from the freezer, a BI was placed inside the chest cavity through the pipette, and 4 carcasses were placed inside a double bag and into a metal pan. Two BIs were placed between the carcasses, and autoclave cycle B was run as indicated for test 2 (Figure 5).

Results

Ferret Carcass Test 1

Of the 18 test BIs that were exposed to the sterilization cycle, 17 passed validation according to the established criteria. The 1 BI that did show growth was placed between the carcasses from the 4-per-bag configuration in the top front pan (Figure 6).

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Figure 6.

Results of ferret carcass test 1. (A) The biological indicator (BI) shown in the red circle failed the sterilization cycle. The control vial is shown at the top right. (B) The location of the failed BI is indicated.

Ferret Carcass Tests 2 and 3

Because of the failed BI from test 1, the validation cycle was altered by increasing the temperature of sterilization from 123.1°C to 125°C, decreasing the pressure of the prevacuum pulses from 20 to 15 psi, and increasing the number of prevacuum pulses to 5 from 3 (see Table 1 for cycle comparison). After completion of the sterilization cycle for both the fresh and frozen carcasses, all test BIs passed and showed no growth. After the sterilization cycle from test 3, the section of serological pipette that was embedded in each carcass was slightly deformed because of the exposure to high temperatures. The BIs that were inside the pipettes remained intact but could not be extracted from the pipettes (Figure 7). The pipette-containing BIs were incubated as is for 48 hours.

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Figure 7.

Results of frozen carcass test. (A) On the left, the control biological indicator (BI) exhibited growth (turbid, yellow color) as expected. On the right, the BIs placed between the carcasses exhibited no growth. (B) The BIs placed inside the pipettes exhibited no growth. Shown here is how the pipette deformed and melted around the BI.

Discussion

Decontaminating potentially infectious waste, including carcasses, is critical in a high-containment (ABSL-3) area, and full sterilization of waste is imperative if select agents are involved. ³ The parameters should be validated to ensure that laboratories are achieving these goals of their decontamination and sterilization protocols. The validated solid-waste cycle that is typically used in the ABSL-3 containment area is sufficient for most noncarcass solid-waste loads, but previous studies conducted at this facility showed that it was insufficient to inactivate BIs placed inside ferret carcasses (data not shown). The importance of running validation studies to confirm decontamination of new and different types of waste is demonstrated here. It is also important to note that users must be familiar with their specific autoclaves and be familiar with manufacturers’ recommendations for selecting and programming cycles.

Ideally, decontamination and sterilization protocols will be validated prior to initial work with the infectious substances and then confirmed with the agent of interest or a proper analog in the actual conditions for which the protocols are being developed. Identifying a proper model for decontamination and sterilization protocols can prove difficult. ⁴ Studies with mice generally provide abundant carcasses with which to work, but medium-sized to large animal models are quite expensive and as such are performed more rarely with much smaller animal numbers. We would prefer to be able to perform these validation experiments cheaply and without the need for animal carcasses. The water tests described above used physical volumes well in excess that of the ferret carcasses, and testing BIs always proved successful in validating the cycle, even in frozen ice blocks.

However, testing in carcasses was not successful with our first cycle in even room-temperature carcasses. One of the BIs placed between the carcasses failed, which we speculate may have been the result of an insulated air pocket created from overlapping fur between 2 of the carcasses. If the BI was located in this air pocket, the steam may not have fully penetrated this area, resulting in viable G stearothermophilus spores. Another possibility is that the double bags used to contain the carcasses could have created insulating air pockets in the space between the bags or between the bags and the carcasses. Previous studies have shown that air trapped in a load can create insulating pockets that interfere with thermal transfer and result in longer cycle times needed to reach sterilizing temperatures.^7,8 Once 2 extra prevacuum pulses were added to the decontamination cycle, all of the BIs passed. Adding extra pulses has been previously shown to reduce the equilibration time needed for thermocouples placed within dry goods loads ⁹ and, in this study, could have better allowed the steam to reach any air pockets between the ferret carcasses. It is also important to note that the physics of thermal conductivity of the tissues and bones compared with that of water may also account for these differences, but this setback highlights the need for better medium-sized to large animal decontamination and sterilization validation models.

The placement of the BIs inside the carcasses was carefully considered. The smallest incisions possible were made to keep the carcasses intact and to mimic an unaltered, whole carcass as much as possible while still adhering to approved euthanasia secondary measure procedures. By keeping the carcasses as intact as possible, the manipulation of infectious carcasses is reduced, therefore mitigating the risk for exposure to personnel.

The decision to test frozen carcasses was made to allow future experiments in which same-day autoclaving of waste is not possible and infectious carcasses must be stored in the containment area for a period of time. Unfortunately, these BIs could not be frozen with the carcasses, because the type of indictor used is no longer efficacious once frozen and could have resulted in a false negative. ¹⁰ One shortcoming of our method to use the 25-mL serological pipette is that during autoclaving, the BI is not completely encapsulated in the frozen carcass with the open tube allowing access of steam to the BI. Depending on when the pipette tube deformed, this could have sealed off the BI and possibly created a better simulation of the BIs that were sealed inside the nonfrozen carcasses. One follow-up for this study would be to identify a method of either including a freezer-stable BI with the carcass before freezing or another form of postfreeze measurement (ie, a real-time temperature probe to ensure that temperature is achieved at the proper length of time). Another possibility could be to seal off the 25-mL serological pipette with the BI inside before beginning the autoclave cycle.

Future directions include repeating the ferret test studies to ensure the results can be repeated. Finding a better method of implanting the BIs into frozen carcasses is also being explored. Additional studies are planned to look at increasing the number of carcasses and pans per load. Testing the cycles described here with other types of carcasses (eg, rabbits and other medium-sized to large animals) is also planned.

Conclusions

The autoclave cycles described here were validated for a total load of 16 ferret carcasses (of approximately 1.5-2 kg each) with up to 4 carcasses per stainless steel pan and 4 pans per load. The cycle was also validated with previously frozen ferret carcasses, demonstrating that the chosen cycle is sufficient to sterilize even fully frozen carcasses.