4th National Symposium on Biosafety

Special Containment Devices for Research Animals

William J. White, VMD, MS
Senior Director, Professional Services
Charles River Laboratories
251 Ballardvale Street
Wilmington, MA 01887
508-658-6000, x1347

How do you evaluate containment devices or systems? Perception is reality. How you perceive a containment system becomes your reality and you end up using it that way. With any luck your perception won't conflict with someone else's reality because, when it does, it is clear that you haven't really understood the containment system. You haven't thought it through. There are some questions to be asked. First, what are we trying to do? Are we trying to keep things in or keep things out? That might sound like not a real good question to ask here, but animal care is really focused on keeping things out, rather than in. So many of the devices are really designed to do that. This brings me to my next little pearl, bio-exclusion, keeping things out of a containment vessel is not equal to bio-containment, keeping things in. A device designed for bio-exclusion is not necessarily applicable to bio-containment. Certainly in many cases, not without modification.

Look at some of the strategies used to contain biohazards in the laboratory and realize that they are at odds with strategies to house animals. When we house animals we like to have high air flows to get a lot of ventilation, perhaps to get rid of odors or perhaps to dilute out infectious organisms. In terms of bio-containment it becomes expensive to dump lots of conditioned air. In animal manipulation in a standard laboratory, you try to get frequent bedding and cage changes, but in bio-containment you'd like to minimize that so you aren't transporting large amounts of materials that can cross the barrier at any given time. We tend to use reusable supplies in a standard animal operation, but in bio-containment, there is a tendency to use disposabl equipment and supplies. Frequent handling is something that can be done in a normal animal situation, but you really don't want to do that in bio-containment if you can help it.

Another thing we've got to worry about is understanding the nature of the hazard. If you don't know what you're dealing with, just try to apply a general solution. Many times that's going to fail. You really need to understand the nature of the organism. Dr. Newcomer and others spent time bringing to our attention things that we need to contain. Remember that containment is more than just air movement. Many of these systems are geared to controlling air movement, but in fact, fomites are a principal means of transmissions of infectious organisms. Also, we must not forget direct contact. If you look at the whole picture, don't focus just on air, but also on other modes of transmission.

The chance of acquiring a significant exposure is proportionate to a lot of things. The concentration, the agent, the environment, the total length of exposure, whether you have a single exposure or multiple exposures, the persistence of the agent in the environment, and the infectivity of the agent, as well as many other factors. You have to appreciate these variables before you select a bio-containment system.

Everything we do has risks. There are risks associated with all containment systems and we need to be aware of what they are. What are some of these risks? Movement of people, materials and animals across the barrier can be a risks. Every time that happens, there's a chance that something can get out. Incomplete disinfection or sterilization of materials, inappropriate decontamination, or improper gowning of personnel are risky behaviors. Remember, people tend to disregard procedures if they're too complex or if they are inadequately trained in the procedure. Also, remember that there can be breaks in gloves, and clothing rips. Do you have a plan to deal with that?

Lack of understanding of the rationale behind this system is often a problem. If people don't understand how the system works, they tend to get creative and that's when the problem occurs. If you don't understand how the system works, it is just a black box, and it is a risk. Don't forget that equipment fails. Don't forget that emergencies occur. Power failures, escaped animals, a break in the integrity of physical barrier, something falls through the roof, and/or inadequate maintenance are risks.

People forget that when you buy a system it doesn't last forever. You have to maintain it. Motors burn out, filters get clogged, etc. Failure to regularly evaluate the performance of the system is something that everybody tends to forget about. Do we really know that the system is working? Remember that no containment system is perfect. There's always a trade off. We're always making compromises. If there was a perfect containment system everybody would use it.

There are a number of other different basic bio-containment strategies to remember: containing organisms may involve filtration; absorption or adsorption; containerization; shielding in the case of radio isotopes; dilution; chemical conversion; and incineration. All of these could be applied to any animal containment system. Autoclaving is important, but not everything has to be completely sterilized. There are other ways to kill life forms that might be sufficient for containment purposes. You need to understand the agent enough to determine whether you need to go through more rigorous procedures or whether others will be appropriate. Sterility is not always required. In many cases it is, but it is not always required.

Do you have a quantifiable measure of success? Once you put the animals in containment, how do you know things haven't gotten out? Are you waiting for somebody to walk by and get the disease? How do you know? Certainly there are a number of strategies you could use. One is to survey for the agent on the non-contaminatedside of the barrier, assuming that some kind of reliable assay is available. This assumes that you have an effective sampling methodology, you know where to sample, how often to sample, and how many samples to take. You almost need to use a sentinel population outside of the barrier, but if you find the agent, you already have indications of infection that's been spreading all over the place.

One could survey for a marker on a non-contaminated side of the barrier. This assumes that some kind of marker can escape. If that is true, then so can the agent. It assumes that the characteristics of whatever marker you use are similar to the agent. Some possible markers are fluorescein label particles. In the case of isolators, one can use inert tracer gases such as helium or cold isotope labeled compounds and particles. Difficult as it is, you really ought to have some measurement of success. Otherwise, it becomes an exercise in faith that the containment device is working.

What about disaster planning? Sometimes things just don't go the way they are supposed to go, so you really need a disaster plan. What are you going to do if there's a breach in the barrier? That can happen, and it is surprising how many people haven't thought about that. So we need to set up evaluation criteria for any system you plan to use.

The first thing I would ask is whether or not it is a complete system. Does whatever device or series of items that you put together provide a physical barrier to all modes of microorganism release? Does it provide a well thought-out method for moving supplies and equipment across the barrier? Or is it some cumbersome task that nobody bothered to think about until after the barrier has been constructed or the piece of equipment has been purchased? Does it provide for a means for decontamination? Does it provide a means for experimental manipulation of the animals without contaminating them or releasing contamination to adjacent units? Does it prevent microbiological contamination of adjacent units which can occur by any of the routes already mentioned? Does it provide a logical means of moving animals of known health status across the barrier? You know how to move supplies easily, but what about a living breathing creature in a containment system that is in operation? Can you introduce new animals and then take them out?

How heavily does the device rely upon the goodwill, training and understanding of operating principles by the personnel using it? Some equipment is user-friendly. Other is extremely technique dependent. Remember that all systems can fail. Every system will fail. I will guarantee that any bio-containment system you put together will fail. I can't tell you when, but it will happen. In selecting a system you need to think about the cost of failure. What's the cost? How easy is it to recycle, to get this back into operation? What's the cost in terms of time to get it back into operation? What are the numbers of animals at risk? What's the value of the lost research? I don't know if anybody could put a value on that, but there probably is.

The costs and risks related to a release of the biohazard must include people in the equation. In North America we're a very litigious society, and an awful lot of lawyers reside here. These are things that we need to think about in terms of total costs of failure.

Purchase costs do not include operating costs. You spend three, four, five, ten, or a hundred thousand dollars on something, but you forget that you really need to put in utilities, peoples' time, and a variety of other things to make it operate. The more complex and cumbersome the device, the more that operating cost skyrockets. You may need to sterilize supplies in or out, or you may need to proide clothing and laundry services. Showers don't work without water, and utilities are not free.

Ease of access. How easy is it to get to the animals for experimental manipulation? If it is difficult people aren't going to use the system. They're going to find a way around it. How easy is it to perform all husbandry task? How easy is it to introduce new animals? The amount of time that animals are in an unprotected setting is important. With some systems you actually take them out of that unprotected setting to manipulate them. You transfer them to a laminar flow hood or other places, and it is during that period that the containment system can easily fail. It is much less expensive to think through how a system will be used and realize its limitations before you commit to it then after the fact.

There are four important types of bio-containment systems: Static micro-isolators, isolators, barrier rooms, and what I call barrier within a barrier. No one system is appropriate for all circumstances. One size does not necessarily fit all. As an aside, some people think that microorganisms are repelled by lab coats, just shed right off them. Wrong!

What we're trying to do is to contain microorganisms within a defined space that we can manipulate. The most commonly considered bio-containment system or device for animals is a barrier room. The principles of operation are: it is a sealed room that can be disinfected or sterilized; all supplies are disinfected or sterilized either in or out; and all animals crossing the barrier are free of agents to be contained or excluded. These are the general characteristics of the barrier room. There are also bio-exclusion barrier rooms, where basically the same operating principles apply. The problem that many people don't seem to realize is that in a barrier room that's designed for bio-exclusion to keep things out, all the mechanical systems function just the opposite of what you need for bio-containment. Air pressure differentials are totally opposite. If you want to keep things in, you want a negative pressure; keep things out with positive pressure. Just because you see a barrier suite in your facility, it doesn't mean that you can automatically throw things in there for bio-containment. Remember that it is employee and technique dependent, and logistically its difficult to operate.

Large volumes of materials must be decontaminated in and out because you're going to have either large animals or large numbers of animals in that room. You need to regularly calibrate/validate disinfection or sterilization procedures. Lest you think that you have validated your cycle if you look at a dial and it says you've reached a certain temperature, or that you have autoclave tape that has changed color on a few items, or you've placed a few spore strips in a load, think again. That's not calibrating the load. Each load really is different, and you need to calibrate it for the materials and the load configuration. That ought to be done once a year. If you don't do that, I can tell you from personal experience that there will be spots in that autoclave that never get above room temperature; so you need a multiple point temperature calibration on a regular basis if you're going to autoclave things.

Also remember that all sanitation procedures are generally done within that barrier; a lot of items move across it, and you may have little or no control of things that move around within that barrier.

Let's talk about another type of containment device that is used quite commonly, micro-isolators. This involves containment at the cage level. One thing to remember about micro-isolators is that there is an imperfect seal of the lid to the cage which allows uncontrolled escape or input of air. It allows inflow of contaminants from the cage surface or, conversely, things going out during manipulation, especially in a laminar flow hood. Proper use of a micro-isolator system is very training dependen. It is technique dependent and everybody has to follow the same technique.

We can demonstrate imperfect seals in the following manner: take a micro-isolator cage and put a heavy rubber band around it to make sure it is seated well; then release smoke from a smoke stick, and you will find smoke inside the cage and not because its coming in through the filter at the top.

When you open a micro-isolator cage in a hood with all the supplies that you need to change the cage, you're going to be removing whatever is in that cage all over the supplies, all over the operator's arms, etc. You need to do the cage changing in a vertical flow hood, not a horizontal flow hood. The operator must be protected from hand and arm contamination. It is usually a two person process if you're dealing with bio-hazardous materials. Depending on the nature of the biohazard, you may need complete personnel gowning and post task decontamination.

Filters on micro-isolators come in different types. Most cage filters are dust filters. They're usually spun bond polyester made with a continuous thread lying down in a random fashion to form a sheet, but the average pore sizes can range all over the place. Those filters get more efficient the longer they're used because the pores get blocked up with dust and other particles. They are not high efficiency particulate air (HEPA) filters. How often you change filters and the type of filter material make a difference in the degree of bio-containment you really have. Organisms such as bacteria can lodge on filters and grow through them; sometimes you can find the bacteria on the other side of the filter.

Isolators hold small groups of cages in a controlled environment. They're easier to monitor than a barrier room or micro-isolators, falling somewhere in between. Isolators are under negative pressure in the bio-containment mode. They are available as flexible films or semi-rigid. There are more designs than you know what to do with. Some are even available for rather large animals, though they are generally used with small or very young animals. Isolators insure minimal operator exposure to the agent. The risks are primarily through breaks in the glove. Exhaust air filtration can be matched to the type and level of the hazard. Generally you need to fit them with HEPA filters.

Let me end with this concept. You can begin to combine various pieces of equipment into what I call a barrier within a barrier. It combines a barrier room with other forms of containment devices, such as isolators or micro isolators, resulting in additional barriers to keep the organism from escaping. These systems are generally expensive to operate and are usually only suitable for small numbers of small animals. It may involve total personnel enclosure into ventilated suits. Husbandry practices and waste handling can become very cumbersome. We have one at Charles River in which everything is done inside the barrier, including examination of tissue cultures, use of incubators, and transfer of materials into bio-containment hoods.

Special Containment Devices for Research Animals Breakout Session
Rapporteur: Michael Huerkamp, DVM President, Southeastern Branch AALAS Division of Animal Resources G81 Rollins Research Center Emory University Atlanta, GA 30322

Our group first covered barrier ooms which were acknowledged to have considerable value. Barrier rooms, by definition, are sealed rooms that contain what is within. They have air and waste handling capability, allow for disinfection and sterilization of equipment and implements, provide for decontamination of personnel and have back up power. Entry and egress are through air locks. Established standard operating procedures appropriate for the degree of risk and for the task, are rigorously followed. Our group believed that barrier rooms were most appropriate for containment of large numbers of animals or of large animals themselves (i.e., livestock). They are also effective for pathogen or chemical containment. The effectiveness of barrier rooms is heightened when one applies additional layers of protection using, for example, barrier cages, such as micro-isolators, or flexible film isolators. This scenario creates a barrier within the barrier. Barrier rooms are especially valuable for containing biohazards associated with large animals. Livestock containment presents challenges related to the sheer size of the animal and the considerable volume of waste products. Large animals discharge feces and urine from relative great height or pressure. When excrement is splashed, biohazards can be disseminated by aerosol droplet formation. From the perspective of the researcher and the animal caretaker, the use of personal protective devices within a barrier room provides the safest and least encumbered access to livestock.

On the other hand, we acknowledged barrier rooms to have problems. They are expensive to build, cumbersome to use, and energy intensive to operate. One also should not take for granted that they can provide containment or exclusion interchangeably. Often, engineering and physical modifications may be necessary to convert the area to the desired use.

Showers are frequently a component of a barrier room or facility. The value of showering as a decontamination procedure was questionable. For example, body orifices, such as the nose, may not be adequately cleansed by showering. This may create a break in a barrier operated to exclude certain microorganisms. Personnel compliance may be a problem. People may avoid taking a shower. Consequently, we agreed that the greatest value of showering was in forcing people to change their attire and thereby prevent fomite transmission of harmful agents on the clothing.

One participant in our session made the point that simply designing or having a barrier room was not sufficient. One needed to oversee the planning and construction of the facility, because contractors may cut corners and one could end with an ineffectual barrier room.

Flexible film isolators received much attention from our group. These containment devices receive filtered air and contain glove ports and an air lock. They are suitable for housing small numbers of small animals, but are versatile. They can be used for containment or exclusion, for studies involving microbial agents or chemicals, and can adapt to BSL2 or BSL3 activities. Isolators can be used to subdivide large spaces into several smaller spaces. The use of multiple flexible film isolators allows animals to be isolated from others of dissimilar microbial or genetic backgrounds, separate sources or different research uses. The down side to isolator use is that they are labor intensive and present problems with dexterity due to the gloves used. However, vendors are developing thin and durable gloves. Isolator technology has been enhanced by the development of prepackaged food, water and bedding that can be easily introduced into the device.

Some members of our group expressed concern about the containment of livestock in flexible film isolators. These devices can only house animals up to a certain size and present challenges in accessing animals, collecting and handling waste, and maintaining an environment that is wholesome for the animal. Isolator durability was a concern, because large animals can exert tremendous forces of wear and tear.

The cost of isolator containment devices was also a concern. The purchase price is clearly high, but considered relative to the cost of purchasing a barrier caging system with appropriate change-out stations (i.e., Class II laminar air flow cabinets) it may be affordable. Costs of consumable supplies, such as gowns, gloves and disinfectant, should also be less when using flexible film isolators.

Several devices or rooms were considered undesirable for pathogen or chemical containment. Mass air displacement rooms and cubicles were inappropriate for the task. Individually-ventilated microisolator caging systems, open-fronted laminar air flow cabinets and positive-pressure laminar air flow racks were not considered containment devices. Vibration, air turbulence and noise from these units could have an effect on the well-being of animals. With respect to laminar air flow racks and cabinets, variability in device performance depending on make, manufacturer and maintenance of the device was a cause of apprehension. A major concern was that laminar air flow systems can have their protective integrity disrupted by eddying of air when doors or cages were opened or when arms, hands or implements were introduced.

BioBubbles are portable, HEPA-filtered, mass air displacement, laminar air flow containment devices that can serve as barriers or clean rooms. They are popular at a number of institutions. We had no group consensus on BioBubbles. One individual regarded them as inappropriate as containment devices. Several other individuals gave endorsements.

In many of our institutions and in many of our containment operations, micro-isolator cages are our primary caging system. Some concerns were expressed whether micro-isolators were truly exclusion or containment devices when eddying could occur when they were opened in a laminar air flow work station. Individually-ventilated micro-isolator cages were not seen as adequate for containment or exclusion. The manufacturers have been innovative in developing cages with solid lids with gaskets to seal the lid-bottom interface, but air balance was not considered reliable and leaks are a risk.

The bottom line is that little can surpass knowledgeable people operating the appropriate equipment. Success in containment is based upon knowing the need, understanding the adopted containment/exclusion system, identifying pitfalls and risks, planning and training to avoid risks, pitfalls and the unexpected, and properly maintaining equipment.

Office of Health and Safety, Centers for Disease Control and Prevention, 1600 Clifton Road N.E., Mail Stop F05 Atlanta, Georgia 30333, USA Last Modified: 1/2/97
Send us your Comments.

OhASIS HOME | Biosafety Information | Safety Manuals ||| Symposium Contents

Special Containment Devices for Research Animals