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Cleaning and Disinfection of Life Systems
C H A P T E R
27 Cleaning and Disinfection of Life Systems Erik Sanders Aquatics Lab Services LLC, St. Peters, MO, United States of America
Abstract A. Understanding the differences between Cleaning, Sanitizing, Disinfection, and Sterilization a. Cleaning Cleaning refers to the removal of both organic and inorganic debris or soils by means of physical impingement, such as scrubbing, scraping, wiping, or brushing. Some common examples of soils in aquatic animal housing include fecal casts, uneaten food, and biological fouling agents, such as bacterial biofilm, algae, diatoms, and bryozoans. The object of cleaning is simply to remove this debris. Scraping tools and abrasive pads are commonly used to remove debris, but the powerful spray of water from commercial cage wash machines, especially when combined with suitable chemical solvents, may be capable of removing some or all of these soils. b. Sanitization Sanitization refers to the process of reducing the levels of pathogens on an object to safe levels and consequently reducing the likelihood of perpetuating a contaminant. c. Disinfection Disinfection refers to the process of destroying or preventing the growth of pathogenic organisms. Disinfection has the specific aim of a four or five log reduction in the number of microorganisms initially present on a surface. Disinfection of aquatic animal housing and equipment should follow the cleaning process and may be achieved using specialized processes including moderate to high heat, high-pressure sprays, and chemical or ultraviolet light exposure. d. Sterilization Sterilization refers to the process through which, statistically, all microorganisms and their spore
The Zebrafish in Biomedical Research https://doi.org/10.1016/B978-0-12-812431-4.00027-0
cells and pathogenic or toxic products are destroyed, sometimes referred to as a log six kill, where we expect a 99.9999% reduction of microorganisms initially present on a surface. Sterilization may be achieved through autoclaving, gas plasma, irradiation, or chemical exposure to compounds, such as sodium hydroxide, chlorine dioxide, hydrogen peroxide, peracetic acid, ozone, or glutaraldehyde. B. Sources and Kinds of Pathogens Relevant to Aquatics Facilities a. Pathogens that may have an untoward effect on the overall health status of an aquatics facility may be introduced from either an aquatic animal or human vector, or from fomites such as a dip net, aquaria components, or apparel worn by husbandry technicians and researchers. b. Some feeds may also be vectors of pathogens. Examples include tubifex worms, Artemia cysts, rotifers, and virtually any of a wide array of wildcaught products commonly used to feed fish cultures. Adequate and regular testing of such feed items is warranted to ensure that the risks of importing a pathogen are minimized. C. Hygiene as a Preventative Medicine Program and Biosecurity Measure a. A sound Quarantine procedure must be in place and strictly adhered to in order to minimize the risks of introducing pathogens from other facilities. This involves taking procedural steps that minimize the risks that fish imported from other sources will infect your colony with a pathogen, parasite, or biofouling agents such as bryozoans, mollusks, or algae. b. Environmental hygiene practices must be designed to effectively target the specific pathogens common to aquatics facilities. Not all pathogens require the same agents or level of
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exposure to disinfecting agents; thus, special care must be taken when developing cleaning practices to ensure that the measures taken are effective. c. While it is uncommon to find a dirty-side/cleanside approach in aquatics facilities, and perhaps even more rare to find barrier facilities, where aquatics species are concerned, implementing proper workflow can have a positive impact toward minimizing cross-contamination from within the facility. d. The use of disposable surgical gloves is common in the laboratory animal field, and aquatics facilities are no exception. Pathogens, including zoonotic species, such as Mycobacterium marinum, may be spread through contact with one’s hands, and often disposable gloves can mitigate these risks. However, gloves that contain powder should be avoided as they may contribute to health risks to your fish. Moreover, the use of disposable gloves should always be accompanied by a rigorous hand-washing policy, which is likely to be more effective at preventing outbreaks in the aquatic facility. e. Chemical footbaths are often found in aquatics facilities. If used, they must be appropriately designed, positioned, and maintained to target the specific pathogens of concern.
Recirculating Aquaculture Systems (RAS) Components Overview a. Fixed Components i. Fish Holding and Spawning Racks are those units, where the fish housing units are placed in order to receive flowing water from the RAS. These are often made from stainless steel and are designed to withstand the substantial weight of water held in the numerous tanks. Once fish holding system racks are set into place, we may anticipate that they will not be moved. In fact, it is not uncommon to find that the racks have been affixed to the floors, walls and/or, ceilings due to building codes relating to seismic zone and occupational health and safety guidelines. Special care will need to be taken to ensure that cleaning around this equipment is both effective and safe for the fish and people. Much of this equipment is either washed or wiped down by hand, and chemicals that are volatile or toxic to fish should be avoided. ii. Supply and Return Plumbing The piping that carries water to, and away from, the fish holding tanks can be differentiated as supply and return plumbing. Cleaning and
sanitizing of this plumbing should only be undertaken when no fish are living on the system. The choice of chemical and method(s) of application chosen will depend on the goals and aims of the task and the identification of any known pathogens and biofouling agents. Developing an action plan should include consulting a qualified fish pathologist. iii. Life Support Systems 1. The pumps used to distribute fish system water may vary in design and location depending on the design of the RAS, but virtually all can be removed for maintenance, including sanitizing the components that come in contact with the RAS water. The design of some of these components can be quite convoluted and may be of specific concern when attempting to eliminate all possible biofilms. It is recommended that you confirm the compatibility of the components with any cleaning and disinfecting agents you may choose to employ. 2. All RAS employ filters to perform various tasks. (a) Mechanical filters remove suspended and settleable solids and may be disposable, re-useable, or permanent components within the RAS design. Regardless of the nature of the mechanical filter, all of them must be adequately sanitized to prevent reinfecting your RAS. (b) Chemical filtration in RAS is often performed using activated carbon/ charcoal media. Activated carbon is most commonly used to remove undesirable odors and colors or stains (e.g., tannins) from the RAS water. This media should be removed during any sanitization procedures as it may reduce the efficacy of some chemical agents. (c) Ultraviolet (UV) filters are often used to minimize the growth of algae, and pathogens, by direct exposure to UV-C radiation. It is critical that these UV reactors and the quartz sleeves that contain the UV-C bulbs are properly sanitized, and that the UV-C is powered off to prevent interfering with any chemical treatment used for sanitizing your RAS. These reactors and bulbs should be maintained on a strict schedule to ensure their efficacy in your regular RAS operations. (d) Biofiltration is a component of all RAS. This is the process whereby bacteria perform the process of nitrification and in
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Types of Soils and Dirt Found in and on RAS Equipment
some cases, denitrification. Most attempts at sanitization of a RAS will kill all of the beneficial bacteria living in the biofilter. Biofilters, by their very design, must have tremendous amounts of surface area in which the bacteria colonize. Please ensure that any sanitization procedure adequately addresses this important concern. And be prepared to properly cycle, or inoculate, the biofilter when you decide to return the RAS to service in the future. 3. Heaters/chillers (a) Heaters are often submerged in sumps and will require special attention when employing any sanitization efforts. (b) Chillers are often stand-alone items through which the RAS water is passed into a circuitous route to cool the water to the desired temperature before it exits the equipment. Consequently, chillers may pose a difficulty when attempting to sanitize any RAS. 4. Aeration (a) RAS often require supplemental aeration, and zebrafish RAS often employ air-stone diffusers for this purpose. These diffusers are very porous, and may be disposable, or may have their own instructions for proper cleaning and disinfection. It is best to consult the manufacturer. b. Modular Components i. Fish Holding Tanks and Parts Fish holding tanks may be constructed of a variety of plastics and glass. While most are truly modular and are routinely moved around for purposes of access to fish and clean tank exchange, some are essentially stationary and will require manual cleaning and sanitization. Modular zebrafish aquaria and their associated components, such as lids, siphons, and baffles, are typically constructed from polycarbonate plastic, silicone rubber, and stainless steel. These items lend themselves to being cleaned and sanitized in industrial cage wash machines that differ very little from those used in a rodent or rabbit facility. Some are constructed from materials that may be autoclaved, but it is advisable to follow the manufacturer’s guidelines concerning the temperature and duration of autoclave cycles. ii. Spawning Chambers and Traps 1. Spawning chambers or cages are typically modular items that can be used as stand-alone units or placed into the top of a fish housing tank. These items are typically constructed from the same materials as the fish housing
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tanks and may be cleaned and sanitized in the same way. 2. Spawning traps are items that are typically placed into a larger fish housing tank with the intent to collect fertilized eggs as the fish spawn over them. These traps may include artificial plants, mesh-screen panels, or a substrate such as marbles. Due to the substantial surface area of these items, special attention will be required to ensure adequate cleaning and sanitization. iii. Culturing Equipment for Live Feeds Equipment used for the production and feed out of living feeds, such as paramecia, Artemia, rotifers, and other planktons represent potential sinks for unwanted microorganisms and pathogens. A well-written and effective SOP for the disinfection and cleaning of this equipment should be followed to ensure that your live feeds do not become the vector for disease in your facility. iv. Mobile Carts and Racks Mobile carts and racks are commonly used as work surfaces and transport vehicles within and without the zebrafish facility. Regular cleaning and sanitizing of this equipment are necessary to prevent the transmission of any possible pathogens and microorganisms from one use to the next. Facility policies should be established to prevent these carts and racks from traveling between facilities and laboratories. c. Room Floors, Walls, Ceilings The floors, walls, and ceilings of your zebrafish holding facility should be designed and built to a wash-down specification. This means that you can apply surface disinfectants and rinse them off as needed without destroying the construction materials. Although you may have a wash-down spec facility, it is still best to test an area with the sanitizing agents you are interested in using before committing to a specific action plan. Further, it is always prudent to consider the volatile nature of some cleaning agents and to err on the side of caution when using them in areas where RAS water may be exposed since this may pose a threat to the health of your fish colony.
Types of Soils and Dirt Found in and on RAS Equipment a. A variety of algae and diatoms may be found in a healthy zebrafish RAS, but efforts should be made to minimize its presence since it may have negative outcomes on animal welfare and system operations.
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While some algae are single cells that remain in the water column, others form colonies that may interfere with the ability to perform proper daily health checks of the fish. These algal colonies are often tenacious and will require mechanical removal or physical impingement with high-pressure sprays. Some algae are toxic or produce toxic substances, so it is a best practice to prevent or minimize the occurrence of algae in a zebrafish RAS. Figs. 27.1e27.3 show tanks with accumulations of algae/diatoms. b. Bryozoans are invertebrate animals known to colonize and encrust surfaces in zebrafish (and other) RAS. In addition to creating a barrier to performing proper health checks of the fish in the tanks, bryozoans also act as biofouling agents that may impede the proper flow of water in and out of the tanks and throughout the entirety of the RAS. Importantly, bryozoans are also a secondary host for one or more myxozoans known to cause Proliferative Kidney Disease (PKD) in aquaculture fish species. Finally, bryozoans are notoriously difficult to remove from tank surfaces and will require prolonged exposure to harsh chemicals such as bleach, and high temperatures to control or eliminate from the RAS. Figs. 27.4 and 27.5 show bryozoans in fish tanks.
FIGURE 27.2
Example of algae on tank surfaces. Photo courtesy of
IWT/Tecniplast.
FIGURE 27.3 Examples of algae on tank surfaces. Close up view from each tank is underneath in inset view. Photos courtesy of IWT/ Tecniplast.
FIGURE 27.1 Examples of algae and diatoms on tank surfaces. Close up view from each tank is on the right in the inset view. Photos courtesy of IWT/Tecniplast.
c. Biofilms form when bacteria adhere to wet surfaces and excrete molecular strands and consequently form three-dimensional communities. Biofilms accumulate over time and are an integral part of all RAS. In zebrafish RAS, the biofilms that pose the most direct obstacle to proper daily operations occur at the tanklevel. Those biofilms impede the proper flow of water in and out of the tanks and create a visual barrier to those who must perform the daily observations of fish in their home tank. A number of factors will influence
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FIGURE 27.4 Images of bryozoans on tanks. Photographs courtesy of IWT Tecniplast.
FIGURE 27.5 Image of bryozoa (Plumatella rugose) on a tank surface with visible statoblasts. Photograph courtesy of Erik Sanders.
the growth of biofilms in your fish tanks, including light intensity, nutrient load, and many consecutive days of operation. Fig. 27.6 shows biofilm accumulated on a tank. d. Fish feces or fecal casts or pellets are a natural consequence of providing your fish with their daily dietary needs. The size, consistency, and amount of this waste is a direct corollary of the amount, type, and frequency of the feeds you provide to your fish. In most cases, the properly functioning tank should
FIGURE 27.6
Image of zebrafish tank with heavy biofilm coating as evidenced by the hazy appearance. Photograph courtesy of Erik Sanders.
be able to remove this waste as it is produced. However, when a tank’s self-cleaning function is impaired, this waste may accumulate rapidly, and a mitigating response from the husbandry provider is required. e. Detritus commonly found in zebrafish RAS will commonly consist of fish waste, uneaten food, and other decomposing organic matter. This detritus must not be allowed to accumulate in excess as it offers a
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FIGURE 27.7 Image of soiled zebrafish tank with accumulated biofilm (hazy appearance), bryozoa, and food water, and fecal detritus. Photograph courtesy of Erik Sanders.
lattice for pathogens and intermediate hosts of pathogens. A properly functioning tank should have no trouble removing this waste, but if not, it may need to be removed manually, and a tank exchange is warranted. Fig. 27.7 illustrates accumulated biofilm, bryozoans, and food/fecal detritus in a tank. f. Suspended solids, in zebrafish RAS, should be well below the size permitted to pass through the mechanical filter in service. If abundant, they may be an indication that other problems need addressing, such as replacing or repairing the mechanical filter. These solids alone should not pose a significant contribution to the needs for cage wash or tank exchange. Settleable solids are those solids that will settle to the bottom of the tank when the velocity of the water is inadequate to keep them in suspension. In zebrafish RAS, these should consist predominately of uneaten food and fish feces. These are often captured in mechanical filters.
Cleaning Agents and Techniques used in Aquatics Facilities A. Cleaning Agents a. In the case of cleaning zebrafish equipment, acids are typically used to neutralize alkalis rather than as a primary cleaning agent. Examples include citric and acetic acid. b. Alkalis are often the primary cleaning agent used to clean and sanitize zebrafish tanks and components. Alkalis hydrolyze organic matter, making it more susceptible to removal and destruction by other chemicals or high temperature. Because of their high pH, alkalis should be neutralized and rinsed away fully before returning an item to service in the RAS. Despite being volatile and highly toxic to fish,
household bleach (sodium hypochlorite) is still commonly used in zebrafish facilities as a cleaning and disinfecting agent. Other examples of alkalis used for cleaning and disinfection in zebrafish facilities include sodium hydroxide and Tosylchloramide sodium (chloramine-T). c. Oxidizers can be powerful cleaning and disinfecting agents in the aquaculture setting. Because the oxidation of a substrate often contributes to the degradation of that substrate, oxidizers are very effective at removing many soils common to aquaculture and zebrafish RAS. Hydrogen peroxide is a readily available, relatively inexpensive, and when used properly, fish-safe chemical oxidizer. This is because, when added to water, hydrogen peroxide breaks down into oxygen and water. Other commonly used oxidizers used in aquaculture cleaning and disinfection include ozone and peracetic acid. d. Soaps and Detergents are both surfactants, which are chemicals that lower surface tension, and may act as foaming agents and emulsifiers. For this reason, they are useful in removing soils from hard surfaces but pose a real danger to both fish and beneficial bacteria cultured in RAS. e. Solvents are a substance, usually a liquid, which dissolves a solute. While not a particularly effective cleaning agent for aquaculture related soils, ethanol (70%) is commonly found in laboratories and zebrafish facilities, where it is often used as a disinfectant and drying agent for common-use equipment, such as microscopes and hand-tools. B. Cleaning Techniques a. Manual washing of RAS equipment and components is perhaps the most commonly employed cleaning technique. When manually washing zebrafish tanks, accessories, and related equipment, there is often a need for pretreatment in baths of water in order to keep the soils wet since, once dried, many of the soils common to zebrafish RAS become tenacious and require additional force to remove. It is common to use household bleach to assist in this process, but this approach will require adequate posttreatment to neutralize the chlorine, which may pose a direct threat if left on the tanks. Sodium thiosulfate is commonly used for this purpose. The manual scrubbing of tanks and accessories is often aided by the use of scrapers, sponges, and so-called nonabrasive pads or electric-powered tools borrowed from the restaurant and bar industry. The use of these tools will ultimately abrade surfaces making tanks that were once optically clear, become foggy and scratched. Fig. 27.8
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FIGURE 27.8 Image of abraded surfaces from cleaning a zebrafish tank. Photograph courtesy of Erik Sanders.
illustrates a tank with abraded inner surfaces. Fig. 27.9 illustrates properly cleaned tanks. This abrasion represents a manifold increase in surface area and will create a surface that will continue to be more and more rapidly colonized by algae and biofilms, effectively decreasing the useful service life of the equipment. From a staffing and labor cost standpoint, manual washing is quite expensive when analyzed on a per-unit basis. Areas of undesirable variability in manual washing include the level of productivity, quality control, and the potential for mistakes with chemicals leading to a risk of human and fish colony health.
FIGURE 27.9 Images of properly cleaned tanks. courtesy of Erik Sanders.
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b. Automated washing, while common in rodent facilities, where it is commonly referred to as cage wash, is relatively new to the field of zebrafish, only becoming benchmarked in 2012. The machines common to this kind of washing are cabinet washers, cage, and rack washers, and tunnel washers. This kind of washing should require a minimum of pretreatment of the tanks and accessories (lids, baffles, etc.) and should render the items washed completely free of any and all soils, and preferably, at a high level of disinfection. All chemicals employed should be free of detergents, soaps, and surfactants since these can cause serious problems by creating foaming in RAS and by damaging the slime-coat of fish and biofilter bacteria. Wash chemicals typically consist of alkalis and oxidizers but may also include acids. While municipal water may be acceptable for the wash phases of the process, the final rinse should be performed only with reverseosmosis or suitably purified water. Importantly, the presence of any chemical residue on the tanks at the completion of the wash cycle is unacceptable considering the highly sensitive nature of aquatic species and biofilter bacteria and the types of research being performed with zebrafish (embryology, toxicology, etc.). In contrast to rodent cage wash standards, the drying phase of the process is not as important for aquatics cage wash standards. Although automated cage wash requires a capital investment, the cost of washing on a per-unit basis is often at least 50% less, and the level of cleaning and disinfection is substantially higher than that which can be expected using a manual approach. Fig. 27.10 illustrates the Calypso Aquatic Cabinet Washer from Tecniplast. Fig. 27.11 illustrates a Tecniplast Presentation Rack for tanks prepared to be rolled into a Tecniplast Cage and Rack Washer. c. Autoclaving, considered to be complete sterilization of an object, is performed using high temperature at elevated atmospheric pressure for an extended period of time. All items to be autoclaved must be fully cleaned before undergoing the process. While many zebrafish housing components are capable of withstanding these conditions, the recommendations of the manufacturer should be carefully followed to avoid damage to the items being autoclaved. C. Cage wash Area Planning a. Design Challenges i. Work Flow While rodent facilities predominantly employ the clean-dirty corridor concept, it is far less common in aquatics facilities. Much of
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FIGURE 27.10
Images of the Tecniplast Calypso Aquatic Cabinet Washer, closed and open, showing crates for accessories, and in service. Photographs courtesy of IWT/Tecniplast.
FIGURE 27.11 Image of Tecniplast cage wash presentation rack loaded with fish tanks and components, prepared for entry to Tecniplast Cage and Rack Washer. Photograph courtesy of IWT/Tecniplast.
this difference can be attributed to the historical design and cost expectations of zebrafish facilities, which often, and mistakenly, are thought to be much less complex and costly than rodent facilities. Dirty aquatics equipment is prone to drip and fall to the floor; thus, it is essential that this be considered when selecting cage wash operations locations. A failure to do so may result in a situation, where researchers are constantly crossing paths with staff carrying out cage wash operations. ii. Space Requirements Because the allotment of space for cage wash in aquatics facilities is not standard practice as
it is in rodent facilities, we must borrow from the rodent facilities in order to arrive at a bestpractice approach. There exist relatively simple formulae for planning throughput and equipment requirements for rodent facilities, which may readily be adjusted for aquatics facilities. Factors for establishing a baseline in a full-capacity facility are the total quantity of cages in use, the percentage of these cages changed daily, the number of days/week cage wash is performed, the number of shifts in a day, and an efficiency factor of a full workday. From these factors, it is possible to define the daily demand. Daily demand will describe how many cages must be processed each day at peak capacity. With this figure, it is possible to specify the type and number of machines needed, and consequently, the space they will require. Additionally, you will be able to determine the total inventory of cages and related components required to maintain the daily operations at full capacity. Considering that colony size may vary wildly from week to week within zebrafish facilities, you should expect to have space to store at least 25% of your total cages and components as clean supply. iii. Finishes Since cage wash operations involve high temperatures, steam, corrosive chemicals, water, and high humidity, all materials in the
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cage wash areas must be constructed from durable materials, such as stainless steel, epoxy resins, fiberglass, etc. Given the high traffic volume, the floors and doors should be specified to handle both a high degree of wear and a wet environment. iv. Waste Handling Most solid waste in a zebrafish facility is disposed of by the RAS mechanical filtration, but some will make its way into the cage wash area. It is important to have the ability to spray down equipment to remove or loosen solid wastes, and that the areas have adequate floor drains to handle its removal. v. Safety Safety of personnel and animal colony are of critical importance. This is especially true where caustic or corrosive chemicals are concerned and where volatile chemicals are used. Always ensure your operational guidelines are aligned with the Environmental Health and Safety (EH&S) coordinators for your institution. The need for eyewash and chemical showers will be dictated by EH&S and will need to be incorporated into your space planning and budget. There will likely be additional requirements from EH&S for personal protective equipment to be worn and used by personnel. b. Operational Philosophy Many variables will impact the time that a zebrafish cage may stay on a rack until it becomes necessary to exchange it for a clean one. It is critical that daily health checks be performed, and if our view of the fish is impeded by the growth of algae, then the tank must be exchanged. If the accumulation of solid waste in the tank has begun to impede the self-cleaning action of the tank or has caused the water level in the tank to rise above its normal operating level, then the tank must be changed. Your daily operations will guide you in this regard, but as a general rule, tanks should be changed at least every 90 days.
Cleaning Validation Techniques A. Microbial/Biological Techniques: These are used to establish the efficacy of the cleaning process as it pertains to removing the biological load. There are several readily available and reliable test methods to consider. a. Replicate Organism Detection and Counting (RODAC) plates are inexpensive and very useful for detecting viable organisms that may be left
FIGURE 27.12 A set of RODAC plates. The top two plates have no growth indicating a complete cleaning and sanitation of the tested surfaces; the bottom two plates show the growth of microorganisms from pre-washed surfaces. Photograph courtesy of Erik Sanders.
behind after cleaning. These plates should be formulated and incubated in a manner that would allow detection of possible organisms of concern in a zebrafish facility. Fig. 27.12 illustrates a set of RODAC plates. b. ATP Detection Swabs are available from several manufacturers. Most utilize a combination of enzymes and buffer which, when combined with ATP, emit light that is detected and enumerated by a handheld device. ATP detection is fast (10second or less), produces recordable and numeric data, is reliable, and is very sensitive, detecting contamination well below the threshold for RODAC plates.
Conclusions Using proper cleaning and disinfection techniques are crucial for the culture of zebrafish for many reasons. Incorrect procedures can result in catastrophic failures, including loss of animals and data. Those working in the field must be well trained, familiar with their systems, and observant to ensure that the systems are kept in a manner that supports optimal animal health and valid research results. For further reading on this topic, please refer to the references listed below.
Further Reading Allen, T. J. T. (2003). 1953-Animal Welfare Information Center (U.S.), information resources for animal facility sanitation and cage wash [electronic resource]/compiled by Tim Allen. Beltsville, Md, USDA, ARS, NAL. Animal Welfare Information Center Resource Series No. 19.
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Collymore, C., Porelli, G., Lieggi, C., & Lipman, N. S. (November 2014). Evaluation of 5 cleaning and disinfection methods for nets used to collect zebrafish (Danio rerio). Journal of the American Association for Laboratory Animal Science, 53(6), 657e660. Feinstein, S. (6/18/2018). What is the difference between sanitation, sterilization, and disinfection? ALN magazine. https://www.alnmag.com/ article/2014/10/what-difference-between-sanitation-sterilizationand-disinfection. Garcia, R. L., & Sanders, G. E. (November 2011). Efficacy of cleaning and disinfection procedures in a zebrafish (Danio rerio) facility. Journal of the American Association for Laboratory Animal Science, 50(6), 895e900. Harper, C., & Lawrence, C. (2010). The laboratory zebrafish. Boca Raton (FL): CRC Press. International Office of Epizootics. (2009). Aquatic animal health standards commission. Chapter 1.1.4: Methods for disinfection of aquacultural establishments, in Manual of diagnostic tests for aquatic animals (6th ed.). Paris, France: Office International des epizooties. Lawrence, C., & Mason, T. (2012). Zebrafish housing systems: A review of basic operating principles and considerations for design and functionality. ILAR Journal, 53(2), 179e191. Leary, S. L., Majoros, J. A., & Tomson, J. S. (June 1998). Making cagewash facility design a priority. Laboratory Animals, 27(6), 28e31.
Liu, D., Behrens, S., Pedersen, L.-F., Straus, D. L., & Meinelt, T. (2016). Peracetic acid is a suitable disinfectant for recirculating fishmicroalgae integrated multi-trophic aquaculture systems. Aquaculture Reports, 36(4), 1e142. Martins, M. L., Watral, V., Rodrigues-Soares, J. P., & Kent, M. L. (February 2017). A method for collecting eggs of Pseudocapillaria tomentosa (Nematode: Capillariidae) from zebrafish Danio rerio and efficacy of heat and chlorine for killing the nematode’s eggs. Journal of Fish Diseases, 40(2), 169e182. https://doi.org/10.1111/ jfd.12501. Policy SC-50-103. (6/10/2016). Animal facility quality assurance and monitoring. UC Davis Office of the Attending Veterinarian Standards of Care. University of Wisconsin-Madison, Office of chemical safety, the chemical safety mechanism: Laboratory glassware cleaning, SOP. Wallace, J. (6/24/2018). Cage processing operations in laboratory animal facilities. ALN magazine. https://www.alnmag.com/article/2012/ 07/cage-processing-operations-laboratory-animal-facilities. Whipps, C. M., Murray, K. N., & Kent, M. L. (February 2015). Occurrence of a myxozoan parasite Myxidium streisingeri N. Sp. In laboratory zebrafish Danio rerio. Journal of Parasitology, 101(1), 86e90. https://doi.org/10.1645/14-613.1.
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27 Cleaning and Disinfection of Life Systems Erik Sanders Aquatics Lab Services LLC, St. Peters, MO, United States of America
Abstract A. Understanding the differences between Cleaning, Sanitizing, Disinfection, and Sterilization a. Cleaning Cleaning refers to the removal of both organic and inorganic debris or soils by means of physical impingement, such as scrubbing, scraping, wiping, or brushing. Some common examples of soils in aquatic animal housing include fecal casts, uneaten food, and biological fouling agents, such as bacterial biofilm, algae, diatoms, and bryozoans. The object of cleaning is simply to remove this debris. Scraping tools and abrasive pads are commonly used to remove debris, but the powerful spray of water from commercial cage wash machines, especially when combined with suitable chemical solvents, may be capable of removing some or all of these soils. b. Sanitization Sanitization refers to the process of reducing the levels of pathogens on an object to safe levels and consequently reducing the likelihood of perpetuating a contaminant. c. Disinfection Disinfection refers to the process of destroying or preventing the growth of pathogenic organisms. Disinfection has the specific aim of a four or five log reduction in the number of microorganisms initially present on a surface. Disinfection of aquatic animal housing and equipment should follow the cleaning process and may be achieved using specialized processes including moderate to high heat, high-pressure sprays, and chemical or ultraviolet light exposure. d. Sterilization Sterilization refers to the process through which, statistically, all microorganisms and their spore
The Zebrafish in Biomedical Research https://doi.org/10.1016/B978-0-12-812431-4.00027-0
cells and pathogenic or toxic products are destroyed, sometimes referred to as a log six kill, where we expect a 99.9999% reduction of microorganisms initially present on a surface. Sterilization may be achieved through autoclaving, gas plasma, irradiation, or chemical exposure to compounds, such as sodium hydroxide, chlorine dioxide, hydrogen peroxide, peracetic acid, ozone, or glutaraldehyde. B. Sources and Kinds of Pathogens Relevant to Aquatics Facilities a. Pathogens that may have an untoward effect on the overall health status of an aquatics facility may be introduced from either an aquatic animal or human vector, or from fomites such as a dip net, aquaria components, or apparel worn by husbandry technicians and researchers. b. Some feeds may also be vectors of pathogens. Examples include tubifex worms, Artemia cysts, rotifers, and virtually any of a wide array of wildcaught products commonly used to feed fish cultures. Adequate and regular testing of such feed items is warranted to ensure that the risks of importing a pathogen are minimized. C. Hygiene as a Preventative Medicine Program and Biosecurity Measure a. A sound Quarantine procedure must be in place and strictly adhered to in order to minimize the risks of introducing pathogens from other facilities. This involves taking procedural steps that minimize the risks that fish imported from other sources will infect your colony with a pathogen, parasite, or biofouling agents such as bryozoans, mollusks, or algae. b. Environmental hygiene practices must be designed to effectively target the specific pathogens common to aquatics facilities. Not all pathogens require the same agents or level of
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exposure to disinfecting agents; thus, special care must be taken when developing cleaning practices to ensure that the measures taken are effective. c. While it is uncommon to find a dirty-side/cleanside approach in aquatics facilities, and perhaps even more rare to find barrier facilities, where aquatics species are concerned, implementing proper workflow can have a positive impact toward minimizing cross-contamination from within the facility. d. The use of disposable surgical gloves is common in the laboratory animal field, and aquatics facilities are no exception. Pathogens, including zoonotic species, such as Mycobacterium marinum, may be spread through contact with one’s hands, and often disposable gloves can mitigate these risks. However, gloves that contain powder should be avoided as they may contribute to health risks to your fish. Moreover, the use of disposable gloves should always be accompanied by a rigorous hand-washing policy, which is likely to be more effective at preventing outbreaks in the aquatic facility. e. Chemical footbaths are often found in aquatics facilities. If used, they must be appropriately designed, positioned, and maintained to target the specific pathogens of concern.
Recirculating Aquaculture Systems (RAS) Components Overview a. Fixed Components i. Fish Holding and Spawning Racks are those units, where the fish housing units are placed in order to receive flowing water from the RAS. These are often made from stainless steel and are designed to withstand the substantial weight of water held in the numerous tanks. Once fish holding system racks are set into place, we may anticipate that they will not be moved. In fact, it is not uncommon to find that the racks have been affixed to the floors, walls and/or, ceilings due to building codes relating to seismic zone and occupational health and safety guidelines. Special care will need to be taken to ensure that cleaning around this equipment is both effective and safe for the fish and people. Much of this equipment is either washed or wiped down by hand, and chemicals that are volatile or toxic to fish should be avoided. ii. Supply and Return Plumbing The piping that carries water to, and away from, the fish holding tanks can be differentiated as supply and return plumbing. Cleaning and
sanitizing of this plumbing should only be undertaken when no fish are living on the system. The choice of chemical and method(s) of application chosen will depend on the goals and aims of the task and the identification of any known pathogens and biofouling agents. Developing an action plan should include consulting a qualified fish pathologist. iii. Life Support Systems 1. The pumps used to distribute fish system water may vary in design and location depending on the design of the RAS, but virtually all can be removed for maintenance, including sanitizing the components that come in contact with the RAS water. The design of some of these components can be quite convoluted and may be of specific concern when attempting to eliminate all possible biofilms. It is recommended that you confirm the compatibility of the components with any cleaning and disinfecting agents you may choose to employ. 2. All RAS employ filters to perform various tasks. (a) Mechanical filters remove suspended and settleable solids and may be disposable, re-useable, or permanent components within the RAS design. Regardless of the nature of the mechanical filter, all of them must be adequately sanitized to prevent reinfecting your RAS. (b) Chemical filtration in RAS is often performed using activated carbon/ charcoal media. Activated carbon is most commonly used to remove undesirable odors and colors or stains (e.g., tannins) from the RAS water. This media should be removed during any sanitization procedures as it may reduce the efficacy of some chemical agents. (c) Ultraviolet (UV) filters are often used to minimize the growth of algae, and pathogens, by direct exposure to UV-C radiation. It is critical that these UV reactors and the quartz sleeves that contain the UV-C bulbs are properly sanitized, and that the UV-C is powered off to prevent interfering with any chemical treatment used for sanitizing your RAS. These reactors and bulbs should be maintained on a strict schedule to ensure their efficacy in your regular RAS operations. (d) Biofiltration is a component of all RAS. This is the process whereby bacteria perform the process of nitrification and in
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some cases, denitrification. Most attempts at sanitization of a RAS will kill all of the beneficial bacteria living in the biofilter. Biofilters, by their very design, must have tremendous amounts of surface area in which the bacteria colonize. Please ensure that any sanitization procedure adequately addresses this important concern. And be prepared to properly cycle, or inoculate, the biofilter when you decide to return the RAS to service in the future. 3. Heaters/chillers (a) Heaters are often submerged in sumps and will require special attention when employing any sanitization efforts. (b) Chillers are often stand-alone items through which the RAS water is passed into a circuitous route to cool the water to the desired temperature before it exits the equipment. Consequently, chillers may pose a difficulty when attempting to sanitize any RAS. 4. Aeration (a) RAS often require supplemental aeration, and zebrafish RAS often employ air-stone diffusers for this purpose. These diffusers are very porous, and may be disposable, or may have their own instructions for proper cleaning and disinfection. It is best to consult the manufacturer. b. Modular Components i. Fish Holding Tanks and Parts Fish holding tanks may be constructed of a variety of plastics and glass. While most are truly modular and are routinely moved around for purposes of access to fish and clean tank exchange, some are essentially stationary and will require manual cleaning and sanitization. Modular zebrafish aquaria and their associated components, such as lids, siphons, and baffles, are typically constructed from polycarbonate plastic, silicone rubber, and stainless steel. These items lend themselves to being cleaned and sanitized in industrial cage wash machines that differ very little from those used in a rodent or rabbit facility. Some are constructed from materials that may be autoclaved, but it is advisable to follow the manufacturer’s guidelines concerning the temperature and duration of autoclave cycles. ii. Spawning Chambers and Traps 1. Spawning chambers or cages are typically modular items that can be used as stand-alone units or placed into the top of a fish housing tank. These items are typically constructed from the same materials as the fish housing
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tanks and may be cleaned and sanitized in the same way. 2. Spawning traps are items that are typically placed into a larger fish housing tank with the intent to collect fertilized eggs as the fish spawn over them. These traps may include artificial plants, mesh-screen panels, or a substrate such as marbles. Due to the substantial surface area of these items, special attention will be required to ensure adequate cleaning and sanitization. iii. Culturing Equipment for Live Feeds Equipment used for the production and feed out of living feeds, such as paramecia, Artemia, rotifers, and other planktons represent potential sinks for unwanted microorganisms and pathogens. A well-written and effective SOP for the disinfection and cleaning of this equipment should be followed to ensure that your live feeds do not become the vector for disease in your facility. iv. Mobile Carts and Racks Mobile carts and racks are commonly used as work surfaces and transport vehicles within and without the zebrafish facility. Regular cleaning and sanitizing of this equipment are necessary to prevent the transmission of any possible pathogens and microorganisms from one use to the next. Facility policies should be established to prevent these carts and racks from traveling between facilities and laboratories. c. Room Floors, Walls, Ceilings The floors, walls, and ceilings of your zebrafish holding facility should be designed and built to a wash-down specification. This means that you can apply surface disinfectants and rinse them off as needed without destroying the construction materials. Although you may have a wash-down spec facility, it is still best to test an area with the sanitizing agents you are interested in using before committing to a specific action plan. Further, it is always prudent to consider the volatile nature of some cleaning agents and to err on the side of caution when using them in areas where RAS water may be exposed since this may pose a threat to the health of your fish colony.
Types of Soils and Dirt Found in and on RAS Equipment a. A variety of algae and diatoms may be found in a healthy zebrafish RAS, but efforts should be made to minimize its presence since it may have negative outcomes on animal welfare and system operations.
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While some algae are single cells that remain in the water column, others form colonies that may interfere with the ability to perform proper daily health checks of the fish. These algal colonies are often tenacious and will require mechanical removal or physical impingement with high-pressure sprays. Some algae are toxic or produce toxic substances, so it is a best practice to prevent or minimize the occurrence of algae in a zebrafish RAS. Figs. 27.1e27.3 show tanks with accumulations of algae/diatoms. b. Bryozoans are invertebrate animals known to colonize and encrust surfaces in zebrafish (and other) RAS. In addition to creating a barrier to performing proper health checks of the fish in the tanks, bryozoans also act as biofouling agents that may impede the proper flow of water in and out of the tanks and throughout the entirety of the RAS. Importantly, bryozoans are also a secondary host for one or more myxozoans known to cause Proliferative Kidney Disease (PKD) in aquaculture fish species. Finally, bryozoans are notoriously difficult to remove from tank surfaces and will require prolonged exposure to harsh chemicals such as bleach, and high temperatures to control or eliminate from the RAS. Figs. 27.4 and 27.5 show bryozoans in fish tanks.
FIGURE 27.2
Example of algae on tank surfaces. Photo courtesy of
IWT/Tecniplast.
FIGURE 27.3 Examples of algae on tank surfaces. Close up view from each tank is underneath in inset view. Photos courtesy of IWT/ Tecniplast.
FIGURE 27.1 Examples of algae and diatoms on tank surfaces. Close up view from each tank is on the right in the inset view. Photos courtesy of IWT/Tecniplast.
c. Biofilms form when bacteria adhere to wet surfaces and excrete molecular strands and consequently form three-dimensional communities. Biofilms accumulate over time and are an integral part of all RAS. In zebrafish RAS, the biofilms that pose the most direct obstacle to proper daily operations occur at the tanklevel. Those biofilms impede the proper flow of water in and out of the tanks and create a visual barrier to those who must perform the daily observations of fish in their home tank. A number of factors will influence
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FIGURE 27.4 Images of bryozoans on tanks. Photographs courtesy of IWT Tecniplast.
FIGURE 27.5 Image of bryozoa (Plumatella rugose) on a tank surface with visible statoblasts. Photograph courtesy of Erik Sanders.
the growth of biofilms in your fish tanks, including light intensity, nutrient load, and many consecutive days of operation. Fig. 27.6 shows biofilm accumulated on a tank. d. Fish feces or fecal casts or pellets are a natural consequence of providing your fish with their daily dietary needs. The size, consistency, and amount of this waste is a direct corollary of the amount, type, and frequency of the feeds you provide to your fish. In most cases, the properly functioning tank should
FIGURE 27.6
Image of zebrafish tank with heavy biofilm coating as evidenced by the hazy appearance. Photograph courtesy of Erik Sanders.
be able to remove this waste as it is produced. However, when a tank’s self-cleaning function is impaired, this waste may accumulate rapidly, and a mitigating response from the husbandry provider is required. e. Detritus commonly found in zebrafish RAS will commonly consist of fish waste, uneaten food, and other decomposing organic matter. This detritus must not be allowed to accumulate in excess as it offers a
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FIGURE 27.7 Image of soiled zebrafish tank with accumulated biofilm (hazy appearance), bryozoa, and food water, and fecal detritus. Photograph courtesy of Erik Sanders.
lattice for pathogens and intermediate hosts of pathogens. A properly functioning tank should have no trouble removing this waste, but if not, it may need to be removed manually, and a tank exchange is warranted. Fig. 27.7 illustrates accumulated biofilm, bryozoans, and food/fecal detritus in a tank. f. Suspended solids, in zebrafish RAS, should be well below the size permitted to pass through the mechanical filter in service. If abundant, they may be an indication that other problems need addressing, such as replacing or repairing the mechanical filter. These solids alone should not pose a significant contribution to the needs for cage wash or tank exchange. Settleable solids are those solids that will settle to the bottom of the tank when the velocity of the water is inadequate to keep them in suspension. In zebrafish RAS, these should consist predominately of uneaten food and fish feces. These are often captured in mechanical filters.
Cleaning Agents and Techniques used in Aquatics Facilities A. Cleaning Agents a. In the case of cleaning zebrafish equipment, acids are typically used to neutralize alkalis rather than as a primary cleaning agent. Examples include citric and acetic acid. b. Alkalis are often the primary cleaning agent used to clean and sanitize zebrafish tanks and components. Alkalis hydrolyze organic matter, making it more susceptible to removal and destruction by other chemicals or high temperature. Because of their high pH, alkalis should be neutralized and rinsed away fully before returning an item to service in the RAS. Despite being volatile and highly toxic to fish,
household bleach (sodium hypochlorite) is still commonly used in zebrafish facilities as a cleaning and disinfecting agent. Other examples of alkalis used for cleaning and disinfection in zebrafish facilities include sodium hydroxide and Tosylchloramide sodium (chloramine-T). c. Oxidizers can be powerful cleaning and disinfecting agents in the aquaculture setting. Because the oxidation of a substrate often contributes to the degradation of that substrate, oxidizers are very effective at removing many soils common to aquaculture and zebrafish RAS. Hydrogen peroxide is a readily available, relatively inexpensive, and when used properly, fish-safe chemical oxidizer. This is because, when added to water, hydrogen peroxide breaks down into oxygen and water. Other commonly used oxidizers used in aquaculture cleaning and disinfection include ozone and peracetic acid. d. Soaps and Detergents are both surfactants, which are chemicals that lower surface tension, and may act as foaming agents and emulsifiers. For this reason, they are useful in removing soils from hard surfaces but pose a real danger to both fish and beneficial bacteria cultured in RAS. e. Solvents are a substance, usually a liquid, which dissolves a solute. While not a particularly effective cleaning agent for aquaculture related soils, ethanol (70%) is commonly found in laboratories and zebrafish facilities, where it is often used as a disinfectant and drying agent for common-use equipment, such as microscopes and hand-tools. B. Cleaning Techniques a. Manual washing of RAS equipment and components is perhaps the most commonly employed cleaning technique. When manually washing zebrafish tanks, accessories, and related equipment, there is often a need for pretreatment in baths of water in order to keep the soils wet since, once dried, many of the soils common to zebrafish RAS become tenacious and require additional force to remove. It is common to use household bleach to assist in this process, but this approach will require adequate posttreatment to neutralize the chlorine, which may pose a direct threat if left on the tanks. Sodium thiosulfate is commonly used for this purpose. The manual scrubbing of tanks and accessories is often aided by the use of scrapers, sponges, and so-called nonabrasive pads or electric-powered tools borrowed from the restaurant and bar industry. The use of these tools will ultimately abrade surfaces making tanks that were once optically clear, become foggy and scratched. Fig. 27.8
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FIGURE 27.8 Image of abraded surfaces from cleaning a zebrafish tank. Photograph courtesy of Erik Sanders.
illustrates a tank with abraded inner surfaces. Fig. 27.9 illustrates properly cleaned tanks. This abrasion represents a manifold increase in surface area and will create a surface that will continue to be more and more rapidly colonized by algae and biofilms, effectively decreasing the useful service life of the equipment. From a staffing and labor cost standpoint, manual washing is quite expensive when analyzed on a per-unit basis. Areas of undesirable variability in manual washing include the level of productivity, quality control, and the potential for mistakes with chemicals leading to a risk of human and fish colony health.
FIGURE 27.9 Images of properly cleaned tanks. courtesy of Erik Sanders.
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b. Automated washing, while common in rodent facilities, where it is commonly referred to as cage wash, is relatively new to the field of zebrafish, only becoming benchmarked in 2012. The machines common to this kind of washing are cabinet washers, cage, and rack washers, and tunnel washers. This kind of washing should require a minimum of pretreatment of the tanks and accessories (lids, baffles, etc.) and should render the items washed completely free of any and all soils, and preferably, at a high level of disinfection. All chemicals employed should be free of detergents, soaps, and surfactants since these can cause serious problems by creating foaming in RAS and by damaging the slime-coat of fish and biofilter bacteria. Wash chemicals typically consist of alkalis and oxidizers but may also include acids. While municipal water may be acceptable for the wash phases of the process, the final rinse should be performed only with reverseosmosis or suitably purified water. Importantly, the presence of any chemical residue on the tanks at the completion of the wash cycle is unacceptable considering the highly sensitive nature of aquatic species and biofilter bacteria and the types of research being performed with zebrafish (embryology, toxicology, etc.). In contrast to rodent cage wash standards, the drying phase of the process is not as important for aquatics cage wash standards. Although automated cage wash requires a capital investment, the cost of washing on a per-unit basis is often at least 50% less, and the level of cleaning and disinfection is substantially higher than that which can be expected using a manual approach. Fig. 27.10 illustrates the Calypso Aquatic Cabinet Washer from Tecniplast. Fig. 27.11 illustrates a Tecniplast Presentation Rack for tanks prepared to be rolled into a Tecniplast Cage and Rack Washer. c. Autoclaving, considered to be complete sterilization of an object, is performed using high temperature at elevated atmospheric pressure for an extended period of time. All items to be autoclaved must be fully cleaned before undergoing the process. While many zebrafish housing components are capable of withstanding these conditions, the recommendations of the manufacturer should be carefully followed to avoid damage to the items being autoclaved. C. Cage wash Area Planning a. Design Challenges i. Work Flow While rodent facilities predominantly employ the clean-dirty corridor concept, it is far less common in aquatics facilities. Much of
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FIGURE 27.10
Images of the Tecniplast Calypso Aquatic Cabinet Washer, closed and open, showing crates for accessories, and in service. Photographs courtesy of IWT/Tecniplast.
FIGURE 27.11 Image of Tecniplast cage wash presentation rack loaded with fish tanks and components, prepared for entry to Tecniplast Cage and Rack Washer. Photograph courtesy of IWT/Tecniplast.
this difference can be attributed to the historical design and cost expectations of zebrafish facilities, which often, and mistakenly, are thought to be much less complex and costly than rodent facilities. Dirty aquatics equipment is prone to drip and fall to the floor; thus, it is essential that this be considered when selecting cage wash operations locations. A failure to do so may result in a situation, where researchers are constantly crossing paths with staff carrying out cage wash operations. ii. Space Requirements Because the allotment of space for cage wash in aquatics facilities is not standard practice as
it is in rodent facilities, we must borrow from the rodent facilities in order to arrive at a bestpractice approach. There exist relatively simple formulae for planning throughput and equipment requirements for rodent facilities, which may readily be adjusted for aquatics facilities. Factors for establishing a baseline in a full-capacity facility are the total quantity of cages in use, the percentage of these cages changed daily, the number of days/week cage wash is performed, the number of shifts in a day, and an efficiency factor of a full workday. From these factors, it is possible to define the daily demand. Daily demand will describe how many cages must be processed each day at peak capacity. With this figure, it is possible to specify the type and number of machines needed, and consequently, the space they will require. Additionally, you will be able to determine the total inventory of cages and related components required to maintain the daily operations at full capacity. Considering that colony size may vary wildly from week to week within zebrafish facilities, you should expect to have space to store at least 25% of your total cages and components as clean supply. iii. Finishes Since cage wash operations involve high temperatures, steam, corrosive chemicals, water, and high humidity, all materials in the
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cage wash areas must be constructed from durable materials, such as stainless steel, epoxy resins, fiberglass, etc. Given the high traffic volume, the floors and doors should be specified to handle both a high degree of wear and a wet environment. iv. Waste Handling Most solid waste in a zebrafish facility is disposed of by the RAS mechanical filtration, but some will make its way into the cage wash area. It is important to have the ability to spray down equipment to remove or loosen solid wastes, and that the areas have adequate floor drains to handle its removal. v. Safety Safety of personnel and animal colony are of critical importance. This is especially true where caustic or corrosive chemicals are concerned and where volatile chemicals are used. Always ensure your operational guidelines are aligned with the Environmental Health and Safety (EH&S) coordinators for your institution. The need for eyewash and chemical showers will be dictated by EH&S and will need to be incorporated into your space planning and budget. There will likely be additional requirements from EH&S for personal protective equipment to be worn and used by personnel. b. Operational Philosophy Many variables will impact the time that a zebrafish cage may stay on a rack until it becomes necessary to exchange it for a clean one. It is critical that daily health checks be performed, and if our view of the fish is impeded by the growth of algae, then the tank must be exchanged. If the accumulation of solid waste in the tank has begun to impede the self-cleaning action of the tank or has caused the water level in the tank to rise above its normal operating level, then the tank must be changed. Your daily operations will guide you in this regard, but as a general rule, tanks should be changed at least every 90 days.
Cleaning Validation Techniques A. Microbial/Biological Techniques: These are used to establish the efficacy of the cleaning process as it pertains to removing the biological load. There are several readily available and reliable test methods to consider. a. Replicate Organism Detection and Counting (RODAC) plates are inexpensive and very useful for detecting viable organisms that may be left
FIGURE 27.12 A set of RODAC plates. The top two plates have no growth indicating a complete cleaning and sanitation of the tested surfaces; the bottom two plates show the growth of microorganisms from pre-washed surfaces. Photograph courtesy of Erik Sanders.
behind after cleaning. These plates should be formulated and incubated in a manner that would allow detection of possible organisms of concern in a zebrafish facility. Fig. 27.12 illustrates a set of RODAC plates. b. ATP Detection Swabs are available from several manufacturers. Most utilize a combination of enzymes and buffer which, when combined with ATP, emit light that is detected and enumerated by a handheld device. ATP detection is fast (10second or less), produces recordable and numeric data, is reliable, and is very sensitive, detecting contamination well below the threshold for RODAC plates.
Conclusions Using proper cleaning and disinfection techniques are crucial for the culture of zebrafish for many reasons. Incorrect procedures can result in catastrophic failures, including loss of animals and data. Those working in the field must be well trained, familiar with their systems, and observant to ensure that the systems are kept in a manner that supports optimal animal health and valid research results. For further reading on this topic, please refer to the references listed below.
Further Reading Allen, T. J. T. (2003). 1953-Animal Welfare Information Center (U.S.), information resources for animal facility sanitation and cage wash [electronic resource]/compiled by Tim Allen. Beltsville, Md, USDA, ARS, NAL. Animal Welfare Information Center Resource Series No. 19.
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Collymore, C., Porelli, G., Lieggi, C., & Lipman, N. S. (November 2014). Evaluation of 5 cleaning and disinfection methods for nets used to collect zebrafish (Danio rerio). Journal of the American Association for Laboratory Animal Science, 53(6), 657e660. Feinstein, S. (6/18/2018). What is the difference between sanitation, sterilization, and disinfection? ALN magazine. https://www.alnmag.com/ article/2014/10/what-difference-between-sanitation-sterilizationand-disinfection. Garcia, R. L., & Sanders, G. E. (November 2011). Efficacy of cleaning and disinfection procedures in a zebrafish (Danio rerio) facility. Journal of the American Association for Laboratory Animal Science, 50(6), 895e900. Harper, C., & Lawrence, C. (2010). The laboratory zebrafish. Boca Raton (FL): CRC Press. International Office of Epizootics. (2009). Aquatic animal health standards commission. Chapter 1.1.4: Methods for disinfection of aquacultural establishments, in Manual of diagnostic tests for aquatic animals (6th ed.). Paris, France: Office International des epizooties. Lawrence, C., & Mason, T. (2012). Zebrafish housing systems: A review of basic operating principles and considerations for design and functionality. ILAR Journal, 53(2), 179e191. Leary, S. L., Majoros, J. A., & Tomson, J. S. (June 1998). Making cagewash facility design a priority. Laboratory Animals, 27(6), 28e31.
Liu, D., Behrens, S., Pedersen, L.-F., Straus, D. L., & Meinelt, T. (2016). Peracetic acid is a suitable disinfectant for recirculating fishmicroalgae integrated multi-trophic aquaculture systems. Aquaculture Reports, 36(4), 1e142. Martins, M. L., Watral, V., Rodrigues-Soares, J. P., & Kent, M. L. (February 2017). A method for collecting eggs of Pseudocapillaria tomentosa (Nematode: Capillariidae) from zebrafish Danio rerio and efficacy of heat and chlorine for killing the nematode’s eggs. Journal of Fish Diseases, 40(2), 169e182. https://doi.org/10.1111/ jfd.12501. Policy SC-50-103. (6/10/2016). Animal facility quality assurance and monitoring. UC Davis Office of the Attending Veterinarian Standards of Care. University of Wisconsin-Madison, Office of chemical safety, the chemical safety mechanism: Laboratory glassware cleaning, SOP. Wallace, J. (6/24/2018). Cage processing operations in laboratory animal facilities. ALN magazine. https://www.alnmag.com/article/2012/ 07/cage-processing-operations-laboratory-animal-facilities. Whipps, C. M., Murray, K. N., & Kent, M. L. (February 2015). Occurrence of a myxozoan parasite Myxidium streisingeri N. Sp. In laboratory zebrafish Danio rerio. Journal of Parasitology, 101(1), 86e90. https://doi.org/10.1645/14-613.1.
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