Maintaining Hygiene in Laboratory Animal Care: Sterilization, Disinfection & Sanitization

1. Chapter Ten Hygiene in the Laboratory Animal Facility

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3. Sterilization, Disinfection & Sanitization • Difference between sterilization, disinfection & sanitization is a matter of degree of cleanliness. • Sterilization = destruction of all organisms on an object • Disinfection = reduction of number of pathogenic microorganisms on an object to a harmless level • Sanitization = reduction of number of microorganisms on an object to acceptable public health standard • Sanitization = make an object aesthetically pleasing & clean

4. Sterilization • Autoclaves sterilize by exposure to moisture at high temperature & pressure. • Autoclaving is fast, reliable, relatively cheap. • Autoclaving avoids the use of toxic chemicals.

5. Autoclaving • Microorganisms, type & size of material being sterilized determines autoclaving time. • large bags of bedding, nested shoebox cages or heavily wrapped surgical instruments • Items should be autoclaved for at least 15 min. at 250°F & with a pressure of 15 psi. • High pressure allows steam to be superheated. • Some items cannot be autoclaved because of sensitivity to heat, moisture or high pressure. • Other sterilization techniques include: • ethylene oxide gas, dry heat sterilization, chemical sterilization, gamma irradiation & liquid filtration

6. Disinfection • Disinfectants too strong for use on living animals. • Classified according to type of microorganism they are most effective against. • End in “–cidal” has killing action. • End in “–static” inhibits microorganism growth. • Bacteriostat prevents growth, does not necessarily kill. • Bactericidal kills bacteria, not necessarily spores. • Sporicidal kills spores & bacteria. • Chemicals, such as phenols, bleach & quaternary ammonia disinfect objects.

7. Disinfection II • Bleach is a superior disinfectant. • It kills many types of bacteria & viruses, is inexpensive & available. • It does not contain dirt-loosening detergents. • < effective if surface not cleaned of organic matter. • Phenolic compounds were popular disinfectants. • High concentrations needed to produce disinfection. • Cats react adversely to phenol. • Quaternary ammonia compounds are weak. • Destroy cell membranes of certain types of microorganisms • Available as virucides, algicides or fungicides • Less effective when mixed w/ detergents or soaps. • Combination neutralizes disinfecting capability.

8. Sanitization • Number of bacteria on inanimate objects reduced enough to prevent disease. • Routine cleaning of items such as floors, cages, walls, feeders, sinks, implements & tables • Removes dirt, hair, dust, saliva, blood, feces, urine. • Wash w/ detergent & rinse w/ water at 180°F. • Deodorants not used in place of sanitization. • Indiscriminate mixing of chemicals can cause reactions hazardous to animals & people.

9. Chemical Guidelines • Guidelines concerning handling of chemicals: • Store chemicals in a cool, central area. • Follow the instructions on label. • Never use a container that is not labeled. • Do not make an assumption as to contents. • Never mix two chemicals together unless authorized by manufacturer. • Mixing ammonia & bleach produces a toxic gas.

10. Cage Changes • The Guide: • Cages should be changed often enough “to provide a healthy environment for animal, in accord with its normal behavior and physiologic characteristics.” • Generally change solid-bottom cages 1-2 x/wk. • Generally drop-bottom cages changed 1x/2 wks. • Number of animals in a cage, cage size & type of bedding affect change cage frequency. • Larger animals (dogs, cats, nonhuman primates) - daily cleaning & bedding replacement. • AWA reg’s set minimum cleaning standards for cages, feeders & water bottles.

11. Equipment Cleaning Techniques • No hand cleaning in animal room. • Airborne microorganisms contaminate environment. • Pass-through washers have clean & dirty side. • Soiled equipment delivered to dirty side. • Cages scraped free of most bedding, feces & debris. • Chemical descalers remove most of urine scale. • Feeders & dishes are scrubbed free of debris. • Bottle brush loosens deposits inside bottles. • Sipper tubes rinsed, washed in a cage washer. • Equipment too large to fit into cage washers should be vigorously hand-scrubbed or pressure washed, using detergents and disinfectants.

12. (Image) Washing Dog Run Flooring

13. Room Cleaning Schedules & Techniques • Sinks - clean, free of clutter & stocked. • Vents and doors - kept free of dust, grime, hair... clogged vents reduce air circulation. • Trash cans - disposable plastic liners, emptied frequently, & disinfected regularly. • Animal rooms - emptied, cleaned & disinfected on fixed schedule. • Clean walls, ceilings, lights & all exposed surfaces. • Clean sink, broom, mop & bucket after each use. • Replace or launder mop heads frequently. • Keep cleaning items in room where they are used. • Use ONLY for cleaning of that room. • Reduces chance of spreading disease from room to room.

14. (Images) Room Cleaning

15. Other Equipment Changes • Racks w/ built-in cages - wash > 2 x/mo. • Cleaning feeders depend on number & type of animals being fed & type of diet. • Critical to inspect water bottles, automatic valves & sipper tubes daily to be sure working. • Replace used water bottles w/ sanitized bottles. • Put refilled bottles back on cages from which they came to prevent cross-contamination. • Watering equipment should be thoroughly flushed before reconnecting rack to room water lines. • Racks should be stored w/ empty water lines.

16. Environmental Monitoring • Temp tapes used to evaluate efficiency of sanitization. • Temp indicator monitors for proper washing & rinsing temps. • One type is a strip labeled w/ heat-sensitive indicator. • Indicator is attached to surface of equipment, which is then sent through washer. • Strip indicates highest water temp on surface. • Rinse water temperature should reach at least 180°F.

17. Environmental Monitoring II • Bacterial culture - testing for bacteria on surface of clean cages & equipment that have been through cage washer is also a common monitoring method. • Small plastic dishes containing a nutrient substance suitable for bacterial growth are pressed onto surface of a clean area. • Small plastic dish is placed in an incubator or allowed to sit at room temp 24 hrs. • Bacteria present on cage surface will grow on nutrient substance & indicate effectiveness of cleaning procedure.

18. Vermin Control • A properly constructed building, good housekeeping program & proper waste disposal help control vermin populations. • flies, fleas, cockroaches, ticks, wild rodents... • Vermin enter on feed, bedding, and humans, and through cracks & small openings. • Keep areas sanitary, dispose of food & bedding, close doors & seal cracks. • Wild rodents that enter a facility must be trapped. • Use of pesticides in animal areas should be strictly controlled.

19. Pesticides • Investigators must consent to their use, as pesticides, just like deodorants, can compromise experimental results. • These chemicals must not be allowed to contact the animals or their feed, bedding or water. • As part of a comprehensive control program, relatively harmless chemicals, such as boric acid and amorphous silica, can help control cockroach infestations.

20. Personal Safety & Hygiene • Protective clothing: • prevents contact w/ infectious, toxic or corrosive agents • type needed depends on procedures being performed • Non-slip bottoms & steel-toed shoes offer protection against slipping as well as from dropped equipment injuries. • Disposable shoe covers prevent cross-contamination in germ free, quarantine & isolation areas. • Only wear work shoes in facility. • Ear protectors recommended in noisy areas in which average noise level is >85 decibels.

21. Goggles & Uniforms • Goggles offer the best protection, because they cover the entire eye and surrounding area. • Facilities should also have eyewash stations in areas where chemical splashes could occur. • Closely fitted face masks are most effective in preventing personnel from inhaling contaminants. • Street clothes should not be worn while working in an animal facility, and uniforms should not be worn outside the facility. • Uniforms protect personnel & environment outside facility against contamination.

22. Gloves • Leather gloves of various lengths are worn to handle animals that bite or scratch. • Some reinforced w/ metal for bite protection. • Heat-resistant to handle hot items or dry ice. • People w/ contact skin allergies to animals should wear disposable plastic or latex gloves. • Special gloves are available for people who have allergies to latex or powder.

23. Personal Hygiene Practices • Safety and personal hygiene guidelines: • Store & consume food in designated areas of facility. • Keep hands away from mouth, nose, eyes, face & hair. • Smoke only in assigned areas. • Always wash hands: • after removing dirty PPE • before applying makeup, smoking or eating • before leaving animal room or cage washing area • Do not wear jewelry that interferes w/ hand washing. • Some facilities require employees to shower before entering and/or exiting animal areas.

24. Occupational Health Program • A pre-employment physical exam, medical history & vaccinations are part of program, • Tetanus - spore-forming bacteria in environment. • Personnel who handle animals or clean up after them are exposed to these spores. • Infection follows deep puncture wounds which are difficult to clean by conventional washing. • Rabies - virus which can occur in any mammal • Personnel who handle animals obtained from animal shelters should be inoculated w/ pre-exposure vaccine. • Hepatitis - virus that infects liver. • Most cases acquired from from nonhuman primates. • Personnel who work w/ primates are often vaccinated

25. Occupational Health Program II • Zoonotic agents are infectious agents that can be transmitted from animals humans. • TB, measles & salmonellosis • Personnel who work with NHP run a higher risk of exposure to TB. • Handlers have tuberculosis tests performed regularly. • All injuries & accidents must be reported to supervisor, regardless of how insignificant. • Immediately wash bite wound w/ soap & water. • Notify supervisor of bite as quickly as possible. • Report location of bite and animal that bit them.

26. Occupational Health Training • In addition to providing protective equipment and vaccinations, a research facility is also required to provide training of personnel in the areas of proper use of equipment, zoonotic diseases, blood-borne pathogens and other areas which can be hazardous to employees.

27. Additional Reading Biosafety in Microbiological and Biomedical Laboratories. HHS Publication No. (CDC) 93-8395. 3rd. Edition US Government Printing Office, Washington, D.C. 1993. Occupational Health and Safety in the Care and Use of Research Animals. National Research Council. National Academy Press, Washington, D.C. 1997.