The Laboratory Environment

1. The Laboratory Environment LAT Chapter 7

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3. Humidity & Temperature • Standard relative humidity = 30 to 70 % • Standard temperature range =19°C to 26°C • Higher temperatures for post-operative recovery for birds, reptiles, many nonhuman primates, and hairless rodents. • Measurement: • thermometer, minimum-maximum thermometer, thermograph • Relative humidity: • amount of water present in air • hygrometer, wet/dry bulb thermometer • Computerized systems automatically record environmental data.

4. Air Exchange • 10 to 15 fresh air room changes / hour standard • Either 100% fresh air or re-circulated air • Air filtered before and after leaving the room • Reduce level of airborne pathogens, odors, chemical contaminants, and particulates • Supply and exhaust measured with anemometer • measures the velocity of air passing through the vent expressed as cubic feet per minute (CFM) • Adjustments to supply and exhaust flows are what determine positive or negative room pressure.

5. Dealing With Ammonia • A gaseous by-product of the bacterial metabolism of urea, a substance found in urine. • Heavier than air and becomes concentrated • Can be a serious problem in filter-top cages. • Mycoplasma pulmonis may cause disease in the presence of a high ammonia. • Some commercial bedding material contains an ammonia inhibitor.

6. Laminar/Mass Air Displacement • Uniform, unidirectional, continuous flow of filtered air • 200 or more air changes per hour • Laminar air flow combined with HEPA filters prevent airborne microorganisms from entering. • HEPA filters are 99.7 percent efficient. • removes particles as small as 0.3 microns. • If HEPA filters are kept dry, bacteria and viruses cannot pass through them. • Laminar air flow cabinets or cubicles - • Air is drawn through a pre-filter and forced into a plenum or distribution chamber, then through HEPA filters and over cages. • With reversal of air flow, devices achieve biohazard containment.

7. Laminar/Mass Air Displacement

8. Ventilated Cage Racks • Provide HEPA-filtered air to each individual cage. • Provide a barrier at the cage level. • Continuous air flow reduces ammonia levels. • Dust and hair can clog a HEPA filter. • pre-HEPA filter for gross contaminants • Laminar flow hoods (work stations) used for manipulations • Most are of the type which protects both the operator and the hood contents from contamination (Class II). • Understand how the hood functions • Stacking cages and other materials inside a hood can disrupt the normal air flow => hood ineffective. • Will not prevent exposure of personnel to chemical agents such as formaldehyde or gas anesthetics.

9. Other Environmental Variables • Intense lighting => retinal degeneration in albino spp. • Most animals tolerate lighting of 35- to 100-foot candles. • 12-hour light/dark cycle - on 12 hrs and off 12 hrs. • Automatic light timing devices prevent lighting variables. • Variations => reduced breeding in some species. • Animals can be sensitive to noises humans can’t hear. • Some rodents susceptible to audiogenic seizures when exposed to sudden loud noises. • Noise stress => enlarged adrenal glands, reduced breeding efficiency, increased blood pressure, auditory damage, and behavioral disorders. • Avoid sudden loud noises greater than 80 decibels (db).

10. Sanitation - Disinfect or Sanitize 4 levels of sanitation: 1. Cleaning: complete removal of visible soil from surface 2. Sanitation: reducing organisms living on inanimate object to an acceptable public health standard 3. Disinfection: reducing number of pathogenic organisms (not necessarily spores) to a harmless level 4. Sterilization: rendering an object free of all living organisms

11. Sanitation - Sanitize • The animal facility is continuously recontaminated by air, water, animals, and people. • Sanitation program = cleaning + sanitizing. • Degree of risk = type and level of contamination + use. • > risk of infection if organisms are resistant +/or highly virulent. • > risk if the animals are particularly susceptible (e.g. immunocompromised).

12. Choosing a Chemical Label Claims: regulated by EPA under the Federal Insecticide, Fungicide, and Rodenticide Act Spectrum of Activity: the specific organisms tested against the product Effectiveness in Hard Water: Hard water ions can inactivate chemical. Stability of the pH: Buffers prevent > pH changes from the concentrated to the diluted form or by additives such as soaps. Use Dilution: Using too much of product is wasteful and using too little may reduce or eliminate the antimicrobial effect. Contact Time: Essential that agent be in contact with surface long enough to kill the most resistant organisms present. Temperature: Heat could cause the evaporation of some of the components of the formulation.

13. Other Attributes Toxicity: thoroughly rinse away Application - Mops and squeegees Sprays Immersion Fogging Fumigation Evaluation Methods: bacterial cultures

14. Sterilization • Moist heat, dry heat, chemicals, and radiation • Steam autoclave is primary means of sterilizing. • Resistant organisms indicators for testing sterility. • Most common biological indicator are spores of the bacterium Bacillus stearothermophilus. • Small vials placed into the autoclave during a sterilizing cycle. • Vial incubated to detect any growth of the spores. • A color change indicates growth of the bacteria. • No growth = sterilizing cycle is operating properly. • Chemical reaction temperature indicators - change color when correct surface temperature reached.

15. Methods: Moist Heat • Hot water is effective only as a sanitizer. • Steam is a good sterilizer. • Steam under pressure - temperature > 100°C (212°F). • Limitations • cutting edges dull • dry fabrics scorch • some wet materials corrode • some rubber and plastics deteriorate • certain materials don’t mix with water • possibility of serious injury • Minimum sterilization time 15 minutes at 121°C (250°F) or 5 minutes at 132.2°C (270°F).

16. Autoclaves 1. Central chamber is surrounded by a jacket. 2. Steam saturated with water vapor and superheated under pressure. 3. Steam baffle prevents load from being saturated. 4. Drain present at the lowest point of the chamber. 5. Valves top and bottom permit the exit of air and steam 6. Safety valve if the steam pressure exceeds a safe level. 7. Air inlet and vacuum air filters remove particulates/ 8. In-line thermometer in the steam drainpipe 9. Door gaskets, joints and seals must be air-tight.

17. To Run an Autoclave • Steam in at top, displaces air out drain in bottom. • Only effective if all the air is removed. • Air pockets prevent steam penetration and heat transfer. • Load equipment to be sterilized. • To start, close door tightly and turn on timer. • When the temperature reaches 121°C and pressure reaches 15 pounds per square inch (psi), the timer begins sterilization time. • Steam is then vented. • Drying cycle reduces residual moisture.

18. Autoclaves Air and Steam In Avoid Trapped Air Chart records the run cycle, times and temperature. Doors can be automatic or manual. Safety tip: Jacket stayshot! Air and Steam Out Make sure the gasket and drain are clear of debris.

19. Dry Heat • Kills most commonly encountered microbes. • Requires long time (one to two hours at 160°C) to effectively sterilize. • Can scorch or burn certain materials. • Most common dry heat method is hot air oven. • Effective on equipment or bedding materials damaged by moist heat or chemicals.

20. Chemicals • Glutaraldehyde • Formaldehyde • Toxic, carcinogenic, corrosive, and has limited penetrability. • It should be used only by specially trained personnel. • Peracetic acid • Chlorine dioxide • Ethylene oxide • Plasma sterilizers

21. Radiation For interesting information on food irradiation, go to: ccr.ucdavis.edu/ irr/what2.shtml • Ionizing = gamma and beta • Non-ionizing = ultraviolet, or UV • Disrupts microorganisms protein structure • Ionizing radiation can be lethal to humans • Gamma rays used on instruments and supplies. • Irradiated diets are nutritionally supplemented. • Non-ionizing radiation less penetrability. • Irradiated items are not radioactive.

22. Vermin Control • Carriers of disease-causing agents • Walls and floors free of cracks and crevices • Pipelines, drains, and air filters well sealed • Inspect incoming supplies for vermin • Keep stored feed, bedding, and caging away from walls • Noninvasive = traps, sticky boards, boric acid, or silica • Eliminating feral or wild rodents = poisoning or trapping in areas outside the animal rooms. • Place traps with triggers or entry ports along walls • Control involving pesticides pest control personnel

23. Safety & Hygiene • Instruct on precautions taken in work area and use of safety equipment. • Advise that use of safety equipment is mandatory. • Equipment must be available for any type of risk or exposure encountered. • Employee’s responsibility to perform in a safe manner. • First aid stations / emergency eye-wash or shower stations / fire extinguishers / spill kits and instructions / emergency evacuation routes • Good personal hygiene needs to be enforced.

24. Research Environment Hazards Radioisotopes, living pathogens, carcinogens, and toxins • Sign information required: • identity of biohazardous agent, the name and telephone number of responsible supervisor, and special requirements for entering • Basic - microorganisms not known to cause disease in healthy adult humans • CDC classifies these organisms as BSL1 (Biosafety Level 1). • Containment - separate environment from public • These organisms are classified as BSL2 (Biosafety Level 2), • High containment - may cause serious or fatal disease • These organisms are classified as BSL3 (Biosafety Level 3).

25. Research Environment Hazards Link to web site on the various uses of radioisotopes. http://www.uic.com.au/peac.htm What is a radioisotope? Isotopes are different forms of an atom of the same chemical element. They have identical chemical properties but a different relative atomic mass. While the number of protons is the same, the number of neutrons in the nucleus differs. Some isotopes are referred to as 'stable' and others as 'unstable' or 'radioactive'. It is the radioactive nature of these unstable isotopes, usually referred to a 'radioisotopes', which gives them so many applications in modern science and technology. see also ANSTO paper on Radioactivity, Radioisotopes etc

26. Primary Barriers Biological safety cabinets • Class I,II & III cabinets • Effective operation of safety cabinets depends on inward flow of air, and any activity that disrupts that flow can result in escape of material from the cabinet. • Cabinets should be tested at installation, any time it is moved to a new location, and at least annually. • A certification label, with the date that the next routine check is due, is attached to the cabinet at the time of testing.

27. Secondary Barriers • Air locks, locker rooms, shower areas, ultraviolet lighting, differential airflow, air filters, and other such facilities outside immediate animal housing environment • Combination of structural barriers plus primary barriers and good technical safety skills routinely protect technicians.

28. Secondary Barriers

29. Secondary Barriers Ultra Violet Irradiation

30. Waste Disposal 1. Research protocol must specify type, amount, route and method of excretion. 2. Isolate hazardous animals from animals that don‘t contain materials. 3. Post sign on room/cages containing hazardous materials. 4. Post “Notice To Employees” and “Emergency Procedures” signs. 5. Under supervision of veterinarian and Occupational Health and Safety. 6. Monitor personnel working with animals that emit gamma radiation. 7. Instruct personnel in proper procedures for handling and disposing of contaminated wastes from the cages. 8. Occupational Health and Safety Office approval of investigators . 9. Occupational Health and Safety Officer central control over methods and records of disposal of hazardous animal carcasses. 10. Instruct personnel in any additional precautionary measures.

31. Environmental Enrichment • Allows animals to engage in “species typical” behavior. • The Guide divides behavioral management into: • Structural environment • Social environment • Activity • Animal Welfare Act mandates written plan and records. • Dogs - exercise schedules outside of cage and technician interaction • Primates - indestructible objects, food treats, puzzle solving, group housing of compatible animals, swings, perches and technician interaction

32. Additional Reading 1. Biosafety in Microbiological and Biomedical Laboratories, HHS publication No. (CDC) 93-8395, 1993. 2. Fox, J.G., Cohen, B.J., and Loew, F.W., eds. Laboratory Animal Medicine. Academic Press, Inc., Orlando, FL, 1984. 3. Occupational Health and Safety in the Care and Use of Research Animals. National Academy Press. 1997.