The Laboratory Environment

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The Laboratory Environment

• LAT Chapter 7

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Humidity Temperature

• Standard relative humidity 30 to 70
• Standard temperature range 19C to 26C
• 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.

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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.

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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.

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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.

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Laminar/Mass Air Displacement
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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 gt hood
  ineffective.
• Will not prevent exposure of personnel to
  chemical agents such as formaldehyde or gas
  anesthetics.

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Other Environmental Variables

• Intense lighting gt 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 gt reduced breeding in some species.
• Animals can be sensitive to noises humans cant
  hear.
• Some rodents susceptible to audiogenic seizures
  when exposed to sudden loud noises.
• Noise stress gt enlarged adrenal glands, reduced
  breeding efficiency, increased blood pressure,
  auditory damage, and behavioral disorders.
• Avoid sudden loud noises greater than 80 decibels
  (db).

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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

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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.
• gt risk of infection if organisms are resistant
  /or highly virulent.

• gt risk if the animals are particularly
  susceptible (e.g. immunocompromised).

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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 gt 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.

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Other Attributes

• Toxicity thoroughly rinse away
• Application -
• Mops and squeegees
• Sprays
• Immersion
• Fogging
• Fumigation
• Evaluation Methods bacterial cultures

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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.

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Methods Moist Heat

• Hot water is effective only as a sanitizer.
• Steam is a good sterilizer.
• Steam under pressure - temperature gt 100C
  (212F).
• Limitations
• cutting edges dull
• dry fabrics scorch
• some wet materials corrode
• some rubber and plastics deteriorate
• certain materials dont mix with water
• possibility of serious injury
• Minimum sterilization time 15 minutes at 121C
  (250F) or 5 minutes at 132.2C (270F).

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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.

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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 121C and pressure
  reaches 15 pounds per square inch (psi), the
  timer begins sterilization time.
• Steam is then vented.
• Drying cycle reduces residual moisture.

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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 stays hot!
Air and Steam Out
Make sure the gasket and drain are clear of
debris.
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Dry Heat

• Kills most commonly encountered microbes.
• Requires long time (one to two hours at 160C) 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.

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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

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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.

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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

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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.
• Employees 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.

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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).

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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
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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.

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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.

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Secondary Barriers
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Secondary Barriers
Ultra Violet Irradiation
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Waste Disposal

• 1. Research protocol must specify type, amount,
  route and method of excretion.
• 2. Isolate hazardous animals from animals that
  dont 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.

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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

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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.