CONTROL OF

MICROORGANISMS &
LABORATORY SAFETY
 ROUTES OF TRANSMISSION
Stages of Development
>Portal of Entry: Mode of Transmission
> Incubation: period of adjustment
> Prodomal: early signs and symptoms
> Clinical/Active Period: Period of Illness
> Phase of Decline: less signs and symptoms
> Convalescent: period to recovery
 Terms
Process that kills all forms of life
Sterilization Examples: incineration, moist or dry heat,
filtration, gamma radiation

Process during which most pathogenic organisms are

destroyed
Disinfection Physical: boiling, pasteurizing, ultraviolet light
Chemical: alcohols, aldehydes, phenolic compounds

Disinfectants used on living

Antiseptic tissue
3
 Factors that influence the degree
of killing
Types of organism
Number of organisms
Concentration of disinfecting agent
Presence of organic material
Nature of surface to be disinfected
Contact time
Temperature
pH
Biofilms
Compatibility of disinfectants and sterilants
TYPES OF ORGANISM
 Number of organisms
Aka microbial load (bioburden)
 Concentration of disinfecting
agent

Amount of disinfectant needed to destroy

microbes

Instruction on preparation, dilution, use:

must be followed
 Presence of organic material
Blood, mucus, pus
Inactivates the disinfecting agent
Coats the surface to be treated
Bleach is easily inactivated by organic
material
 Nature of surface to be
disinfected
Certain medical instruments are made up of
materials that exclude the use of certain
disinfection or sterilization methods
because of possible damage.

Eg. Endoscopic instruments

 Contact Time
Amount of time a disinfectant or sterilant is
in contact w/ the object

Determines whether an agent is a

disinfectant or sterilant
eg. glutaraldehyde
 Temperature
Disinfectants are generally used at rm temp
(20OC to 22OC)
Increase in temperature will increase
activity of disinfectant, decrease in temp
will decrease the activity of disinfectant
Too much increase or decrease in temp will
render a disinfectant inactive.
 pH
The pH of the material to be disinfected or
sterilized can affect the activity of the
disinfecting or sterilizing agent. It is critical
to make sure at what pH the agent is active
and what the pH of the material to be
exposed to the agent is at the time the
process will be done.
 Biofilms
Group of bacteria that have a protective
material over them that shields them from
the outside environment
To disinfect materials that may have a
biofilm present, the concentration of the
disinfectant may need to be increased, the
contact time may need to be increased, or
both.
 Compatibility of disinfectants
Some disinfectants may inactivate other
disinfectants

eg;. Bleach and quaternary ammonium

compounds
 Type of water
Hard water may reduce the rate of killing
microbes

70% etOH more effective disinfectant than

90%: increased water hydrolizing bonds in
protein molecules make the killing of
microbes more effective
 Methods of sterilization
PHYSICAL METHODS
1. Incineration
2. Moist heat
3. Dry heat
4. Filtration
5. Ionizing (gamma) radiation
 Incineration
Most common method of treating infectious
waste

Hazardous materials are burned to ashes at

temp 870 OC to 980 OC

Safest method to ensure that no infective

materials remain in the samples or
containers
 Moist heat
Steam under pressure
Uses autoclave
To sterilize biohazardous waste and heat-
stable objects
1 atm (15 psi) : pressure
121 OC (250OF) for 15 mins or 132 OC
(270OF) for 30-60 mins
Principle: gravity displacement type
 Dry heat
Longer exposure times (1.5 to 3 hours) and
higher temperatures than moist heat (160 OC
and 180OC )

Glasswares, oil, petrolatum, powder

 Filtration
For heat sensitive: antibiotic solutions, toxic
chemicals, radioisotopes, vaccines, and
carbohydrates
Liquids: cellulose acetate or cellulose
nitrate membrane w/ vacuum
Air: HEPA filter
 Ionizing radiation
In the form of gamma rays or e- beams
Short wavelength and high energy
Used in microwaves & radiograph
machines
Also for disposables: plastic syringes,
catheters, or gloves
 Chemical sterilants
Ethylene oxide: most common, used in
gaseous form for sterilizing heat sensitive
objects
Formaldehyde vapor/vapor phase H2O2:
sterilize HEPA filters in BSC
Glutaraldehyde: sporicidal in 3 to 10 hours,
used for medical equipment
Peracetic acid: for surface sterilization of
surgical instruments
 Methods of disinfection
Physical Methods
1. Boiling at 100OC for 15 mins: kills
vegetative bacteria
2. Pasteurizing at 63OC for 30 mins or 72OC
for 15 secs: kills food pathogens w/o
damaging the nutritional value/flavor
3. Using UV light
 Methods of disinfection
Chemical methods
1. Alcohols
2. Aldehydes
3. Halogens
4. Heavy metals
5. Quaternary ammonium compounds
6. Phenolics
 Chemical disinfectant
Alcohol
EtOH/isopropyl alcohol: non sporicidal
Act on microbes by denaturing CHONS
Excellent in vitro bactericidal activity
against most gram-positive and gram-
negative bacteria.
Also kill Mycobacterium tuberculosis,
various fungi, and certain enveloped
viruses;
Aldehydes
Formaldehydes and glutaraldehydes: not
generally used as surface disinfectant due to
irritating fumes

Glutaraldehyde: used as a 2% solution, it is

germicidal in approximately 10 minutes and
sporicidal in 3 to 10 hours. Its killing activity is
due to inactivation of DNA and RNA
through alkylation of sulfhydryl and amino
groups.
Halogens
Chlorine and iodine: most often used

Iodine: either tincture w/ alcohol or an

iodophor coupled to a neutral polymer
(povidone-iodine)
Iodophors: contact time is more that 30
secs.
 chlorine
Chlorine: NaOCl (bleach); 1:10 dilution

Killing activity is based on the oxidative

effects of hypochlorous acid, formed when
chloride ions are dissolved in water

Used as surface disinfectants

Heavy metals
Mercury is toxic; no longer recommended
Eye drop solution with 1% silver nitrate :
used for newborns eyes

Quaternary ammonium compounds

To disinfect bench top or other surfaces in
the lab
Phenolics
Amphyl: derivatives of carbolic acid
(phenol)
Addition of detergent: cleans and disinfects
Concentration is 2-5%: for cleaning bench
tops
 Phenolics
Chlorhexidine gluconate
Hexachlorophene
Chloroxylenol
Triclosan
 Chlorhexidine gluconate
Disrupts cell membrane and precipitates
cell content
(0.5%-4%) : more effective against gram
positive than gram negative
Less activity against fungi and tubercle
bacilli
 Hexachlorophene
Effective against gram positive bacteria
A chlorinated bisphenol: interrupts bacterial
electron transport, inhibits membrane-
bound enzymes, ruptures bacterial
membranes at high concentrations
Unsafe for OTC distribution
 Chloroxylenol
Parachlorometaxylenol
Halogen-substituted phenolic compound
Acts by microbial cell wall disruption and
ENZ activation
Good activity against gram (+), less in gram
(-), fungi, viruses, M. tuberculosis
 Triclosan
Diphenyl ether that disrupts the cell wall
Good activity against gram positive, gram
negative, viruses
Fair activity against M.tb
Poor activity against fungi
LAB SAFETY
 STANDARD PRECAUTIONS
1. Consider ALL patients body fluids to be
bio-hazardous regardless of diagnosis.
2. Always wash hands before and after
contact with patients.
- hospital prescribed hand sanitizer
3. Wear gloves when handling body fluids,
tissues or contaminated surfaces.
 STANDARD PRECAUTIONS
4. Wear gloves and waterproof lab gown,
mask and goggles if splashing can occur or
during procedures ( e.g. surgical pathology
cutting, postmortem examination).

5. Dispose all needles in a puncture-proof

box/container.
 STANDARD PRECAUTIONS
6. Minimize mouth-to-mouth transmission by
keeping mouth pieces (mask) readily
available.
7. Clean blood and body fluid spills with a
solution of bleach (10%) and water or
with a hospital prescribed disinfectant.
8. Immediately report all needle sticks and
accidental splashes.
 BODY FLUIDS
Blood Urine
Semen Feces
Vaginal secretions Nasal secretions
Synovial fluid Vomitus
Amniotic fluid Perspiration
Cerebrospinal fluid Sputum
Pleural fluid Saliva
Peritoneal fluid
Pericardial fluid
 UNIVERSAL
PRECAUTIONS/STANDARD
PRECAUTIONS
CDC Recommendation
Applied to all patients >> potential
for infection is unknown
TREAT ALL SPECIMENS AS
INFECTIOUS
Exceptions: feces, sputum, tears,
urine, nasal secretions, vomitus,
saliva and breast milk (UNLESS
THEY CONTAIN VISIBLE BLOOD)
 PERSONAL HYGIENE
1. Laboratory gown must be worn in all
laboratory working areas.
- should offer protection from splashes and
spills, should easily be removed in case of
accident.
2. Laboratory clothing should be kept clean
and replaced when necessary.
 PERSONAL HYGIENE
3. Laboratory gowns are not to be worn outside the
laboratory, especially in canteen or break
facilities.
4. No sandals, open toed shoes shall be worn by
laboratory personnel.
5. DO NOT drink, eat, smoke or apply cosmetics in
the laboratory or storage areas.
6. No food, beverage, tobacco or cosmetic products
are allowed in the laboratory at any time.
 HOUSEKEEPING
1. The area must be kept as clean as the work
allows.
2. Each laboratory employee shall be responsible
for maintaining the cleanliness of his/her area.
3. Reagents and equipment should be returned to
their proper places after use.
4. Chemical, especially liquids, should never be
stored on the floor, except in closed cabinets
suitable for the material to be stored.
 HOUSEKEEPING
6. Stored items or equipment shall not block access to
the fire extinguishers, safety equipment or other
emergency items.
7. Hallways, passageways and access to emergency
equipment must be kept dry and not be obstructed in
any fashion.
8. All working surface and floors must be cleaned
regularly.
9. All containers must be labeled with at least the identity
of the contents and the hazards those chemicals
present to users.
GENERAL SAFETY EQUIPMENT

1. Fire extinguishers
2. Ventilation hoods
3. Storage cabinets
4. Emergency shower
5. Eye wash station
6. First Aid Kit
 FIRE EXTINGUISHERS
1. Class A ordinary combustible materials,
such as wood, cloth, paper, rubber and
plastics.
2. Class B flammable fluids, oil, greases,
paints, and flammable gases.
3. Class C- energized electrical equipment
4. Class D- combustible metals such as
magnesium, titanium, zirconium, sodium,
lithium
 FUMEHOODS
1. Toxic, odoriferous, volatile or
harmful specimens.
2. Purpose toxic or irritating vapors
and fumes out of laboratory working
area.
3. Purpose shield between the worker
and equipment. Done by lowering the
sash.
BIOLOGICAL SAFETY CABINETS
BIOLOGICAL SAFETY CABINETS

-engineering control; type of

containment barrier that protects
the worker from the aerosolized
transmission of organisms
Class I: open fronted, negative-
pressure, ventilated cabinets
-unsterilized air and enters and
circulates within the cabinet, and
the exhaust air from the cabinet is
 BIOLOGICAL SAFETY
CABINETS
Class II: Laminar Flow; commonly
used
-sterilize both the air entering and
circulating within the cabinet and
the exhaust air
IIA: fixed opening
IIB: variable sash opening
 BIOLOGICAL SAFETY
CABINETS
Class III: highest level of safety
-all air entering and leaving the cabinet is
sterilized with a HEPA filter.
-supply air is drawn through a HEPA filter
while exhaust air is filtered through 2 HEPA
filters
 Airflow of not less than
60 ft/min 120 ft/min
BIOLOGICAL SAFETY CABINET

CLASS I SAFETY CABINET

- open-fronted, negative
pressure, ventilated
cabinet.
- Unsterilized air enters and
circulates within the
cabinet
- Exhaust air from the
cabinet is filtered by a
HEPA filter.
BIOLOGICAL SAFETY CABINET

CLASS II SAFETY
CABINET
- Sterilize both the air
entering and circulating
within the cabinet and the
exhaust air.
BIOLOGICAL SAFETY CABINET

CLASS III SAFETY

CABINET
- All air entering and
leaving the cabinet is
sterilized with a HEPA
filter.
- Three HEPA filters
- One for supply air
- Two for exhaust air.
 Biosafety Levels
Examples
Level Precautions of Agents

Biosafety level 1 No known potential for infection. Bacillus subtilis

Standard laboratory techniques. Nigeria gruberi

Biosafety level 2 Include common agents of disease Human

Primary containment equipment is used. immunodeficiency
virus (HIV)
Salmonella

Biosafety level 3 Organisms are transmitted by aerosol. Mycobacterium

Air movement is closely controlled. tuberculosis
Coxiella burnetii

Biosafety level 4 Includes exotic agents. Marburg virus

Maximum containment is required. Congo-Crimean
hemorrhagic fever
60
 BIOSAFETY LEVELS
BSL 1: No known pathogenic
pathogens for normal individuals.
Ex. Bacillus subtilis.
- most undergraduate labs
BSL2: Level 1 plus wearing of gowns,
gloves and other protective
equipment, limited access,
decontamination of all infectious
wastes, inclusion of BSC 1 and 2. -
HBV, HIV, Staphylococcus,
 BIOSAFETY LEVELS
BSL 3: Level 2 plus special lab clothing and
controlled access for handling M. tuberculosis,
Brucella, rickettsiae
-air movement must be controlled
BSL 4: :Level 3 plus entrance through a separate
room where street clothes are removed
-maximum containment
-research facilities
-Exotic viruses such as Filovirus and Arenavirus
FLAMMABLE LIQUID STORAGE
CABINET
EMERGENCY SHOWER
EYEWASH STATION
FIRST AID KIT
 FIRST AID KIT
Alcohol prep pads
Antibiotic cream
Scissors and forceps
Cotton
Gauze pads (4 x 4 and 2 x 2)
Compression bandage
Band-aids
Sterile gloves
First aid booklet
 PERSONAL PROTECTIVE
EQUIPMENT (PPE)
1. Eye Protection
2. Clothing
3. Aprons
4. Gloves
5. Lab gowns
6. Hair net
 EYE PROTECTION
SAFETY GLASS WITH SIDE SHIELD
EYE PROTECTION
GOGGLES
EYE PROTECTION
FACE SHIELD
 CLOTHING
Guidelines:
1. Loose nor torn clothing should be avoided
unless wearing a laboratory gown.
2. Dangling jewelry and excessive long hair
3. Tight jewelry should be avoided.
4. Laboratory gown should be worn at all
times in the working areas.
 CLOTHING
Guidelines:
5. The laboratory gown should be snapped-
type , long sleeved that are buttoned or
garterized.
6. Shoes shall be worn at all times in the
laboratory. Sandals, open-toed shoes are
prohibited.
APRONS (PLASTIC OR
RUBBER)
GLOVES
 LABORATORY WASTE
MANAGEMENT
1. Black trash bag for collection of non-infectious
dry waste
- located in all units, offices
2. Green trash bag biodegradable waste.
- Pantry/ staff room
3. Yellow trash bag infectious and pathological
waste
4. Punctured-proof sharp container
5. Broken glass container
 Infectious and Pathological
Waste
- Used or contaminated gloves, aprons,
gowns, towels, papers.
- Used syringe
- Tissues
- Excreta
- Culture and stocks of infectious agent
(disinfect first)
 ELECTRICAL SAFETY
All electrical equipment shall be properly
grounded.
Extension cords shall not be used as a substitute
for permanent wiring.
Electrical cords or other lines shall not be
suspended unsupported across rooms or
passageways.
Multi-outlet plugs shall not be used unless they
have a built-in circuit breaker.
 CHEMICAL SAFETY
Do not pipette by mouth.
Read the chemical safety information
Material Safety Data Sheet (MSDS)
Dress appropriately
Identify the safety equipment
Dont taste or sniff chemicals
Dont casually dispose chemicals down the drain
Dont eat or drink
 MATERIAL SAFETY DATA
SHEET (MSDS)
-- a written document that provides product users
and emergency personnel with information and
procedures needed for handling and working with
chemicals.
-- Familiarity with MSDSs allows for precautions to
be taken for potentially dangerous products;
seemingly safe products may be found to contain
unforeseen hazards.
 MSDS
- OSHA (USA)
16 SECTIONS
Identification
Hazard(s) identification
Composition/information on ingredients
First-aid measures
Fire-fighting measures
Accidental release measures
MSDS
Handling and storage
Exposure controls/personal protection
Physical and chemical properties
Stability and reactivity
Toxicological information
Ecological information
Disposal considerations
Transport information
Regulatory information
Other information
1. Identification of substance:
Trade name: Benzene
Manufacturer/Supplier:
Alfa Aesar, A Johnson Matthey Company
Johnson Matthey Catalog Company, Inc. 30
Bond Street Ward Hill
 2. Composition/Data on
components

Benzene (CAS# 71-43-3); 100%

Identification number(s):
EINECS Number: 200-753-7
EU Number: 601-020-00-8
 3. Hazards identification
Hazard description:
T Toxic F Highly flammable
Information pertaining to particular dangers for
man and environment
R 45 Can cause cancer - Group I (extremely
hazardous)
R 11 Highly flammable.
R 48/23/24/25 Toxic: danger of serious damage to
health by prolonged exposure through inhalation,
in contact with skin and if swallowed.
 4. First Aid Measures
After inhalation: Supply fresh air. If
required, provide artificial respiration. Keep
patient warm. Seek immediate medical
advice. After skin contact .
 5. Fire fighting measures
Suitable extinguishing agents Carbon
dioxide, extinguishing powder or water
spray.
6. Accidental release measures
Person-related safety precautions: Wear protective
equipment......
Measures for environmental protection..... Do not
allow material to be released to the environment
without proper governmental permits. Measures
for cleaning/collecting: Absorb with liquid-
binding material (sand, diatomite, acid binders,
universal binders, sawdust). Dispose contaminated
material as waste according to item 13
 7. Handling and storage
Information for safe handling:
Keep container tightly sealed. Store in cool, dry place in
tightly closed containers. Ensure good ventilation at the
workplace. Information about protection against
explosions and fires: Keep ignition sources away. Protect
against electrostatic charges. Fumes can combine with air
to form an explosive mixture.
Storage
Requirements to be met by storerooms and receptacles:
Store in a cool location. Store away from oxidizing agents
 8. Exposure controls and
personnel protection
Additional information about design of technical
systems: Properly operating chemical fume hood
designed for hazardous chemicals and having an
average face velocity of at least 100 feet per
minute. Components with limit values that require
monitoring at the workplace: Benzene mg/m3
ml/m3 ACGIH TLV short term 1.6 0.5 ACGIH
TLV long term 8 2.5 B VME 1,6 0,5
 8. Exposure controls and
personnel protection
Personal protective equipment General protective
and hygienic measures The usual precautionary
measures for handling chemicals should be
followed. Keep away from foodstuffs, beverages
and feed. Remove all soiled and contaminated
clothing immediately................ Wash hands before
breaks and at the end of work. Breathing
equipment:...........
Protection of hands: Impervious gloves
Eye protection: Safety glasses, Full face protection
9. Physical and chemical property
Form: Liquid
Color: Colorless
Odor: Aromatic
Change in condition
Melting point/Melting range: 5.51 C
Boiling point/Boiling range: 80.1 C
 10. Stability and reactivity
Thermal decomposition / conditions to be
avoided: Decomposition will not occur if used and
stored according to specifications.
Materials to be avoided: Oxidizing agents
Dangerous reactions No dangerous reactions
known
Dangerous products of decomposition: Carbon
monoxide and carbon dioxide
11. Toxicological information

Oral: LD50: 3306 mg/kg (rat)

Dermal: LD50: 48 mg/kg (mus)
Inhalative: LC50/7H: 10.000 ppm/7H (rat)
 12. Ecological information
General notes: Do not allow material to be
released to the environment without proper
governmental permits.
 13. Disposal consideration
Consult state, local or national regulations
for proper disposal.
 13. Disposal consideration
Consult state, local or national regulations
for proper disposal.
 14. Transport information
DOT regulations:
Hazard class: 3 Identification number:
UN1114 Packing group: II
 15. Regulations
National regulations (This may include a variety of
country-specific detail) All components of this product are
listed in the U.S. Environmental Protection Agency Toxic
Substances Control Act Chemical Substance Inventory .....
This product contains a chemical known to the state of
California to cause cancer or reproductive toxicity.
This product contains benzene and is subject to the
reporting requirements of section 313 of the Emergency
Planning and Community Right to Know Act of 1986 and
40CFR372.
 16. Other information
Employers should use this information only
as a supplement to other information
gathered by them, and should make
independent judgement of suitability of this
information to ensure proper use and
protect the health and safety of
employees ....
Manuals and vials found on top of the ref.
Boxes are found on top of the autoclave.
Boxes and plastic containers found on
the floor.
Gallons of flammable materials
found on the floor.
Gallons of highly flammable/ hazardous
materials stored in wooden cabinets; also
found on the floor.

Xylene
Hazardous/ Flammable materials
stored inside the cabinet; with
alcohol spill.
Junk/Old supplies found inside the
cabinets.
Cabinet handles left hanging.
A safety cabinet must be available for
handling potentially infectious
specimens.
Lab equipment found on the floor.
The end