Biosafety and Waste Management in Molecular Laboratory
THE LABORATORY QUALITY SYSTEM
Why is Safety Important?
• Coming in contact with human blood or blood products
(plasma, serum, etc.), or with certain chemicals used in the
laboratory, is potentially hazardous.
• What’s inside the laboratory should remain inside, without
any leakage.
• Coming in contact with human blood or blood products
(plasma, serum, etc.), or with certain chemicals used in the
laboratory, is potentially hazardous.
• Safety involves taking precautions to protect you and
coworkers against infection, injury or poisoning.
HAND HYGIENE
*The first important to prevent any contamination is through
Proper Hand Hygiene.
*If working with your bare hands you are at risk of contracting
any disease, especially the Respiratory Infections: Influenza,
colds, CoronaVirus. Other Infectious Agents: Salmonella
*Before wearing gloves, we must practice proper hand
hygiene.
*It is better to use liquid soap, rather than bar soap, because
there is a lesser chance to transfer the infectious
agents.
• Persons must wash their hands after working potentially
hazardous materials and before leaving the Laboratory.
• Lather and Scrub: 20 seconds
• Rinse: 10 seconds
• Do not use Hand Dryers!
• Performance of entire handwashing: 40-60 seconds
DECONTAMINATION
• Decontamination – Process of removing or neutralizing
chemical or Biological Agents so that they no longer pose a
hazard. (https://ncbi.nlm.nih.gov)
3 Forms:
• Sterilization – Removal or Destruction of all forms of life,
including bacterial spores.
• Killing all types of microorganisms.
Examples:
o Autoclaving
o Gamma Radiation (for heat sensitive; e.g., Pasteur
pipette; requires a facility for radiation containment)
o Hydrogen Peroxide Gas
o Ethylene Oxide
o Incineration
• Disinfection – Removal, inhibition, or killing of
microorganisms including potential pathogens by using
chemical agents usually on inanimate objects; does not
remove all bacterial spores.
• Only kill the vegetative organisms but not bacterial
spores; only reduces the microbial contamination
• Antisepsis – Antimicrobial substances typically applied in
the skin to reduce possibility of sepsis formation or
putrefaction.
o Injection sites (Germicidal solutions)
o 70% Ethanol
o Iodine Tincture (Iodine + Alcohol)
o Iodophor (Iodine + Detergent)
o Hydrogen Peroxide
o Used in living tissues
• Question: Alcohol, 70% ethanol. Is it a disinfectant or an
antiseptic solution?
• Answer: Both
• Ratio: Alcohol can be used in the skin and for inanimate
objects.
• Iodine: Only an antiseptic solution.
STANDARD PRECAUTIONS
• Previously called as Universal Precautions (with blood
only)
• Include all body fluids, except for sweat. Regardless of the
presence of blood or not, as long as it is a body fluid, it is
considered as infectious.
• Treat all as infectious.
• Minimum infection prevention practices that apply to all
patient care, regardless of suspected or confirmed infection
status of the patient, in any setting where health care is
delivered (CDC)
a) Avoid touching eyes, nose, or mouth with gloved or
unwashed hands
b) ALL SPECIMENS shall be treated as infectious
c) Avoid wearing jewelry
d) Refrain from using mobile electronic devices inside the
Laboratory
e) Keep work area tidy, clean, and free of cluster and
materials not necessary for the work being done
DECONTAMINATION/DISINFECTION
*Germicidal solution
*Before starting any molecular techniques; you must
decontaminate.
• Use of decontamination solutions with proven activity
against enveloped RNA viruses
➢ 70% Ethyl Alcohol
➢ 10% Sodium Hypochlorite: Spills (1:10)
➢ can increase up to (1:5) dilution.
➢ 1% Sodium Hypochlorite: General Surface
Decontamination
• Contact Time of Bleach: 10-15 minutes
• All surfaces and equipment must be decontaminated
before and after usage.

CHEMICAL AGENTS
Efficacy is based on different factors:
• Organic Load (active component)
➢ type of chemical agent that we are using
➢ Sodium hypochlorite (active component of bleach),
ethanol
• Microbial Load
➢ Type of Organism
➢ Condition of surface to be decontaminated
➢ Disinfectant concentration, pH, temperature, contact
time, environmental humidity

SODIUM HYPOCHLORITE
*Also known as household bleach. Sodium hypochlorite is the
active component of the chlorine/ bleach that we are using. If
mixed with incompatible chemicals, it can produce toxic by-
products and gases. It should be diluted first in water.
*It is routinely used in the laboratory to decontaminate surfaces
and to inactivate vegetative bacteria, fungi, lipid and non-lipid
viruses.
• Contact Time: 10-15 Minutes
o Exposure to air (↓ free Chlorine concentration due to
• Bought in concentrations of 5-8.25%, most common:
Chlorine evaporation)
5.25%; (amount of sodium hypochlorite in the bleach.)
o Storage in direct sunlight and varying temperatures
*It is highly corrosive and reactive. Thus, it must be diluted.
(NaOCl solutions can be stored up to 6 months if this
• Preparation: 1:10 or 1:100
is followed!)
o At least 5000 ppm (parts per million), no more than
• Bleach loses 20-50% of its concentration (deteriorates)
10,000 ppm Chlorine for decontamination
after 6 months
• CORROSIVE: Needs to be washed after (Steel); It is an
• Different Cleaning Jobs Require Different Bleach Solutions:
oxidizer (can cause damage to living tissues).
• General lab use: Hypochlorite Solutions
• DO NOT AUTOCLAVE BLEACH SOLUTIONS!
*Example: Prepare 50 ml of 1:10 and 1:100 dilution.
o toxic fumes; damaging to lungs when inhaled.
*10% of 50 mL= 5 mL of NaOCl and 45 mL of water
• Effect: Oxidative effect of Hypochlorous acid
• Avoid the following:

ALCOHOL
• Best preparation: 60-90% Alcohol (↑ Water conc while
maintaining high Alcohol concentrations ↑ contact time of
Alcohol to microorganism)
o For highest effectiveness- 70% (achieved the required
contact time of 2-5 minutes)
o 95% (lesser contact time since highly
concentration=highly volatile)
• If used as a disinfecting agent:
– Without Bleach:2-5minutes
– With Bleach: No need for contact time
• Bactericidal, Fungicidal, Virucidal, non-Sporicidal
– Lipid containing- easy to kill
– Lipophobic (non-lipid)- variable.
• Allowed to evaporate from the surface to which they were
applied to achieve maximum effectivity.
• Effect (not commonly used): Cause
denaturation/aggregation of protein and dissolution of lipid
membrane
• Should not be used as a lone decontaminant for
MOLECULAR TESTING (sodium hypochlorite or Lysol is
used) – causes aggregation of nucleic acids not eradicating
them
ULTRAVIOLET LIGHT
*Disinfectant only; not sterilization procedure
*Comparing UV Light (longer wavelength=low energy=lesser
penetration) to Gamma Rays (shorter wavelength= high
energy=penetrate through walls).
*It can kill organisms floating in the air. Example: As a Room
Disinfectant: Exposing laboratory rooms with UV light to destroy
the bases of the nucleic acid of the organisms floating in the air.
• A type of non-ionizing radiation that causes damage to
cellular DNA by producing Thymine dimers
– Most lethal wavelength: 260nm
– Longer wavelength (>1 nm) and low energy
• Microorganisms are destroyed when water is passed under
the UV Lamps
• Quality Control Indicator: Bacillus pumilus
o (+) growth: incomplete disinfection
o (-) growth: complete disinfection
• Disadvantage: Can only destroy those under its direct
contact. Eyes and skin (reddening, sunburn, and skin
cancer).
*Wears out overtime. Has little to no effect on RNA.
LYSOL
*Phenolic compounds, very active vegetative bacteria and lipids
containing viruses. But cannot kill spores.
• 2 Active Ingredients: Benzalkonium Chloride or Hydrogen
Peroxide
*Even if these two active ingredients are antiseptic, it cant
be used in the skin as it contain other ingredients such as
phenol, an organic solvent that can cause obvious damage
in the skin (corrosive) and is carcinogenic.
• KNOWN EFFECTIVE AGAINST MANY
MICROORGANISMS
Effects:
o Benzalkonium Chloride: Disruption of cell membrane
(create a hole), resulting in leakage of cell contents.
o Hydrogen Peroxide: Degrades Organic compounds by the
highly reactive Hydroxyl radical (-OH). It results to oxidative
burst.
SPILL CONTROL
• General Steps:
1) Add concentration bleach (>5.25% available Chlorine) to a
final concentration of 10% Bleach (1:10; can also use 1:5,
depending on the severity)
2) Let the solution sit for 30-60 minutes (variable; depends on
the amount of spill: smaller spills=10-15 minutes).
3) Larger volumes may require longer contact times.
4) Dispose of the solution down the sanitary sewer.
INACTIVATION METHOD FOR PRIONS
*It is a combination of chemical disinfection and
autoclaving.
• Instrument: (applied to reusable apparatus such as test
tubes)
➢ Immerse in 1N NaOH or 2.5% NaOCl for 1 hour;
remove and rinse in water, and then transfer to an open
pan and treat in a gravity displacement (121C) or porous
load (134 C) autoclave for 1 hour.
• Surfaces:
➢ Spray or pour 1N NaOH or 2.5% NaOCl on surface and
let sit for 1 hour. Ensure surfaces stay wet for the entire
period. Surfaces should be clean of any gross
contamination as organic material can reduce the
effectiveness of the solutions.
INACTIVATION OF MICROORGANISMS:
❖ AUTOCLAVE
*Most effective method of sterilizing laboratory equipment
especially for liquid handling products (e.g., glasswares).
• Principle: Steam under pressure (boiling point of water
increases as the pressure increases)
• Fastest and simplest method of sterilization – all organisms
(except Prions) and spores are killed within 15 minutes.
• Example culture media in Bacteriology.
• Biological Indicator: Geobacillus stearothermophilus
• Properties: psi: pounds per square inch
o 121C, 15 psi, for 15 minutes: Media, Liquids, Utensils,
Glass Pipettes, and Instruments for Assay (to be used
materials)
o 132C, 15 psi, 30-60 minutes: Decontaminating Medical
Wastes (used materials/contaminated=higher
temperature, longer time)
QUALITY CONTROL FOR AUTOCLAVE
❖ Biological Indicator:
➢ After autoclaving, incubate the tubes at 37 C for 24 hours.
➢ No color change if sterilization is complete=
VIOLET/PURPLE
➢ Turns color YELLOW if growth happens=contaminated
❖ Autoclave Tape:
➢ Turns black (dead) if Sterilization is complete
➢ Geobacillus stearothermophilus: spore forming organism
(inactivated= non-infectious
BIOSAFETY CABINET
• Calibrated and Certified before use
• Practice proper usage, placement, and decontamination
(UV light)
• The Biosafety Cabinet is a requirement to process
infectious specimens.
*Commonly used: Class II
*Highest Level of Containment: Class III
*Providing clean recirculating air inside
HOW BIOSAFETY CABINETS PROVIDE
CONTAINMENT/ PROTECTION
*Exhaust system: The air coming inside and outside is
already clean.
1) HEPA Filter – Standard for Biosafety
➢ High Efficiency Particulate Air Filter
➢ 99.97% efficiency at 0.3 microns (pore size)
➢ Made from Pleated borosilicate glass, arranged in
random fibers
*Trap all known infectious agent; ensures that the air that
will be discharged is microbe-free.
2) ULPA Filter
➢ 99.999% efficiency at 0.12 microns
➢ Ultra-Low Penetration Air Filter
*The amount of air that is filtered is extremely low.
3) SULPA Filter
➢ 99.9999% efficiency at 0.12 microns
• Super Ultra-Low Penetration Air Filter
• Curtain of Air” at the opening- protection of the product.
• Laminar flow of the filtered air within the BSC- Protection of
the personnel
• Filtration of exhausted air- protection of the environment.
*There are holes at the bottom, and if you pat your hands,
you can feel that the air is pulled downward. Thus, there is
a tendency that the room air coming inside will be pulled
downwards (negative pressure) and will go to the blower.
The air from the front portion of the blower is pushed, will
the air from the back portion is pulled. As the blower pulls
the air (negative pressure; red arrow), the air will be
pushed (positive pressure; pink arrow; still contaminated)
on the filters. The air will either go outside or will go inside
again.
BIOSAFETY MANAGEMENT
*Managerial factors: Responsibility of the head of the
laboratory to ensure. Concrete plan, protocol, employees
should be well aware.
1) Ensure the development and adoption of a biosafety
management plan and a safety or operations manual.
2) Ensure that regular training in laboratory safety is provided-
Biosafety seminar (3 days)
3) Personnel should be advised of special hazards, and
required to read the safety or operations manual and follow
standard practices and procedures
4) There should be an arthropod and rodent control
programme.
➢ can be a vehicle of disease transmission
5) Appropriate medical evaluation, surveillance and treatment
should be provided for all personnel in case of need, and
adequate medical records should be maintained. (Annually)
• Active or Passive immunization
• Hep B Vaccine
• Active: own forming of Ab
• Passive: Immunoglobulin
• Example, Needle prick: Hep B Ig and Anti-tetanus
HEALTH AND MEDICAL SURVEILLANCE
1) Provision of active or passive (Ig already) immunization
where indicated.
*Usually, when there is an accident, medtechs or interns
are given with HbIg and anti-tetanus as passive
immunization.
2) Facilitation of the early detection of laboratory- acquired
infections.
3) Exclusion of highly susceptible individuals (e.g., pregnant
women or immuno-compromised individuals) from highly
hazardous laboratory work.
4) Provision of effective personal protective equipment and
procedures (by the laboratory).
APPLY SAFETY PRACTICES THROUGHOUT THE TESTING
PROCESS
1) Before Testing (Pre-analytical)
o Specimen collection
o Specimen preparation
o Specimen transport
*Prevent contamination
2) Testing (Analytical) -Testing
*Must apply safety rules (standard precaution)
3) After Testing (Post-analytical) -Disposal
➢ clean working area; yellow bag
DEVELOP PERSONAL SAFE WORK HABITS
• Wash hands before and after entering the lab
• Change gloves frequently
• Wear lab coat or apron
• Dispose of contaminated sharps and waste immediately
after testing
• Pipetting by mouth is strictly forbidden
• Never eat, drink or smoke at the test site
• Keep food out of the laboratory/testing site refrigerator
MAINTAIN CLEAN AND ORDERLY WORK SPACE BIOLOGICAL WASTE
• Keep work areas uncluttered and clean • Exposure: Ingestion, Inoculation, Tactile Contamination,
• Disinfect work surfaces daily Aerosolization, Inhalation of Infectious materials
• Restrict or limit access when working • Blood (yellow bag)
• Keep supplies locked in a safe and secure area o HBV, HCV, HIV
• Keep emergency eye wash units in working order and within o Blood-borne pathogens
expiry date o Inactivated and Autoclaved (if incinerated, no needed
to inactivate)
PROPER DISPOSAL OF BIOWASTES • Swabs
• Good Safety Practices o Respiratory infections/
➢ Identify Hazards o Inactivated (Heat, Chemical) and Autoclaved before
➢ Implement safety strategies to contain hazards (must disposal
inactivate waste before disposal) • Tissues
➢ Audit existing practices to determine whether new ones o Should be fixed with a fixative
are needed o Unfixed tissues must be inactivated and autoclaved
• Other body fluids
➢ Sputum: Tuberculosis, other respiratory infections –
Inactivated and Autoclaved

PREVENTION
• Gloves – Primary barrier protection
➢ Not reused or washed for reusing
• Masks/Respirators, Protective Eyewear, Face Shield– for
exposure from splashes to the mouth, eyes, nose
*Depends on the biosafety level (e.g., BSL-3:N95; BSL-
4:respirators)
• Decontaminate work area regularly

CHEMICAL HAZARDS
• Stored in certain sections of the Laboratory
➢ Not in use, packaging with leakage or corrosion:
Replaced
➢ Water reactive chemicals: No contact with water
*Acids and bases must be separated.
• MATERIAL SAFETY DATA SHEET (MSDS) – Document
that gives detailed information about a material and about
any hazards associated with the material or latest: Material
Safety Sheet (MSS)
*Included here are the composition, preparation, and
antidote of the chemical. Inventory of chemicals must
be done.

HANDLING CHEMICALS
• How to properly handling different kinds of chemicals in the
workplace

CHEMICAL HAZARDS IN THE GENOTYPING LABORATORY

Why is it hazardous?
• Ignitable
• Corrosive
• Reactive
• Toxic
Examples
*From left to right: Flammable, Carcinogenic, Toxic, Corrosive
*CMRA Chemicals (Carcinogenic, Mutagenic, Respiratory,
Allergenic): must be careful in handling chemicals with this label.
• Check label for substance verification
• Chemical-resistant gloves
• Containers held away from the body during transferring
• Do not heat with direct flame

• ACID TO WATER (to prevent sudden reaction)

• Do not touch, taste, or smell chemical
• Use a Laboratory Chemical Hood.
• Clean spills properly and promptly, dispose accordingly.
CHEMICAL HAZARDS IN THE GENOTYPING
LABORATORY
Examples:
• Guanidinium thiocyanate (GITC)- commonly used in lysis
buffers
➢ used in DNA RNA isolation/extraction
➢ carcinogenic and corrosive
➢ Strong denaturing agent
➢ DO NOT MIX WITH Bleach; gas production; very toxic
• Ethidium Bromide (mutagen)- DNA staining in agarose
gels, buffers
➢ as a stain or dye
➢ DNA; attach itself to hydrogen bonds between bases=
causing mutation or cause base changes.
• Acrylamide (neurotoxin)- part of gel electrophoresis
➢ Neurotoxic in its pure form (powdered or solution)
➢ Polyacrylamide gel: pre-cast already; not toxic
ETHIDIUM BROMIDE
• For Agarose Gel Electrophoresis
➢ Intercalating agent, Nucleic Acid stain
➢ Fluorescence: Orange
➢ Not directly mutagenic: metabolites are mutagenic
CARCINOGENS
• Any substance that can cause cancer.
➢ Changes the metabolic processes by damaging the
genomes
• Different types
➢ Decontaminating Agents – Dyes
➢ Probes and Labels
➢ Preparing enough; we are not storing working
solutions.
DYES, PROBES, LABELS
• SYBR Green
➢ Replacement for Ethidium Bromide
➢ Can still bind to DNA with high affinity to the side of
nucleic acid (minor groove) =potential carcinogen
• Acrylamide
➢ Used in PAGE (Polyacrylamide Gel Electrophoresis)
➢ Cross-linking agent for gel chromatography and
➢ electrophoresis
➢ Can cause peripheral neuropathy, possible carcinogen
• Phenol
➢ Used in Phenol-Acetic Acid-Urea Polyacrylamide Gel
Electrophoresis (PAU-PAGE)
• Chloroform
➢ solvent for DNA extraction; organic extraction
(hazardous)
➢ Extraction of Proteins
➢ Eye damage, fainting, life-threatening
*Both phenol and chloroform are used for DNA extraction.
IN CASE OF A SPILL OR SPLASH
*Don’t panic.
1) Evacuate Room and notify others to leave the room and
post a warning sign for No entry
2) Remove all contaminated clothing and /or lab coat and
place in a biohazard bag
3) Wash all exposed skin with antiseptic soap and water
(running water for 15 minutes)
4) Inform supervisor
5) Decontaminate the area: Assemble clean-up materials
➢ Large spill - Cover with paper towels and soak with
10% household bleach and allow to stand for 10-30
minutes
➢ Small spill - Wipe with paper towel soaked in 10%
bleach
*If necessary, check other neutralizer on MSDS.
6) Dispose all contaminated towels in biohazard bag and
dispose it properly
IN CASE OF AN ACCIDENT
What types of accidents can happen?
❑ Potential Injury, i.e., needlesticks, falls
❑ Environmental, i.e., splashes or spills
❑ Equipment damage
What should you do?
❑ Report to supervisor immediately
❑ Assess & take action
❑ Record using form
❑ Monitor situation
ACTION PLAN FOR IMPLEMENTING SAFETY PRACTICES
• Identify hazards
• Establish and implement safety policies and procedures
(accessible by all staff)
• Conduct safety specific training
❑ Must be a priority
❑ Communication is key
• Perform regular audits or assessments (by laboratory head)
POLICIES AND PROCEDURES
• Safety reminders (biohazard, chemical, physical) should be
included in lab SOPs
• Refer to general lab or institutional procedures and policies
for safe handling and waste disposal
DEFINITIONS
• Many different terms are used for disinfection and
sterilization. The following are among the more common in
biosafety:
• Antimicrobial- An agent that kills microorganisms or
suppresses their growth and multiplication.
• Antiseptic- A substance that inhibits the growth and
development of microorganisms without necessarily killing
them. Antiseptics are usually applied to body surfaces
• Biocide- A general term for any agent that kills organisms
• Chemical germicide- A chemical or a mixture of chemicals
used to kill microorganisms