[1S] UNIT 1.1: Introduction to Molecular Biology and Diagnostics Flashcards
“Field involved in the examination of the origins of diseases at the molecular level, primarily by studying nucleic acids”
Molecular Biology & Diagnostics / Diagnostic Molecular Biology
Molecular Biology and Diagnostics is also called as?
Diagnostic Molecular Biology
Studies the behavior of living cells in terms of their morphology, anatomy and physiology
Cell Biology
Techniques used in Cell Biology
Microscopy, Scanning, etc.
Importance: identifying the behavioral patterns of a cell and its morphological features in response to various metabolic, environmental and chemical exposures
Cell Biology
Deals with the understanding of the relationship between DNA, RNA and proteins
Molecular Biology
Techniques used in Molecular Biology
Gel electrophoresis, Polymerase chain reaction, cloning techniques, Sequencing, etc.
Importance: Confirmation of most morphological and metabolic conditions. It is also an important diagnostic tool
Molecular Biology
Containment principles technologies and practices implemented to prevent unintentional exposure to pathogens and toxins or their accidental release
Biosafety
Protocols and procedures that we do so that the things that we analyze will not be taken out from the laboratory grounds
Biosafety
● An agent of biological origin that can cause disease in humans
● It could be the ff:
○ Microorganisms
○ Viruses
○ Allergens or Toxins
Biological Hazard/Biohazard
Primary specimen for Molecular Biology
Blood
BIOSAFETY
Universal Precautions should be followed, that is, ….
All blood samples and other body fluids should be treated as potentially infectious for the transmission of blood borne pathogens like HIV, hepatitis, and others.
Applied to blood and body fluids containing visible blood for all patients.
Universal Precautions
Apply the principles of work practice controls and exposure controls to all patients and all patient specimens to prevent exposure of workers to potentially infectious agents and to protect patients.
Standard Precautions
Donning & Doffing Sequence
Donning:
1) Gown
2) Mask
3) Goggles
4) Gloves
Doffing:
1) Gloves
2) Goggles
3) Gown
4) Mask
BIOSAFETY LEVELS
agents are not known to cause disease
Biosafety Level 1
BIOSAFETY LEVELS
Use this BSL when working with:
• Well-characterized agents
• Agents that are of minimal hazard to lab personnel and the environment
Biosafety Level 1
BIOSAFETY LEVELS
• E . coliJM109, DH5a
• Saccharomycescerevisiae
• Bacillus subtilis
Examples of BSL-1 Agents
BIOSAFETY LEVELS: STANDARD WORK PRACTICES
• Use mechanical pipetting devices
• Wash hands frequently
• Minimize splashes and aerosols
• Decontaminate work surfaces daily
• Handle wastes properly
• Maintain insect and rodent control program
BSL-1
PPE for BSL-1
• Lab coat or apron
• Safety glasses or goggles
• Gloves as needed
BIOSAFETY LEVELS
agents are associated with human disease
BSL-2
BIOSAFETY LEVELS
Use this when working with agents of moderate potential hazard to personnel and the environment
BSL-2
BIOSAFETY LEVELS AGENTS
• Human blood or body fluids
• E . coli0157:H7
• Clostridium botulinum
• Retroviral vectors
• Human cells in cell culture
BSL-2
BIOSAFETY LEVELS FACILITY
• Adequate illumination
• Eyewash facility
• Negative air pressure
• Autoclave available
• Biological safety cabinet
• Lab must be separated from public areas
BSL-2
Are red in color, marked with biohazard symbol, leak proof & puncture resistant
Sharps Container (BSL-2 Special Work Practices)
BIOSAFETY LEVELS SPECIAL WORK PRACTICES
• Place used slides and coverslips in sharps containers, never in any other receptacle.
• Sharps containers
BSL-2
PPE for BSL-2
• Lab coat or apron
• Safety glasses or goggles
• Gloves
• Biosafety cabinet
Aerosols or splashes; Large volumes; High concentrations
Biosafety Cabinet
BIOSAFETY LEVELS AGENTS
• HIV
• H1N1 Flu
• Tuberculosis
• Rabies
• Yersiniapestis
• West Nile Virus
• SARS-Cov2
BSL-3
BIOSAFETY LEVELS
agents are associated with human disease and are potentially transmitted as aerosols
BSL-3
BIOSAFETY LEVELS FACILITY
• Controlled access
• Physical separation from access corridor
• Anteroom; two-self closing interlocked door
• Directional airflow with pressure
• HEPA filtered exhaust
• Change room and shower
• No windows;
• Autoclaves available
• Seamless floors
• Chemical resistant fixtures
BSL-3
BIOSAFETY LEVELS SPECIAL WORK PRACTICES
Specific training in handling agents
BSL-3 & 4
PPE for BSL-3
• Specialized clothing
• Respiratory protection (masks)
• Gloves
• Biosafety cabinet
BIOSAFETY LEVELS
For agents of life threatening nature
BSL-4
BIOSAFETY LEVELS AGENTS
• EBOLA
• Small pox
BSL-4
BIOSAFETY LEVELS FACILITY
• Controlled access
• 2 laboratory providing absolute separation of the worker from the infectious agents (suit laboratory and cabinet laboratory)
• Filtered air
• Physical separation from access corridor
• Directional airflow with pressure
• Special waste
• Airlock entry
BSL-4
PPE for BSL-4
• Lab coat or apron
• Positive Pressure suits
• Double ended autoclave
• Biosafety cabinet Class III
T/F: Current WHO guidelines no longer recommend the prescriptive use of Biosafety Levels. Instead, biocontainment are based on 3 categories of requirements anchored on a robust and site-specific risk assessment. However, there are still existing guidelines that still use BSLs as its phaseout is fairly recent
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○ To protect user from the sample
○ To protect the environment from the sample
○ To protect the samples from external elements
Biosafety Cabinets
BSCs key aspects
• Amount of air exhausted
• Amount of employee protection
BSC CLASS
Personnel & environment
Class I
BSC CLASS
Personnel, product, environment; most common in labs
Class II A & B
BSC CLASS
Specialized conditions
Class III
BSC CLASS: DIRECTION OF AIRFLOW
Inward (≥75)
BSC Class I
BSC CLASS APPLICATION
Air intake is through the front face of the cabinet; exhaust exits through a HEPA to the outside or into the room
BSC Class I
BSC CLASS APPROPRIATE FOR USES OF VOLATILE TOXIC CHEM & RADIONUCLIDES
Acceptable if hard ducted
BSC Class I
BSC CLASS APPROPRIATE FOR USES OF VOLATILE TOXIC CHEM & RADIONUCLIDES
Minute amounts only if thimble connected to exhaust*
BSC Class II Type A1 & A2
BSC CLASS APPROPRIATE FOR USES OF VOLATILE TOXIC CHEM & RADIONUCLIDES
Limited amounts*
BSC Class II Type B1 & Class III
BSC CLASS APPROPRIATE FOR USES OF VOLATILE TOXIC CHEM & RADIONUCLIDES
Acceptable
BSC Class II Type B2
BSC CLASS APPROPRIATE FOR USES OF VOLATILE TOXIC CHEM & RADIONUCLIDES
Acceptable, must be hard ducted to lab exhaust and operated in Type B mode
BSC Class II Type C1
BSC PROTECTION
Maximum Product, personnel, and environment
BSC Class III
BSC PROTECTION
Product only
Laminar Flow Clean Bench
BCS DIRECTION OF AIRFLOW
Outward (100)
Laminar Flow Clean Bench
BCS DIRECTION OF AIRFLOW
Inward (75)
BSC Class II Type A1
BSC DIRECTION OF AIRFLOW
Inward (100)
BSC Class II Type A2, B1-2, C1
BCS APPLICATION
Air intake is through a HEPA filter; 70% of airflow is recirculated to the cabinet work area through HEPA; 30% balance can be exhausted through HEPA back into the room or to the outside through a canopy unit. Plenum is under positive pressure
BSC Class II Type A1
BCS APPLICATION
Similar to Class II. A1, but has 100 lfpm face velocity and plenums are under negative pressure to room; exhaust air can be ducted to outside through a canopy unit
BSC Class II, Type A2
BCS APPLICATION
Air intake is through HEPA filter; 30-40% of airflow is recirculated in the cabinet and the remaining 60%-70% is exhausted through a HEPA filter and a dedicated duct to the outside
BSC Class II, Type B1
BCS APPLICATION
Air intake is through HEPA filter; 100% of the air is exhausted to the outside via a HEPA-filtered hard duct. No air recirculation in the cabinet.
BSC Class II, Type B2
BCS APPLICATION
This type of cabinet can be operated in different modes depending on whether the worker protection from hazardous vapors is a consideration. The mode can be set to approximate either a Type A or Type B biosafety cabinet.
BSC Class II, Type C1
BCS APPLICATION
Totally enclosed, gas-tight, glove ports for manipulation of pathogens. Supply air is HEPA filtered. Exhaust air passes through two HEPA filters in series and is exhausted to the outside via a hard connection. Airflow can be turbulent inside the cabinet
BSC Class III
BCS PROTECTION
Product only
Laminar Flow Clean Bench
Laboratory Safety Cabinet Vs Fume Hood and Laminar Flow Center
Closes completely: either horizontally or vertically
Chemical Fume Hood
Laboratory Safety Cabinet Vs Fume Hood and Laminar Flow Center
- Not meant for sitting
- Negative pressure
- May have solvent/chemical storage underneath
Chemical Fume Hood
Laboratory Safety Cabinet Vs Fume Hood and Laminar Flow Center
- Used for strong acids and bases, volatile chemicals
- Protects the analyst from chemically volatile compounds
Chemical Fume Hood
Laboratory Safety Cabinet Vs Fume Hood and Laminar Flow Center
- Fixed sash opening (8 inch.) (alarmed)
- Sash moves up but does not close completely
- Designed for seated work
- Negative pressure - pulls air towards inside the cabinet
BSC
Laboratory Safety Cabinet Vs Fume Hood and Laminar Flow Center
- HEPA filter visible in rear or top of unit
- Usually no sash or sash is fixed
- Useful for preparation of reagents in PCR
Laminar Flow Clean Air Center
Laboratory Safety Cabinet Vs Fume Hood and Laminar Flow Center
- Positive pressure - air blowing into face or breathing zone
- Protects the samples especially in procedures like amplification or putting samples in the PCR template
Laminar Flow Clean Air Center
Developed by Occupational Safety and Health Administration (OSHA) for employees who may be exposed to hazardous chemicals in the workplace
Hazard Communication Standard – Right to Know Law
Need to:
■ Have written hazard communication program
■ Obtain SDSs for each compound
■ Educate employees to interpret labels, SDSs, and health hazards
■ Maintain hazard warning labels
Hazard Communication Standard – Right to Know Law
● Major source of safety information for employees who may use hazardous materials
● Obtained from manufacturers
Safety Data Sheet
T/F: In handling biosafety cabinets, you will observe some holes. Avoid blocking those holes. If failed to do so, the BSC will lose its efficiency and will be useless, because it works via the filtration of the air.
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substances that affect cellular development of a fetus or embryo
Teratogens
cancer causing substances
Carcinogens
chemicals with flashpoint below 37.8°C
Flammable
chemicals with flashpoint at or above 37.8°C
Combustible
chemicals injurious to the skin or eyes by direct contact or to the tissue of the respiratory and GIT if inhaled or ingested
Corrosive
substances that spontaneously explode or ignite or that evolve heat or flammable or explosive gases
Reactive
Safety when using laboratory equipment, materials, and supplies
Mechanical Safety
● Direct hazards: death, shock, burns
● Indirect hazards: fire, explosion
Electrical Safety
ELECTRICAL SAFETY
T/F: Use only explosion-rated (intrinsically wired) equipment in hazardous atmospheres.
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ELECTRICAL SAFETY
T/F: Be particularly careful when operating high-voltage equipment, such as electrophoresis apparatus.
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ELECTRICAL SAFETY
T/F: Do not use properly grounded equipment (three-prong plug) and do not check for frayed electrical cords.
F; use only properly…, check for frayed…
ELECTRICAL SAFETY
T/F: Promptly report any malfunctions or equipment
producing a “tingle” for repair. Do not work on “live” electrical equipment.
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FIRES
Ordinary combustibles, wood, paper, cloth etc
Class A
Pressurized Water & Dry Chemical (PASS)
FIRES
Flammable, Liquid, Grease, Gasoline, Paints, Oils,
etc.
Class B
Dry Chemical & Carbon Dioxide (PASS)
FIRES
Electrical Equipment, Motor Switches
Class C
Carbon Dioxide, Halon, Dry Chemical (PASS)
FIRES
Flammable Metals, Magnesium
Class D
Metal X
Cover burning material with extinguishing agent (scoop, sprinkle)
Destroying all microbial life including spores
STERILIZATION
Destroying majority of microbial life; but not necessarily spores
DISINFECTION
Chemical disinfectants which can be safely applied to the skin or mucous membrane and are used to prevent infection by inhibiting the growth of bacteria
ANTISEPTICS
TYPES OF STERILIZATION PROCESS
- Physical Methods
- Chemical Methods
- Radiation
TYPES OF STERILIZATION PROCESS
● ___ heat: red heat; flaming; incineration; hot air oven
● _____ heat: Pasteurization; boiling; autoclave (steam under pressure)
● Using nanopore filter paper
Physical Methods
- Dry
- Heat
TYPES OF STERILIZATION PROCESS
bleach, ethanol, hydrogen peroxide, ethylene oxide, paraformaldehyde
Chemical methods
PHYSICAL METHOD
● steam pressure rises inside and when it reaches the desired set level, the safety valve opens and the excess steam escapes
● From this point, the holding period is calculated. The holding period is 121° C at 15 pound pressure for 15 minutes
● When the holding period is over, the heater is turned off and the autoclave allowed to cool till the pressure inside is equal to the atmospheric pressure
Autoclaving
PHYSICAL METHOD
● A holding period of 160°C for one hour is used to sterilize glassware, forceps, all-glass syringes, swabs etc.
● Glassware should be perfectly dry before being placed in the oven. Test tubes and flasks should be wrapped in paper.
● The material should be arranged so as to allow free circulation of air in between the objects.
Hot Air Oven (Dry Heat)
PHYSICAL METHOD
● Membrane filters are widely used to remove unwanted microorganisms from liquids such as sera and solutions during preparation of culture media
● Made of cellulose esters and other polymers with an average pore diameter of 0.22 mm
Filtration
CHEMICAL METHOD: Using 70% Alcohol or 10% Bleach
T/F: Ideal disinfectant should be:
● Wide spectrum of activity and must be effective against all microorganisms
● Have fast action; high penetrating power; stable
● Not corrode metals
● Not causing irritation
● Cheap
● Easily available and easy to handle
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TYPE OF STERILIZATION
● Ultraviolet (UV) for sterilization: very high penetrative power that can be highly lethal to all cells including bacteria
● Used for:
• Cleaning surfaces (inoculation hoods)
• Laboratory benches
• Laboratory apparatus
Radiation
Three conditions for a successful aseptic technique
- The work area must be disinfected
- The transfer instruments or glasswares must be sterilized
- The work must be accomplished quickly and efficiently
DISPOSAL OF WASTES
To be lined with clear autoclave bags and should be decontaminated inside the biosafety cabinet and/or autoclaved
Biohazard containers
DISPOSAL OF WASTES
Contaminated culture tubes and glasswares that are for washing and decontamination
Decontamination bins
DISPOSAL OF WASTES
Should contain sharps, slides, coverslips, broken glasswares; disposable pipettes and Pasteur pipettes
Sharps containers
DISPOSAL OF WASTES
Non-contaminated materials
Trash cans
