Laboratory Safety Manual

2023-2024

The Department of Medical Laboratory Technology, School of Health Sciences, The Neotia University, Kolkata

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Introduction:
Safety, security and health considerations are as important as any other materials taught in
curriculum for science students. Working with chemicals, microbes, etc. poses continuous
challenge to one’s health and to the environment. Because each lab is different, and
processes change routinely, each lab has the responsibility to maintain lab-specific safety
information and documentation.
The purpose of this guide is to promote safety awareness and encourage safe working
practices in the laboratory. These brief guidelines should serve as a reminder of things you
can do to work more safely and are applicable to all users of the laboratory.
The requirements and recommendations of this ‘Laboratory Safety manual’ will not fully
protect you unless you exercise diligence in your daily work, or at least stop periodically to
assess your environment.
Step back and look carefully at your laboratory environment, looking at it as a first-time
visitor and check:
Does it look safe, neat, and
orderly? Are chemicals stored
properly?
Are you and other personnel taking appropriate
precautions? Can you see ways to make the lab safer?
Following certain discipline and working as a responsible human being, one can maintain a
total hygiene working with chemicals. Below are few guidelines to ensure maximum safety
of our fraternity and its properties as well as of our society.

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TABLE OF CONTENT
General Laboratory Protocols
Basic Rules
Good Personal Habits & Behavior
Housekeeping
GHS (Globally Harmonized System)

GENERAL SAFETY
General Safety and Operational Rules
Electrical Safety
Vacuum operations
Handling glassware
Fume Hood Safety and Ventilation

SAFE HANDLING OF CHEMICALS

General guidelines
Gathering general information on chemicals
Handling and transportation of chemicals
Chemical storage
Chemical Spills
Guidelines for Ethidium Bromide (EtBr) Waste Management & Disposal
Guidelines for Bis-Acrylamide
Compressed Gas Safety
Safe Handling of Cryogenic liquids
Handling of Dry Ice

BIOLOGICAL SAFETY
Biological Safety Level I
Biological Safety Level II
Biological Safety Level II+
Basic working Principal in Bio-safety
Laboratories Biohazard spills

SPECIALTY LABORATORIES
Working with radioactive materials
Laser Laboratory

WASTE DISPOSALS AND MANAGEMENT

EMERGENCY RESPONSE
Fires
Accident Reporting
Institute policy regarding safety
Committees

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I. General Laboratory Protocols

Basic Rules
 Safety goggles are mandatory for all the lab workers
 Lab workers should not work alone inside the laboratory
 Everyone while working in the lab should wear lab coats and full pants.
 Lab-coats are not allowed in common areas like cafeteria, restrooms etc.
 Lab worker should be aware of the location and proper operation of
laboratory safety equipment
 Know the exits in the laboratory and in the building
 Use of blast shields is strongly recommended while doing potential dangerous
reactions (such as dealing with peroxides, diazo-compounds, high pressure vessels,
distillation of high boiling substances).
 Gloves shall be worn while working in the lab and should be removed before
touching surfaces outside the work area (i.e., computers etc).
 Feet should be covered completely with shoes containing reasonable heel heights.
 Open toe sandals and half pants/shorts should be avoided in the lab.

Good Personal Habits & Behavior

 Students should act in a professional manner at all times.
 Eating, drinking, gum and tobacco chewing, are not permitted in the laboratory.
 Using mobile phone is strictly prohibited inside the laboratory.
 Ice from the ice machines for laboratory use shall not be used for beverages, food
or food storage.
 Do not smell or taste chemicals.
 Before handling any unknown chemicals/biological samples,
students/researchers should consult respective lab in charge.

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  Skin contact with chemicals should be avoided.
 Hands should be washed thoroughly before leaving the lab.

Housekeeping
 Lab areas are to be kept clean and uncluttered.
 Spills should be cleaned up immediately from work areas and floors.
 Equipment and instrumentation shall be cleaned to remove spillage and
contamination before repair or calibration service is requested.
 Personnel must be able to see clearly through the protective glass sashes on fume
hoods.
 Clear aisles, exits, and hallways for obstructions leading to slipping or tripping
hazards (e.g., boxes, electrical cords or other items on the floor).
 Ensure unblocked access to all of the following:
 Eyewash/safety showers
 Electrical panels
 Fire extinguishers
 Chemical storage cabinets
 Fume hoods
 Waste containers
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 Possible ways to avoid clutters in Labs
 Laboratories that are cluttered present a variety of safety hazards to researchers,
students, and visitors. Some common methods for controlling clutter in laboratories
are as follows:
 Properly dispose of chemicals and equipment that are no longer needed
 Do not buy chemicals, solvents or other inventories in bulk unless there is space
available to safely store the material.
 Before purchasing chemicals in bulks explore its availability in other concerned
labs. Students should go through the lab inventory developed by each lab.
 Regularly schedule “lab clean-up days”
 Remove clutters from fume hoods, and ensure that they are not used for long- term
storage of equipment, chemicals, or supplies that are not regularly used in the fume
hood.
 Empty containers of unwanted materials (including trash) on a regular basis, and
never allow them to overflow.
 Store excess materials in a neat, secure manner that provides easy access and
reduces the potential for falling, collapsing, rolling, or spreading of the material.
 Limit overhead storage to lightweight, non-hazardous items.
 Chemical containers, supplies, and equipment are to be stored away from the edges
of benches and shelves.
 Never stack chemical containers directly on top of one another (unless in original
boxes that can be safely stacked) and/or with incompatible chemicals (such as acids
with bases or flammables with oxidizers).
 Containers holding chemicals should not be stored on the floor. When this is
unavoidable, store containers in plastic tubs or other secondary containment.
 Clearly mark bench areas containing radioactive materials with radiation tape and
sorbent pads.
 Clean up all spills promptly. Never leave puddles, powders, or unknown materials
on floors or work surfaces.
 Do not use leg space beneath benches and desks in a way that prevents proper
ergonomic posture.
 Keep personal desk spaces and other areas clean (free of all hazardous research
materials).
 Neatly store lab coats and safety glasses away from potential sources of
contamination.
 Never store hazardous materials in refrigerators that contain food.
 Only eat or store food in designated areas. Maintain sufficient open space within the
laboratory to manage the acquisition and disposition of materials.

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GHS (Globally Harmonized System)
GHS stands for the Globally Harmonized System of Classification and Labelling of
Chemicals that defines and classifies the hazards of chemical products, and communicates
health and safety information. The goal is that the same set of rules for classifying hazards,
and the same format and content for labels and safety data sheets (SDS) will be adopted and
used all around the world. It is important that those working with chemicals are aware of all
the signs and symbols on the chemical containers. The following is the symbol and related
hazard information that are commonly encountered in research labs.

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II. GENERAL SAFETY

People who work in Medical laboratories are exposed to various hazards. Most workplaces
have hazards that are well recognized (those of ordinary fire, for example) with well-defined
actions to control the situation. Laboratories, however, involve a greater variety of possible
hazards and some of these hazards need precautions not ordinarily encountered. An
introduction to safe practices for a variety of widely used laboratory procedures is listed
below.

- GENERAL SAFETY AND OPERATIONAL RULES

1. No running or jumping in a laboratory is permitted. Stored items or equipment shall not

block access to the fire extinguisher(s), safety equipment, or other emergency items.
Stairways, hallways, passageways/aisles and access to emergency equipment and/or exits
must be kept dry and unobstructed; i.e., no storage, no equipment, phone or other wiring.
No combustible material such as paper, wooden boxes, pallets, etc., shall be stored under
stairwells or in hallways. Hallways shall be kept free of boxes and materials so that exits
and normal paths of travel are not blocked.

2. Eating or drinking within laboratories is not permitted. In all laboratories specific office
areas may be designated for food in coordination with the Safety Committee. They must
be physically separated from any laboratory operations. In the specified office areas no
consumables, reagents or any tools should be shared with work areas.

3. No food or beverage may be stored in the cold rooms/Laboratory refrigerators and

freezers.

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4. Working core hours are 9 20 am – 5:20pm (Monday-Friday). No employee shall work
alone in a laboratory or chemical storage area outside the core working hours. Permission
for students and staff to work outside of the core time has to be granted by in charge in
writing.
5. Clothing worn in the laboratory should offer protection from splashes and spills, should
be easily removable in case of an accident. Nonflammable, nonporous aprons offer the
most satisfactory and the least expensive protection. Lab jackets or coats should have
snap fasteners rather than buttons so that they can be readily removed. These coats are to
be fastened closed while working and removed prior to exit from the laboratory. It is
highly recommended that no sandals or open-toed shoes shall be worn by laboratory
technician in the laboratory. Laboratory clothing should be kept clean and replaced when
necessary.
6. Mouth pipetting is never allowed.

A. Electrical Safety
Electrical equipment and wiring comprises a major part of the laboratory, thus posing a new
set of possible laboratory hazards. Periodic laboratory inspections should pay particular
attention to electrical safety.

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A list of possible wiring hazard are as follows:
 Spliced cables
 Worn-out cables
 Tripping hazards from poorly draped cables near hot plates etc.
 Sliced cables near sinks or other wet locations.

Should you be concerned with high voltage or high current? In fact, it is a bit of both! If the
voltage is not high enough, it may be relatively safe to touch an electrical circuit which can
deliver high current. On the other hand, if the voltage is very high, but the current that the
supply can deliver is very low, you might still be safe. Let us see why!
The electricity is nothing but the flow of charged particles. In most of our everyday life
situations, it is the flow of electrons. The electrons flow from a high potential to a lower
potential. What happens when you get a shock is that the electricity flows through your
body from an electrical circuit carrying a high potential to the ground.
Your body has certain electrical resistance. From the tip of your finger to your feet, it is
about 100 kΩ under normal circumstances. However, if you are wet, the resistance can drop
to about one kilo-ohm! Thus, if you are touching a 100 V terminal, the current that can flow
through your body is about one milliamp under normal conditions. The resultant shock is
barely perceptible! However, once the current passes through the body, its resistance
decreases and more current starts flowing, which is indeed dangerous. If your body is wet,
the initial current can be up to 100 mA, which is indeed fatal! However, if the power supply
can deliver a maximum of one milliamp of current, and if it is not faulty, you are very likely
safe! If there is some failure in the grounding of the power supply, the resultant current can
be lethal! Therefore, do not touch any electrical terminals that look suspicious.

One important thing to note at this point is that once the electricity starts flowing through
your body, your resistance will decrease drastically facilitating more current to flow. Given
below in a box is a description of how human body responds to various amounts of currents
[Source: Fish, R. M. & Geddes, L. A. Conduction of electrical current to and through the
human body: a review. Eplasty 9, e44 (2009).]

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 Lethal voltages present in the labs can be identified by the following symbol.

Electricity can kill. Respect it!

B. Vacuum Operations

In an evacuated vacuum system, the higher pressure is on the outside, rather than the inside,
so that a break causes an implosion rather than an explosion. The resulting hazards consist of
flying glass, spattered chemicals, and possibly fire.

1. When working with a vacuum be aware of implosion hazards. Apply vacuum only to
glassware specifically designed for this purpose, i.e., heavy wall filter flasks, desiccators, etc.
2. Never evacuate scratched, cracked, or etched glassware. Always check for stars or cracks
before use.
3. Vacuum glassware which has been cooled to liquid nitrogen temperature or below should
be annealed prior to reuse under vacuum.
4. Rotary evaporator condensers, receiving flasks, and traps should be taped or kept behind
safety shields when under a vacuum.
5. When a vacuum is supplied by a compressor or vacuum pump to distill volatile solvents, a
cold trap should be used to contain solvent vapors. Cold traps should be of sufficient size and
low enough temperature to collect all condensable vapors present in a vacuum system. If such
a trap is not used, the pump or compression exhaust must be vented to the outside using
explosion proof methods.
6. After completion of an operation in which a cold trap has been used, the system
should be vented. This venting is important because volatile substances that have been
collected in the trap may vaporize when the coolant has evaporated and cause a pressure
buildup that could blow the apparatus apart.

7. After vacuum distillations, the vessel must be cooled to room temperature before it
is vented.
8. All desiccators under vacuum should be completely enclosed in a shield or wrapped with
friction tape in a grid pattern that leaves the contents visible and at the same time guards
against flying glass should the vessel collapse. Various plastic (e.g., polycarbonate)
desiccators now on the market reduce the implosion hazard and may be preferable.

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C. Handling Glassware

1. Glass breakage is a common cause of injuries in laboratories. Only glass in good

condition should be used.
2. Clean all glassware before sending for repair. Glassware that has been in contact with
infectious agents shall be disinfected before disposal or repair.
3. Protect hands with leather gloves when inserting glass tubing. Hold elbows close to the
body to limit movement when handling tubing.
4. Use glassware of the proper size. Allow at least 20% free space. Grasp a three-neck flask
by the middle neck, not a side neck.
5. Conventional laboratory glassware must never be pressurized or used with vacuum.

D. Fume Hood Safety and Ventilation

General laboratory ventilation shall provide airflow into the laboratory from non- laboratory
areas and out to the exterior of the building. Laboratory doors should remain closed, except
for exit and entrance. All reactions must be performed within a fume hood. The hood sash
should remain closed or at minimal safe height while working in the lab. Ventilation
problems or fume hood alarms should be reported to the concerned lab supervisor who shall
submit repair requests to facilities maintenance.

III - Safe Handling of Chemicals

General Guidelines.
Working with potentially harmful chemicals is an everyday occurrence in a laboratory.
Employees are requested to inform themselves about toxicological information and
procedures for handling and storage of chemicals used. For most commercially available
substances, detailed instructions are available in the Material Safety Data Sheet (MSDS). A
hardcopy of MSDS for each lab is required.

A. Gathering General Information on Chemicals

The Material Safety Data Sheet (MSDS) describes properties, reactivities, potential
chemical hazards, and safe handling procedures for commercially available chemicals you
are working with. These sheets must be archived in a specified folder; all lab personnel
must know where it is kept. This MSDS log must be updated at regular intervals.
Information that is contained in the Material Safety Data Sheets is also required by law to
be conveyed to employees on a chemical-by-chemical basis.
MSDSs are generally written for chemicals that are used in the industrial setting therefore
some of the information provided on the MSDS may not be applicable to laboratory usage.

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 The use of chemicals in a laboratory is generally in a more controlled environment than in
the industrial setting and much smaller quantities of the chemical are being used at any one
time. Nevertheless, a great deal of information on hazards associated with laboratory
chemicals can be obtained by reading the MSDS.

Familiarize yourself with the pictograms and Hazard codes widely used to mark risks.

Risk and Safety Phrases are widely used and should be known by any user when
handling chemicals in the course of laboratory procedures.

B. Handling and Transportation of Chemicals

1. Many laboratory accidents occur by carrying chemicals from one place to another or
transferring them from one container to another. The chemicals used in a laboratory
are often corrosive, toxic or flammable and any accident involving these has the
potential for personal injury. Therefore, it is good practice to assume that all chemicals
are potentially hazardous.
2. When large bottles of acids, solvents, or other liquids are transported within the
laboratory without a cart, only one bottle should be carried at a time. The bottle should
be carried with both hands, one on the neck of the bottle and the other underneath. Do
not hook a finger through the glass ring on top of the bottle, allowing it to dangle
while being transported. Never carry or attempt to pick up a bottle by the cap.
3. Large quantities of concentrated mineral acids, e.g., sulfuric, nitric and hydrochloric
acids, shall be kept in specific storage rooms or cabinets for corrosive substances.

C. Chemical Storage
Proper storage of chemicals is necessary to maximize employee safety with regard to
chemical compatibility, spill control, fire/explosion control, to provide security,

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 identification, and provide a "user friendly" system with respect to point-of-use.
1. All containers used for storage (even short term) shall be labeled.
2. Quantities greater than one litre of highly flammable liquids (Methanol, Chloroform)
should be stored in specified metal cabinets. Quantities smaller than or equal to one litre
of chemicals may be held at individual work stations but only one of the same kind.
Chemicals should be stored as close as feasible to the point of use in order to maximize
efficiency and minimize transport distance.
3. Out-of-date chemicals shall be disposed of on a periodic basis to reduce overall hazard
potential and minimize inventory tracking and updating.

4. Reduce large inventory of chemicals in the laboratory.

5. Never pipette by mouth. Always use a bulb to pipette.
6. Chemical containers that have been emptied have to be cleaned off remaining residues
by triple rinsing with water or other suitable solvent and air-dried before disposal.

D. Chemical Spills
Any chemical is a possible threat to your personal health and your colleagues. In case of
accident causing the release of hazardous chemicals a calm and determined action is required
to prevent an escalation of the emergency situation.
Thus, for any individual incident, isolation of the spill and/or securing the area is best prior to
or simultaneously with contacting concerned personnel. This should be done according to all
available information on the chemical nature of the spill. Under all circumstances, a
laboratory coat, safety glasses, and gloves should be used for self- protection.

A.Spill Kits may be obtained from Stores

1. Always send for help first and report the necessary information.
a) Where did the incident happen?
b) How many people are injured?
c) What is the chemical?
d) Is there an intermediate risk of fire, explosion, intoxication or suffocation known?

2. If the spill presents an immediate danger, leave the spill site and warn others, control
entry to the spill site, and report to reception.

3. Remove contaminated clothing. Flush skin/eyes with water at least 15 to 30 minutes; use
soap for intermediate and final cleaning of skin areas.
4. Protect yourself, then remove injured person(s) to fresh air, if safe to do so.
5. Notify nearby persons and evacuate as necessary. Prevent entry, as necessary, by posting a
guard in a safe area and/or shutting doors.

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 6. If flammable vapors are involved, do not operate electrical switches unless to turn off
motorized equipment. Try to turn off or remove heat sources, where safe to do so. TURN
OFF ELECTRIC AT THE MAINS, NOT AT SWITCHES INSIDE THE LABORATORY.
7. Do not touch the spill without protection, gloves etc.

B. Where the spill does not present immediate personal danger, try to control the spread or
volume of the spill. This could mean shutting a door, moving nearby equipment to prevent
further contamination, repositioning an over-turned container or one that has a hole in the
bottom or side, creating a dike by putting an absorbent around a spill or opening the sashes
on the fume hoods to facilitate removal of vapors.

C. Never assume gases or vapors do not exist or are harmless because of lack of smell.

D. Increase ventilation by opening closed fume hood sashes to the 12 inch or full open
position. Exterior doors may be opened to ventilate non-toxic vapors.

E. Use absorbents to collect substances. Reduce vapor concentrations by covering the surface
of a liquid spill with absorbent. Control enlargement of the spill area by diking with
absorbent.

F. Spilled Liquids
1. Confine or contain the spill to a small area. Do not let it spread.
2. For small quantities of inorganic acids or bases, use a neutralizing agent or an absorbent
mixture (e.g., soda ash or diatomaceous earth). For small quantities of other materials,
absorb the spill with a nonreactive material (such as vermiculite, clay, dry sand, or
towels).
3. For larger amounts of inorganic acids and bases, flush with large amounts of water
(provided, the water will not cause additional damage refer to MSDS for this information).
Flooding is not recommended in storerooms where violent spattering may cause additional
hazards or in areas where water-reactive chemicals may be present.
4. Mop up the spill, wringing out the mop in a sink or a pail equipped with rollers.
5. Carefully pick up and clean any cartons or bottles that have been splashed or immersed.
6. If the spilled material is extremely volatile, let it evaporate and be exhausted by the
laboratory hood (provided that the hood is authorized for use with the spilled chemical).

G. SpilledSolids
Generally, sweep spilled solids of low toxicity into a dust pan and place them into a container
suitable for that chemical. Additional precautions such as the use of a vacuum cleaner
equipped with a HEPA filter may be necessary when cleaning up spills of more highly toxic

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solids.

1.Dispose of residues according to safe disposal procedures. Remembering that personal

protective equipment, brooms, dust pans, and other items may require special disposal
procedures.
2. Report the chemical spill in writing as required above to laboratory incharge.

- Guidelines for Ethidium Bromide (EtBr) Waste Management & Disposal

Ethidium bromide (3,8 diamino-5-ethyl-6-phenyl phenanthridinium bromide, dromilac, CAS
#1239-45-8), is a compound used in many laboratories. Ethidium bromide is available as a
dark red, crystalline, non-volatile solid and is moderately soluble in water. Since it fluoresces
readily with a reddish-brown color when exposed to ultraviolet (UV) light and with increased
brightness when bound to double stranded-DNA and single-stranded RNA, it is commonly
used in gel electrophoresis applications for visualization of these molecules. For these
applications, liquid ethidium bromide solutions are incorporated into the electrophoresis gel
as a dye for the DNA, RNA, or other molecules to be visualized.

Ethidium bromide is mutagenic and moderately toxic and must be handled with care. The

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powder form is considered an irritant to the upper respiratory tract, eyes, and skin.
Preparation of stock solutions and any operations capable of generating ethidium bromide
dust or aerosols should be conducted in a fume hood to prevent inhalation. Nitrile gloves, a
lab coat, and eye protection must be worn at all times.

When working with ethidium bromide, minimize the potential for spills. Where practical,
purchase pre-mixed stock solutions from chemical manufacturers in lieu of preparing
solutions. If solutions of ethidium bromide must be prepared, perform this process in a fume
hood. Perform all processes that generate ethidium bromide dusts or mists inside the fume
hood to minimize inhalation exposures. Prevent accidents by transporting small quantities of
ethidium bromide in secondary containment.

*Do Not Use sodium hypochlorite (bleach) to treat ethidium bromide. Bleach treatment can
produce mutagenic products and leave behind up to 20% of the original ethidium bromide.

Ethidium Bromide Waste Disposal

Ethidium bromide waste should NOT be poured down the drain or thrown in the trash, unless
the waste has been deactivated or filtered. The following are the recommended disposal
procedures for ethidium bromide.
NOTE - Ethidium bromide DOES NOT go in red bags or red containers, should not be
labeled with a biosafety symbol, and especially should not be treated in an autoclave.

A. Electrophoresis Gels, Contaminated Gloves and EtBr contaminated Solids

Handle ethidium bromide gels, contaminated gloves just as you would chemical hazardous
waste. Use sealable, disposable plastic baggies to store ethidium bromide gel waste.
Minimize free flowing liquids in these bags when they are brought for disposal.

A bin is kept for gloves and gels contaminated with EtBr

- Guidelines for Bis-Acrylamide

When handling Bis-Acrylamide formulations gloves, eye protection and Lab coat are
required.
Dispose remaining acrylamide in original chemical containers by polymerization. For that
add 100µl TEMED and 100µl of 10% Ammonium Persulfate (APS) and shake it
thoroughly and allow to react over night. Dispose the container in the red biohazard bin.

- Guidelines for Phenol/Chloroform

Handle Phenol or Phenol/Chloroform formulation only with gloves, eye protection and
Lab coats. These liquids have to be disposed in dedicated organic solvent containers.
The tubes should be collected separately and allowed to dry in the chemical fume
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hoods in common equipment rooms.

- COMPRESSED GAS SAFETY

A. Identification
1. The contents of any compressed gas cylinder shall be clearly identified for easy, quick, and
complete determination by any laboratory worker. Such identification should be stenciled or
stamped on the cylinder or a label, provided that it cannot be removed from the cylinder. If
the labeling on a cylinder becomes unclear or an attached tag is defaced to the point the
contents cannot be identified, the cylinder should be marked "contents unknown" and
returned directly to the manufacturer. Users are requested to inform themselves through the
Material safety data sheets (MSDS) about safe handling of the specific technical gas.

Examples of color code used in India for identification of gas cylinders:

1. Argon : Peacock Blue
2. Nitrogen : French Grey with a black band on the shoulder of the cylinder
3. Helium : Light Brown
4. CO2 : Black with a silver band on the shoulder of the cylinder
5. Oxygen : Black
6. Zero Air : French Grey

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(Ambient air filtered to contain less than 0.1 parts per million (PPM) of total hydrocarbons.)
7. Hydrogen : Signal Red
8. D.A : Maroon (Dissolved Acetylene)

B. Safe Use of High pressure cylinders

1. Gas cylinders shall be secured at all times to prevent tipping. Cylinders may be
attached to a bench top, individually to the wall, placed in a holding cage, or have a non-tip
base attached.
2. When new cylinders are received, they should be inspected; insure the proper cap is
securely in place and the cylinder is not leaking. Cylinders shall have clear labels
indicating the type of gas contained. If the cylinders are acceptable, they shall be stored in a
proper location.
3. Standard cylinder-valve outlet connections have been devised to prevent mixing of
incompatible gases. Cylinders should be placed with the valve accessible at all times.
Cylinder valves should be opened slowly. Main cylinder valves should never be opened all
the way. Valves should not be closed with force.
4. Regulators are gas specific and not necessarily interchangeable. Always make sure
that the regulator and valve fittings are compatible. Never use oil or grease on the regulator
of a cylinder valve.
5. For transportation of high pressures cylinders general precautions are required. To
protect the valve during transportation, the cover cap should be screwed on hand tight and
remain on until the cylinder is in place and ready for use. When moving large cylinders,
they should be strapped to a properly designed wheeled cart to ensure stability. Cylinders
should be moved only with dedicated trolleys. Cylinders should never be rolled or dragged.

C. Leaking of compressed gas cylinders

If a leak is suspected, do not use a flame for detection; rather, a flammable-gas leak detector
or soapy water should be used. If the leak cannot be remedied by tightening a valve gland or
a packing nut, emergency action procedures should be initiated. Laboratory workers should
never attempt to repair a leak at the valve threads or safety device; rather, they should consult
with the supplier for instructions.

- Safe handling of Cryogenic Liquids (liquid Nitrogen)

The transfer of liquefied gases from one container to another should not be attempted for the
first time without the direct supervision and instruction of someone experienced in the
operation. In all procedures involving liquid nitrogen cryo-gloves and eye protection
(preferably a face shield) should be worn at all times. Gloves should be chosen that are
impervious to the fluid being handled and loose enough to be tossed off easily.

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Fire/Explosions; Neither liquid nitrogen nor liquid air should be used to cool a flammable
mixture.
Adequate ventilation must always be used to avoid suffocation or the possibilities of build up
to explosive gas mixtures.
Only appropriate impact-resistant containers must be used that have been designed to
withstand the extremely low temperatures for handling and storage of larger quantities of
cryogenic liquids. Only dedicated Polyurethane Foam Ice Buckets are permitted for usage
with liquid nitrogen. Styrofoam boxes (thermo col) are NOT allowed for handling liquid
nitrogen at any time.

- Safe Handling of Dry Ice

Dry ice is solidified carbon dioxide (CO2). Dry ice sublimates (changes directly from solid to
gas), releasing CO2. Carbon dioxide vapor is substantially heavier than air. In confined,
poorly ventilated spaces it can displace air, causing asphyxiation. It is even possible for CO2
vapor to accumulate in low-lying areas, out-of-doors. Gloves and eye protection (preferably a
face shield) should be worn at all times when handling dry ice.
Dry Ice is obtained in blocks and has to be crushed for usage. Use only provided tools, during
this procedure eye protection and gloves are a must.
Never store dry ice in glass or other sealed (airtight) containers or coolers. Storage in a sealed
container can result in a rupture or explosion of the container from over-pressurization.

A. Disposal of Unused Dry Ice

Allow the dry ice to sublimate or evaporate to the atmosphere in a well-ventilated area where
no build-up of carbon dioxide vapor can occur.
Do not dispose of dry ice in sewers, sinks or toilets. The extreme cold will harm sink
disposal, toilet parts and pipes. Do not dispose of dry ice in garbage receptacles or garbage
chutes.

IV. BIOLOGICAL SAFETY

Four bio-safety levels are established to regulate laboratory practices, techniques and safety
equipment appropriate for handling of biological agents. Biological agents in that context are:
a)Microorganisms
b)Arthropods
c)Toxins of different origin and those produced using rDNA technology
d)Viruses

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e)Allergens of different origins and those produced using rDNA technology

At lab, the area of biological safety is under the guidance of the Institutional BioSafety
Committee (IBSC) and Bioethics Committee. For Biological Safety levels 1 and 2,
researchers are bound to follow the Committee guidelines and inspections.

Bio-safety Level I:
Practices and safety facilities are appropriate for working with well-defined biological agents.
These biological agents are not known to cause disease in healthy adult humans and pose
only a minimal potential hazard for laboratory personnel and the environment. Bacillus
subtilis, Naegleria gruberi, infectious canine hepatitis virus and non infectious E. coli are
representative of those microorganisms meeting these criteria. Many agents not ordinarily
associated with disease processes in humans are, however, opportunistic pathogens and
may cause infection in the young, the aged, immunodeficient or immunosuppressed
individuals.

Bio-safety Level II:

Practices and safety facilities must meet the perquisites to deal with a broad spectrum of
indigenous moderate-risk agents. These agents are known to cause diseases but immunization
or antibiotic treatment is available. Examples are Hepatitis B virus, the Salmonellae,
Toxoplasma spp. and infectious E. coli strains. Primary hazards to personnel working
with these agents may include accidental autoinoculation, ingestion, and skin or
mucous membrane exposure to infectious materials. Procedures with aerosol potential
that may increase the risk of exposure to personnel, must be conducted in primary
containment equipment or devices.

Bio-safety Level II+:

The term ‘Infectious Material’ applies to blood, any other body fluid, and other potentially
infectious material like biopsies. All body fluids shall be considered as infectious materials.
Research involving Biological Safety Levels II+ shall contact the Biological Safety
Committee for appropriate authorization, guidelines and inspections.
Such work can only be performed in the Biohazard laboratory.

Bio-safety level III:

Biosafety level III (BSL-3) is applicable to clinical, diagnostic, teaching, research, or
production facilities where work is performed with agents that may cause serious or
potentially lethal disease through inhalation, to the personnel, and may contaminate the
environment. It requires that laboratory personnel receives specific training in handling
pathogenic and potentially lethal agents, and be supervised by scientists competent in
handling infectious agents and associated procedures. All work is performed in biocontained

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 environments using appropriate engineering controls.

Bio-safety level IV:

Biosafety level 4 (BSL-4), the highest level, is required for working with dangerous and
exotic infectious agents that pose a high individual as well as environment risk of life-
threatening disease, aerosol transmission, or a related agent with unknown risk of
transmission. Laboratory personnel receive specific training in handling pathogenic and
potentially lethal agents, and have to mandatorily work wearing positive pressure BSL-4
suits.

V. Basic Working Principles in Bio-safety laboratories

The primary principle of biological safety is containment. This refers to a series of safety
procedures which have to be conducted to reduce or eliminate human and environmental
exposure to potentially harmful biological agents. To minimize exposure, observe universal
precautions when handling any biological specimen.

A. Wash your hands thoroughly:

1.Before and after working with any biohazard
2.After removing gloves, laboratory coat, and other contaminated protective clothing
3.Before eating, drinking, smoking, or applying cosmetics
4.Before leaving the laboratory area
5.Do not touch your face when handling biological material
6.Never eat, drink, smoke, or apply cosmetics in the work area
B. Clothing Guidelines:
1.Always wear appropriate lab clothes and gloves when working with biological
agents. 2.Wear gloves over gown cuffs.
3. Remove gloves by peeling them from the inside out.
4. Never wear contact lenses when dealing with infectious agents.
5. Do not wear potentially contaminated clothing outside the laboratory area.
6. Additional appropriate protective clothing should be selected and worn based upon the
task and degree of exposure anticipated.

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C. Handling Procedures liquid infectious materials:
1.Use mechanical pipetting device (examples; pipette aid, pipetteman or bulb).
2.Minimize aerosol generation. Decanting culture supernatants, opening of culture and
streaking of plates should only be done in Safety cabinets or in a circular area around a
burner of 0.5 meter radius. Decanting/Transferring of cultures in common equipment
rooms outside of safety cabinets is forbidden.
3.Add disinfectant to water baths for infectious substances.
4.Use only closed tubes for centrifuging procedures. Inspect the tubes before use.
5.Use secondary leak-proof containers when transporting samples, cultures, inoculated
petri dishes, and other containers of biohazardous materials within the institute.
6.Avoid using syringes and needles whenever possible. Special care has to be taken when
usage of needles is not avoidable: Use a needle-locking or disposable needle unit.
 Take care not to stick yourself with a used needle.
 Place used syringes into a pan of disinfectant without removing the needles.
 Do not place used syringes in pans containing pipettes or other glassware that
require sorting. Do not recap used needles.
 Dispose of needles in an approved sharps container.

D. Work Area:

1.Keep laboratory doors shut when experiments are in progress.

2.Limit access to laboratory areas when experiments involving biohazardous agents are
being performed.
3.Ensure that warning signs are posted on laboratory doors. These signs should include the
universal biohazard symbol and the approved biosafety level for the laboratory.
4.Transport contaminated materials in leak-proof containers.
5.Keep miscellaneous material (i.e., books, journals, etc.) away from potentially
contaminated working areas.
6.Follow a rigorous disinfection plan:
 Completely decontaminate equipment before having maintenance or repair work
done. Decontaminate work surfaces daily and after each spill.
 Decontaminate all potentially contaminated equipment.
 Decontamination should only be performed with these disinfection solutions such
as 70%Ethanol or 5-10% Sodium hypochlorite (bleach) solutions

Work of Bio-safety Level II has to be performed at assigned workbenches. Ensure that

warning signs are posted accordingly. All procedures generating aerosols have to be
performed in safety cabinets. Minimize traffic in the area where Bio-safety Level II work is
done.
E. Safety Equipment (Containment Barriers)

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Safety equipment includes a variety of personal protection items and laboratory devices
which provide the ability to keep infectious agents in a specified contained area that is easily
accessible. The biological safety cabinet is the principal device used to provide containment
of infectious aerosols generated by many laboratory procedures.

F.Disposal of liquid cultures:

1.All culture supernatants in conical plastic tube never more than 25ml can be disposed in
the red biohazard bin.
Tubes have to be closed but not overly tight.

2.All cultures ≥ 25ml must be put in a glass beaker or Erlenmeyer flask and treated to
become 10% with bleach in a biosafety cabinet. Examples; 30 ml culture/3-4 ml of
straight bleach, 250 ml culture/25-30 ml straight bleach, let stand at least 30 minutes.
Dispose off in sink: Turn on the water faucet, pour treated culture in, keep water running
for a minute or two after you have finished pouring the culture.

3.Cultures with other hazardous chemicals and/or heavy metals must be disinfected as
above and then disposed of according to the method prescribed in the MSDS.

VI. Biohazard spills

Biological spills outside biological safety cabinets will generate aerosols that can be
dispersed in the air throughout the laboratory. These spills can be very serious if they involve
microorganisms that require Biosafety Level II and above containment. The Biosafety lab has
its own procedures to follow.

General reaction plan for a biological spill:

1. Cordon off the area to stop anyone from spreading the contamination throughout the
laboratory.
2. Cleaning procedures should be started in a timely manner by a person from the lab where
the spill has occurred. Before starting to clean the spill, Personal Protection Equipment
(gloves, face mask, safety goggles, long sleeve lab coat and shoe covers) must be
obtained and put on.
3. Disinfect the area, all surfaces using 70% Ethanol in a spray bottle. Any material used to
wipe up the spill must be placed in a biohazard bag and decontaminated using an
autoclave.

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Decontamination should only be performed with these disinfectants: 70%Ethanol or 10%
Chlorox
A. Spills on the Body
1. Remove contaminated clothing.
2. Apply disinfection solution. Vigorously wash exposed area with soap and water for one
minute.
3. Obtain medical attention.
4. Report the incident to the Laboratory in-charge

B. Bio-safety Level I Organism Spill decontamination

procedure 1.Wear disposable gloves.
2. Soak paper towels in disinfectant (70% ethanol or 10% chlorox) and place over spill.
Allow a 30-minute contact period on the spill.
3. Place towels in a biohazard bag and decontaminate using an autoclave before
disposal. 4.Clean up spill area with fresh towels soaked in disinfectant.
C. Biosafety Level 2 Organism Spill
1. Shut down the air conditioning units for your laboratory or section of the building.
2. Alert people in immediate area of spill.
3. Put on protective equipment, a laboratory coat with long sleeves, back-fastening gown or
jumpsuit, disposable gloves, disposable shoe covers, safety goggles, mask or full-face
shield.
4. Cover spill with paper towels or other absorbent materials.
5. Carefully pour a freshly prepared disinfectant around the edges of the spill and then into
the spill.
Avoid splashing. Allow a 30-minute contact period. We recommend using a spray bottle.
6.After the spill has been absorbed, clean up the spill area with fresh towels soaked in
disinfectant.
7.Place towels in a biohazard bag and decontaminate in an autoclave.

VII. Specialty Laboratories

- Working with Radioactive Materials
Each lab should take permission from the authority before starting any research related to

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Radioactive Materials. The process to become a Radioactive Laboratory user will be to fill
out an application form available with lab in-charge/reception, pass labsafety. One of the
most important aspects of the orientation program and tests is that the candidates must be
aware of are the specific protocols for disposal of waste materials generated during
experiments performed in the Rad Lab. Once cleared the test and passed through the
orientation, the user must obtain permission before starting to use the Radioactive
Laboratory. Initially for 1-2 months, student must be under the supervision of a senior Lab
user.

VIII. Waste Disposal and Management in IASST

A. Types of waste
With its usual day to day activities, following four types of waste are generated at lab.

1. Organic waste: Organic materials generally make up the largest portion of waste.
Organic wastes are created during every meal. Clearing of jungles, weeds and trimming
of plants also produces green wastes. When properly disposed of, these materials can be
picked up by trash removal services and transported to a facility where it will be turned
into compost, which can actually be used as fertilizer. In some occasion this includes
paper and paperboard products which cannot be used for compost production due to the
presence of unwanted chemicals.
2. Recyclables: Recyclables are types of waste that are non-biodegradable and can be
converted into reusable material. Things like plastics, metals, and glass are all harmful to
the environment when placed into landfills, but proper disposal can eliminate the need to
manufacture even more of these materials, which are instead be reused in more products.
3. Toxic Chemical Waste: This section includes EtBr removal procedure. Since it
fluoresces readily with a reddish-brown colour when exposed to UV light and with
increased brightness when bound to double stranded-DNA and single-stranded RNA, it is
commonly used in gel electrophoresis application for visualization of these molecules.
EtBr may present a hazard if it is poured down the drain untreated or placed in the trash.
4. E-waste: This type of waste has become far more of an issue in recent years with the
surge in technology, such as computers and other sophisticated instruments with
electronic circuits. This is related to regular recyclables in that most of these products are
composed of plastics, metals, and glass.

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B. Procedure of waste disposal & management system
It has been decided to follow a specific and well laid down wastes collection and
management system till the classified materials are disposed by the authority.
1. For Bio-degradable waste.
2. Toxic Chemical Waste
3. For Plastics
4. Sharp materials like metal and glass.

C. Management of bio-degradable waste

Organic and bio-degradable wastes are produced from dry leaf of trees, vegetable waste, food
material remaining etc. Effort is to be made to educate people so that they grow the habit of
depositing this type of waste in the specific bin excluding food materials. Dry leaves and
vegetable waste collected from these bin are to be weighed using balance and to be processed
to produce vermin compost. The i/c Vermin compost unit will guide the workers for the
purpose.
Foods remaining are to be stored in a special type of covered trench with cement lining and
metal container behind the existing Lab. Food waste from Student and Scientist Home are
also to be placed here and for this special hand trolley will be provided to the caterer. After
few days, when this food waste are reduced and ideal for making compost, it will be shifted
to vermin compost pit for further processing.
D. Management of toxic chemical waste- Ethidium Bromide
1. Any EtBr waste should NOT be poured down the drain, or thrown in the trash, unless the
waste has been deactivated or filtered.
2. Handle EtBr gels, contaminated gloves similar to chemical hazardous waste. Use sealable
disposable plastic begs to store ethidium bromide gel waste.
3. Minimize free flowing liquids in these bags when they are brought for disposal. A bin is
kept for gloves and gels contaminated with EtBr.
4. The liquid EtBr waste should be collected in an appropriately labeled 2-5 ltrs glass
reagent bottles.A bind-ET ethidium bromide (EtBr) removal system (Elchrom Medical)
for final disposal of liquid EtBr waste is kept near the wash basin of the laboratory. The
Bind-ETTM is a closed system which removes EtBr from aqueous solutions in the safest
and simplest way. The most important part of the system is an ion exchange column with
a binding capacity of more than 2g of ethidium. The aqueous solution flowing out of the
column contains no detectable amount of EtBr and can be treated as normal effluent.
Because it is closed, the ion exchange column can be sent for incineration. This
convenient way of disposing EtBr solution is not only safe, efficient and cost effective,

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 but fulfils ecological demands and regulations by completely removing EtBr from
solutions.
5. Caution: Gel particles in the solution (usually from the agarose gels) would clog any
packed column. Make sure that the solution is poured into the reservoir through the sieve.
Never change the outlet position by attaching silicon tubes to the outlet port.
6. Change the cartridge after six months of continuous use, since after six month its capacity
might get exhausted. Any maintenance/ service should be performed by authorized
personnel only.

E. Management of plastic wastes

All types of recyclable plastic are to be kept in this specified bin.
Management of Sharp Materials Like Metal and Glass
Any sharp material, other than organic/biodegradable and plastic material are to be stored
here mainly of metal and glasses. These are to be collected and transferred to the Central big
sized dustbin.

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F. Wastes in the Laboratory Works
There are two types of wastes generated in the laboratories and disposal of these wastes will
be done as follows:
1. Wastes of plastics, glass, sharp materials etc. will be kept in a box/bin inside the lab
and at the end of the day these will be placed in designated bins placed in the corridor
by the lab worker.
2. Waste materials of chemicals, reagents, toxic substance, gel, radioactive substances
etc. will be disposed by the scientists concerned following prescribed technical
procedure.

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IX. EMERGENCY RESPONSE

A. FIRES

- Fire Safety

Fire is the most common safety hazard in any organization. Therefore, it is very essential
for everyone to know how to survive a building fire and what to do in case of a fire. The
following section briefly explains some common protocols and procedures that may be
followed during a fire emergency.

How to Survive a Building Fire

 Go out of the building immediately through the nearest exit.
 Always use stairs, not an elevator
 Close doors from outside in case of severe fire to prevent the fire from spreading
 In case of heavy smoke, crawl low and if possible tie a wet cloth on your nose
 Use a fire extinguisher, if the fire is very small and you know how to use it safely
If you are on fire - Stop, Drop and Roll If you get trapped:
 Close the door
 Open the windows if safe
 Do not jump out of a tall building
 Signal for help and call 101

Fire Extinguishers:
Fire, depending on its origin, is classified as:

1. Class A: Combustible materials: Wood, paper, furniture etc.

2. Class B: Flammable Liquids
3. Class C: Flammable Gas
4. Class D: Metal Fire
5. Class E: Electrical equipment
6. Class K: Oil, greases

If FIRE occurs:
It may not be necessary to evacuate the building for a small fire. If, however, there is any
chance that the fire may endanger others or may cause serious damage, confine or control
the fire only if possible.

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 Use an appropriate extinguisher:

 Dry Powder (for all type of fire): Dry powder containing extinguishers are
recommended for putting off all types of fire.
 CO2 (for B and C type fire): Because the use of dry powder leaves a messy
surrounding, CO2 based extinguishers are often recommended for small fire of
chemical origin.
 If fire breaks out close to a flammable gas supply or close to electrical power source,
turn off gas supplies and electrical power sources.

If a solvent in a beaker catches fire, covering the beaker and depriving the fire of oxygen
can easily extinguish the fire than using a fire extinguisher on the same beaker, which may
cause the solvent to spill, thus increasing the hazard!

ACTION TO BE TAKEN IN CASE OF FIRE

Photo source: ThoughtCo / Nusha Ashjaee

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Revision of Safety Manual
Safety Manual will be reviewed whenever there is a necessity for a certain policy

change. A quick run through of lab safety rules/policies:

 Report "All" accidents, no matter how minor, to the Supervisor/Safety In- Charge
immediately
 Do not work alone in the laboratory.
 Know the location of the (i) "Emergency Exits in the lab and instrument room
and (ii) fire extinguishers.
 Student with medical/Health concerns should seek the advice of a Doctor before
attending labs.
 Wear safety goggles and lab coat at all times. If you have spilled chemical in
your eyes, flush with water in an eye wash station for 10 to 15 minutes. Use
safety shower in case of chemical spillage on body. Notify the incident to
Supervisor and Safety In-Charge.
 Always wear full sleeves and a lab coat while working in the lab
 Wear appropriate shoes while working in the lab. Feet must be adequately
covered. Open toed shoes or sandals are not permitted in the laboratory.
 Confine long hair whenever working in the laboratory.
 NO tobacco products in the laboratory.
 Ensure safe handling of chemicals by referring to Material Safety Data Sheet
(MSDS) or ask the supervisor
 Report all spills especially mercury spill to Supervisor and Safety in Charge.
 Segregate the waste solvents and solid wastes appropriately for proper disposal.
 Do not use broken or chipped glassware and dispose them in the glass disposal
box.
 Used syringe needles should be dropped in syringe disposal box, and do not
dump waste paper in the broken glass/needle disposal boxes.
 Do not perform unauthorized experiments in the lab.
 Avoid crowding in lab benches (not more than 6 in each work bench)
 Do not use earphones/headphones while working in lab
 Follow all the special instructions and be careful while handling & disposing bio
hazardous samples.

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XII. Committees

Institutional BioSafety Committee:

1. The Dean- Chairman
2. Dr. M V Simha- member Secretary
3. Mr Swarna Biswas- Biosafety Officer
4. Mr Tamal B- Member
5. Mrs Jagriti B- Member
6. Mr Sagar D- Member
7. Ms Chitra Bag- Member

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