BT1006 LAB SAFETY AND

ANALYTICAL TECHNIQUES
UNIT I
General rules
Personal precautions , lab safety
Good laboratory practices
Lab maintenance
Apparatus
Materials and reagents, test systems
Test and reference substances
Standard operating procedures

Lab safety is the key to reduce injury and illness (health hazard) to the lab users.
Several agencies are involved in safety compliance at the university.
1. National institute of health (NIH)- incharge of the labs that use r-DNA.
2. Centre for disease control and prevention (CDC)- for infectious agents.
3. Occupational safety & health administration (OSHA) for hazardous materials &
chemicals, blood borne pathogens
4. Environmental protection agency (EPA)- for hazardous waste ( that pose a treat to
human, animal , plant and environment) disposal.
5. Environmental health & safety office (EHS) overseeing various activities in the
campus.

General Rules
Any work inside laboratory must be performed under the
supervision of any person authorized for that lab.
Remain at your lab station while performing an experiment.
An unattended experiment can result in an accident.
Eating, drinking, and chewing gum in the lab room is
prohibited.
Keep hands away from eyes and mouth in the lab.
Wash hands before and after performing any activity in the
lab.
Only necessary things should be brought to the work area.
Do not use contact lenses, rather prefer glasses inside the lab.
No open toed- shoes inside the lab

Personal Precautions
Clothing should be appropriate for working in the lab. Fully
covered shoes must be preferred. Do not wear loose or
baggy clothes.
Personal protective equipment(PPE): It refers to
protectiveclothing,helmets,goggles, or other garments or
equipment designed to protect the wearer frominjury. The
proper PPE will protect you from hazards of physical,
electrical, heat, chemicals, biohazards, andairborne
particulate matter.
Never handle broken glass with your bare hands. Use a brush
and dustpan to clean up broken glass and dispose in proper
container.
Do not taste, touch, or smell any reagents unless
directed to do so by your teacher. All chemicals can be a
potential danger. Handle chemicals carefully.

Lab Safety
Lab safety includes:
1.General lab safety
2.Glassware safety
3.Chemical safety
4.Electrical safety
5.Heating safety
6.First aid
General lab safety
All general rules for lab safety must be followed.

Glassware safety
1. Chipped or cracked glassware should not be used.
2. Broken glassware should not be disposed of in a class room
trashcan. It should
be disposed in a proper way.
3. When pouring liquids into glassware, make sure the container
you are pouring into is resting on a table at least a hands
breadth from the edge.
4. Do not place hot glassware in water. Rapid cooling may make
it shatter.
Chemical safety
1. Wear protective goggles and a lab apron whenever heating or
pouring hazardous chemicals.
2. Never mix chemicals together unless you are told to do so (and
then only in the manner specified).
3. Never taste any chemicals (you should never taste anything in
the lab).
4. If you need to smell the odor of a chemical, waft the fumes
toward your nose with one hand. Do not put your nose over the
container and inhale the fumes.

Electrical safety
1. Be sure your hands and your lab area are dry before using
electrical equipment.
2. Unplug all electrical equipment (not the refrigerator , shakerincubator etc.) at the end of the lab period.
Heating safety
1. Let burners and hotplates cool down before touching them.
2. Use tongs and/or protective gloves to handle hot objects.
3. Never reach across an open flame or burner.
4. The only type of glassware that may safely be heated is either
Kimax or Pyrex.
5. Always point the top ends of test tubes that are being heated
away from people.
6. When heating a test tube, move it around slowly over the
flame to distribute the heat evenly.
7. Never leave a burner or hotplate unattended.

Electrical Safety

First aid
Definition: emergencyaidor treatment given to
someone injured, suddenly ill, etc., before
regular medical services arrive or can be
reached.
Injury: Burns
To do: Immediately flush with cold water until
burning sensation is lessened.
Injury: Cuts, bruises
To do: Do not touch an open wound without
safety gloves. Pressing directly on minor cuts
will stop bleeding in a few minutes. Apply cold
compress to bruises to reduce swelling.
Injury: Fainting
To do: Provide fresh air and have the person

Injury: The eyes

To do: Flush eyes immediately with plenty of water
for several minutes. If a foreign object is lodged in
the eye, do not allow the eye to be rubbed.
Injury: Poisoning
To do: Find out what substance was responsible for
the poisoning and alert the teacher immediately.
Injury: Spills on the skin
To do: Flush with large quantities of water. For acid
spills apply baking soda solution. For base spills
apply vinegar or boric acid.
Injury: Electrical shock
To do: Shut off the current at the source.
Remove wire with rubber gloves. Alert the teacher

Good Laboratory Practices (GLPs)

GLP or cGLP(current good laboratory practices) is an
FDA (food and drug administration) regulation.
Definition: GLP embodies a set of principles that
provides a framework within which laboratory
studies are planned performed, monitored, reported
and archived.
GLP makes sure that the data submitted are a true
reflection of the results that are obtained during the
study.
GLP also makes sure that data is traceable and
promotes international acceptance of tests.
If GLPs are not followed the facility can be

GLP stresses the importance of the following main

points:
1. Resources: Organisation, personnel, facilities
and equipments.
(Organisational charts and job descriptions)
(spacious facility, adequate and stable utilities,
equipment condition)
2. Characterisation: Test items and test
systems;
(identity, potency, composition, stability, impurity
profile, etc. should be known for the test item)
3. Rules: Protocols, standard operating
procedures (SOPs);
(latest protocols and SOPs must be known)
4. Results: Raw data, final report and archives;
(Raw data are the original data collected during
the conduct of a procedure. The study report must be
true. All the data must be stored in written format)

Lab Maintenance
Few points which should be considered for laboratory maintenance:
Bench:Cleanandclearbenchtopsafteranexperiment.Keepthematerialsattheirright
location.
Floor: Sweeplabflooraftereveryexperiment.
Eyewash Station:Checkwhethertheeyewashworksornot.
Fire and Emergency Equipment:Thisisveryimportant.Thefireextinguishers,firstaidkit,fire
blanketshouldbepresentandinorder.
Broken Glass and Sharp Containers :Whenthebinishalffilledwithbrokenglassesandsharp
containers,sealit,andthrowitinthedisposableareaortothedumper.
Refrigerators & Freezers:Throwawayitemsthatareoutofdatebutthisshouldbedone
throughappropriatewastestreams.Ifthereareflammablesthatshouldnotbeideallykeptina
refrigerator,removethemandstoretheminappropriateareas.Defrostifnecessary.Checkthe
schedulefordefrosting.
Centrifuges:Washandcleanrotor,adaptersandbuckets.RemoverotorandchecktheO-rings.
Balances:Balancesshouldbeproperlycleaned,bothinandout,andcheckthebalanceislevel.

 pH Meter:Switchoffifnotinuse.
Chemical Waste & Storage:Checkthechemicaldiscardcontainers.Iftheyarealmostfull,take
themtotheproperdisposalarea.Similarly,allchemicalsshouldbeproperlystacked,labelled
andcorrectlysegregatedinthestoringcabinet.
Fume Cupboard & Biosafety Cabinet:Removeitemsfromworkzone.Checkthecontrols.
Checkthattheunitperformssatisfactorily.
Animal Waste:Theanimalwastes,whennotused,shouldbepackedindoublebaggedinblack
andthenstoredinayellowbiohazardbag.Thisshouldbeproperlysealedandlabelledand
shouldbekeptinappropriatestoragearea.
Hand Disinfectant:Replaceorrefillthehanddisinfectantbottle.
Paper Towel:Makesurethatthepapertowelrollisatleasthalffull.Asparerollshouldbekept
besidethedispenser.
Radioactive Wastes:Checktheexpirydate.Discardintheindustrialwaste.
Water Bath:Changewaterinbath.Addbacterialinhibitor.
Gas Cylinder:Returnunusedoremptycylinderstostore.
Media Plates and Flasks:Throwawaytheusedplates,bottles,smallcontainersetc.inthe
properdisposalarea.

Chemical Safety General Measures

Treat every chemical as if it were hazardous.
Make sure all chemicals are clearly and currently
labelled with the substance name, concentration, date,
and name of the individual responsible.
Never return chemicals to reagent bottles. (Try for the
correct amount and share any excess.)
Use volatile and flammable compounds only in a fume
hood.
to prevent inhalation of hazardous material.
Never allow a solvent to come in contact with your skin.
Always use gloves.
Never "smell" a solvent!! Read the label on the solvent
bottle to identify its contents.

Material Safety Data

A Material Safety
Sheets
Data Sheet (MSDS) is
a document that
contains information
on the potential
health effects of
exposure to
chemicals, or other
potentially dangerous
substances, and on
safe working
procedures when
handling chemical
products.

READING
THE
MSDS
Identity Thechemicalname,tradenameandmanufacturersname,

addressandemergencyphonenumbercanbefoundhere.
Ingredients Includes:substance,
%content,CASNumber,Classification,EINECS
Hazards Identification Dangersforhumansandtheenvironment
suchas: Mostimportanthazards&Specifichazards
First Aid givesinstructionsonwhattodoincaseofeyecontact,skin
contactoringestion.
Fire Fighting Suitableextinguishingmedia,Unsuitable
extinguishingmedia,Specialhazardsinfire,Requiredspecial
protectiveequipmentforfire-fighters
Accidental Release Measures/Spill Clean-up Personal
precautions,Environmentalprecautions,Methodsforcleaning
Handling and Storage Exposure Controls and Personal
Protection

InformationonproperPPEtouse,howtostoreandtemperature
limits

READING THE MSDS

contd

Physical and Chemical Properties Appearance,Odor,pH,Boiling

point,Meltingpoint,Flashpoint,Explosiveproperties,Vaporpressure,
Relativedensity,Solubility
Stability and Reactivity Conditionstoavoid,Materialstoavoid,
Hazardousdecompositionproducts
Toxicology Acutetoxicity,Localeffects.Excessiveexposuremayaffect
humanhealthasfollows:Skincontact,Eyecontact,Inhalation/ingestion.
Ecological Information Listsanydangerstotheenvironment
Disposal Listsanyspecialdisposalmethods

Transport Information

listscodesindicatingthedangers
andthetypeoftransportwhichmaybeneeded

Regulations Listsanyagencythatmayregulatethisproduct
Other Information Recommendations/restrictions,Sourcesof

keydatausedtocompileSafetyDataSheet

Types of Hazards

A health hazard has

the following
characteristics:

A physical hazard has

the following
characteristics:

Carcinogen
Toxic or highly toxic
Reproductive Toxins
Irritants
Corrosives
Sensitizers
Hepatotoxins
Nephrotoxins
Neurotoxins

Explosive
Flammable
Oxidizer
Pyrophoric
Organic peroxide
Compressed gas
Combustible liquid
Unstable (Reactive)
Water-reactive

Types of Biohazardous Waste

Solid
Labware (flasks, tubes, plates, bottle,
vials)
Lab waste (stocks, specimens, cultures,
swabs, tissue, plastic pipettes or pipette
tips)
Contaminated gloves, apparel, wipes

Liquid
Sera, body fluids

Sharps
Anything with a point or edge capable of
piercing or cutting (razors, needles,
syringes, glass slides)

Biohazardous Waste

Dry Solids

Collect in a rigid leak resistant container with a lid

and labeled with the Biohazard Symbol that is lined
with a RED bag and labeled with:
The words Biohazard
Must have International Biohazard symbol
Liquid
If you are not trained, do not handle
biohazardous liquid waste.
Sharps
Must not contain any liquid.
Collect in a rigid, puncture and leak resistant,
properly labeled sharps container
Incinerationis awaste treatment process that
involves the combustionof organic substances
contained in waste materials. Incineration and
other high-temperature waste treatment
systems are described as "thermal treatment".

Types of Chemical in Labs

Flammable Chemicals
A number of highly flammable and combustible substances are
commonly used in campus laboratories.
The hazard of a flammable or combustible chemical is based on its flash
point, and, in the case of a flammable chemical, its boiling point as well
Flammable liquids include those chemicals that have a flashpoint of less
than 140 degrees Fahrenheit.
These materials must be stored in flammable storage cabinets.
Flame-resistant laboratory coats must be worn when working with
flammable materials.
Flammable liquids are of 3 classes :
Class IA liquids shall include those liquids that have a flash point below
73F (22.8C) and a boiling point below 100F(37.8C).
Class IB liquids shall include those liquids that have a flash point below
73F (22.8C) and a boiling point at or above 100F(37.8C).
Class IC liquids shall include those liquids that have flash points at or
above 73F (22.8C) but below 100F (37.8C.

Reactive Chemicals
Reactive and explosive substances are materials that
decompose under conditions of mechanical shock,
elevated temperature, or chemical action, and release
of large volumes of gases and heat.
These materials must also be stored in a separate
flame resistant storage cabinet or, in a laboratory
grade refrigerator or freezer that is designed for
flammable and reactive chemicals.
Examples of reactive chemicals are : Acetaldehyde ,
Acetic acid( greater than 6M), Acetone , Acetylene ,
benzene compounds, propane , pentane , ethyl and
methyl alcohol etc.

Combustible liquids
Class II shall include those liquids that have a flash point at
or above 100F (37.8C) and below 140F (60C).
Class IIIA shall include those liquids that have a flash point at
or above 140 (60C) and below 200F (93C).
Class IIIB shall include those liquids with a flash point at or
above 200F (93C).
Chemicals

Class

Glacial acetic acid

II

Formic acid

II

Benzaldehyde

IIIA

Nitrobenzene

IIIA

Cyclohexanol

IIIB

Benzyl alcohol

IIIB

Ethylene glycol

IIIB

Classes of fires

Class A - Ordinary
combustibles or fibrous
material, such as wood,
paper, cloth, rubber, and
some plastics.
Class B - Flammable or
combustible liquids such as
gasoline, kerosene, paint,
paint thinners and propane.
Class C - Energized
electrical equipment, such
as appliances, switches,
panel boxes and power
tools.
Class D - Certain combustible
metals, such as magnesium,
titanium, potassium, and
sodium

Types of Fire Extinguishers

Different types of fire extinguishers are designed to
fight different classes of fire.
The 3 most common types of fire extinguishers are:
1. Water (APW)
2. Carbon Dioxide (CO2)
3. Dry Chemical (ABC, BC, DC)

3thingsneededforfire

Corrosive Substances
As a health hazard, corrosive substances cause destruction of, or
alterations in, living tissue by chemical action at the site of contact.
Major classes of corrosive substances include:
Strong acids e.g., sulphuric, nitric, hydrochloric and hydrofluoric acids
Strong bases e.g., sodium hydroxide, potassium hydroxide and
ammonium hydroxide
Dehydrating agents e.g., sulfuric acid, sodium hydroxide, phosphorus
pentoxide and calcium oxide
Oxidizing agents e.g., hydrogen peroxide, chlorine and bromine.

Acid burns

Base burns

Chemical
burns

Symptoms And Storage

Symptoms of exposure for inhalation include a burning
sensation, coughing, wheezing, laryngitis, shortness of
breath, nausea, and vomiting.
For eyes, symptoms include pain, blood shot eyes,
tearing, and blurring of vision.
For skin, symptoms may include reddening, pain,
inflammation, bleeding, blistering and burns.
As a physical hazard, corrosive substances may corrode
materials they come in contact with.
In most cases, these materials should be segregated
from other chemicals and require secondary
containment when in storage.

Hazardous Substances with Toxic

Effects on Specific Organs
Substances included in this category include:
Hepatotoxins i.e., substances that produce
liver damage, such as nitrosamines and
carbon tetrachloride.
Nephrotoxins i.e., agents causing damage to
the kidneys, such as certain halogenated
hydrocarbons.
Neurotoxins i.e., substances which produce
their primary toxic effects on the nervous
system, such as mercury, acrylamide and
carbon disulfide.
Agents which act on the hematopoietic system

Reduce exposure to hazardous

chemicals
There are four
primary routes of
exposure for
chemicals which have
associated health
hazards :
1. Inhalation
2. Ingestion
3. Injection
4. Skin absorption

Safety Controls
Safety controls are divided into two main
classifications:
1. Engineering Controls; and
2. Protective Apparel and Equipment
3. Chemical Controls
ENGINEERING CONTROLS
Engineering controls include all built in safety
systems. These controls offer the first line of
protection.
They are highly effective in that they generally require
minimal special procedures or actions on the part of
the user except in emergency situations.

Good lab Ventilation

All laboratory rooms involving hazardous materials use
must have fresh air ventilation with 100% of the
exhaust venting to the outside.They should not be part
of recycled air systems.

Fume hoods
It is advisable to use a laboratory hood when working
with all hazardous substances.
A properly operating and correctly used laboratory hood
can reduce or eliminate volatile liquids, dusts and mists.

Chemical Controls

Acids and Bases

Acids and bases are corrosive and will destroy body tissue.
The extent of injury depends on factors such as the type and
concentration of the chemical, the route of exposure, the
type of tissue contacted, and the speed used in applying
emergency measures.
The eyes are especially susceptible to acids and bases and must
be immediately flushed with water for at least 15 minutes if
exposure occurs.
Transfer containers of acid and base solutions in bottle carriers.
Do not pour water into acid. Slowly add the acid to the water and
stir.
Use a mechanical aid or a pipette bulb for pipetting.
Wipe drips from containers and bench tops. Be especially careful
to wipe up visible residues of sodium hydroxide and potassium
hydroxide from all surfaces. Skin contact with dry residue will
result in burns.

Laboratory fires

F la mm a ble & C om b us tib le M at er ial s

Store in NFPA approved

flammable liquid containers
(safety cans) or flammable
liquid storage cabinets or in
designated areas.
When dispensing from metal
or other conductive containers
proper grounding of the
containers is necessary.
Ensure fire extinguishers
and/or sprinkler systems are
in the area.
Flammable liquids should be
stored away from strong
oxidizers, direct sunlight, and
heat sources, such as hot
plates.

Cryogenic Liquids

They should be stored at very

low temperatures like liquid
nitrogen at -196 degree
Celsius.
Liquid nitrogen nor liquid air
should not be used to cool a
flammable mixture in the
presence of air because
oxygen can condense from the
air and lead to a potentially
explosive condition.
Adequate ventilation is
required when using gases,
such as nitrogen, helium, or
hydrogen, since oxygen can be
condensed out of the
atmosphere creating a
potential for explosive
conditions.

Lab Apparatus and Safety

Glassware
Inserting and removing glass tubing from rubber stoppers can be
dangerous.
Always lubricate glassware (tubing, thistle tubes, thermometers,
etc.) before attempting to insert it in a stopper.
Always protect your hands with towels or cotton gloves when
inserting glass tubing into, or removing it from, a rubber stopper.
Pipette
Mouth pipette is based on the principle of suction , used to draw a
solution upwards to the desired volume using the tension created by
the reduced air pressure.
Do not allow the solution to be drawn up into the bulb
Mouth pipetting should be done only for non-hazardous and neutral
solutions like water, NaCl solution etc.

Test-tubes

A test tube with a

stopper or a cotton
plug is often used
for temporary
storage of chemical
or biological
samples.

Test-tube holder

Always hold a testtube with a testtube holder while

heating on a
Bunsen burner.
Always use a test-

Heating test-tubes

 Never mouth pipette an acid ,

base , corrosive
solution, microbe
culture , volatile
solution or any
hazardous and toxic
solutions.
Always use a
mechanical pipette
or a micropipette
for the above
mentioned

Heating Instruments
Light gas burners only as instructed by the
teacher.
If you wish to turn off the burner, do so by
turning off the gas at the gas outlet valve
first, then close the needle valve and
barrel.
SHOULD THE FLAME OF THE BUNSEN
BURNER GO OUT, IMMEDIATELY TURN OFF
THE GAS AT THE GAS OUTLET VALVE.
Never reach over an exposed flame. Never
leave a lit burner unattended. Never leave
anything that is being heated. Hold
apparatus at proper angle.
Hair should be secured and gloves used
with hot tongs or test-tube holder while
heating any test-tube or apparatus.

Hazardous Practices

Apparatu
s
When first entering a science room,do not touch any equipment, chemicals, or other
materials in the laboratory area until you are instructed to do so.
Observe good housekeeping practices.Work areas should be kept clean and tidy at all
times.
Labels and equipment instructions must be read carefully before use. Set up and use
the equipment as directed by your teacher.
Apparatus used for the generation of data should be suitably located and of
appropriate design & adequate capacity.
Apparatus used in a study should be periodically inspected, cleaned, maintained &
calibrated according to standard operating procedures (SOP). Record of procedures

should be maintained.

Materials
Apparatus & materials used in the studies
systems.

should not interfere with the test

Reagents
Reagents should be labelled appropriately to indicate source, identity, concentration
& stability information, preparation date, earliest expiration date and specific storage
instructions.

Test & Reference

Gatheringtheknowledgeabouttestsystemandtestsubstanceisknownas
characterization.
Thetestfacilityshouldhavesufficientnumberofrooms/areastoensurethe
isolationoftestsystems&isolationofindividualprojectsinvolvingsubstances
knownorsuspectedofbeingbiohazardous.
Thetest item/substance mightbeanactiveingredientforamedicine,apesticide,
afoodadditive,avaccine,achemicalcompoundusedindustrially,abiomass,an
extractionfromplanttissue,etc.Thetestitemismostfrequentlycharacterizedby
itsanalyticalprofilee.g.chemicalidentity,impurity,solubility,stability,etc.Testitem
mustbeprotectedfromcrosscontaminationfromotherchemicalsandfrom
pollutionbyexternalfactorssuchasbacteria,dust,water,etc.
Thetest system couldbeananimal,aplant,abacterium,anisolatedorgan,a
fieldorotherecosystemoranalytical equipment,etc.Thecharacterizationofthe
testsystemcanvarywidely.
Oneshouldpreventwronglevelorwrongtestitemadministrationtothetest
system.

Test Substance

Beforestartinganyactivitythestudentshouldhaveknowledge
aboutallthesubstancestobeusedforthatparticularactivity.
Theidentity,activityandbioavailabilityofthetestitemarekey
factorsinthestudy.Thetestmaterialmustbeidentifiedfirstand
then,ifnecessary,itsactivityandchemicalnaturemustbe
checkedbyappropriatetestprocedures.
Storageandhandlingoftestsubstancesmustbeappropriate.
Forexampleallacidsandacidicsolutionsshouldbekeptatone
placeinthelab.Itshouldonlybekeptonground,overalayerof
sand.Acidsshouldnotbekeptathigherlevels.Alsoacidsmustbe
storedinpyrexglasscontainers.
Thelabelsofnewcontainersmustbecheckedforthefollowing
details:
testitemname
batchnumber
expirydate
storageconditions
totalweight

Test And Reference

Substances

Test system
Physical/chemical:Apparatususedforgenerationofphysical,chemicaldatashouldbe
suitablylocated&ofappropriatedesign&adequatecapacity.
Reference substancesshouldbeusedtoassistinensuringtheintegrityofthetestsystems.
Biological:Properconditionsshouldbeestablished&maintainedforhousing,handling&care
ofanimals,plants,cellular/subcellularsystemssoastoensurethequalityofthedata.
Conditionsshouldcomplywiththeappropriatenationalregulatoryrequirements.Thetest
systemshouldbeisolateduntiltheirhealthstatushasbeenvaluated.
eachtest&referencesubstanceshouldbeappropriatelyidentified(eg.Code,name,chemical
abstractnumber(CAS).
Thestabilityofthetest&referencesubstancesunderconditionsforstorageshouldbeknown
forallstudies.
Thestabilityofthetest&referencesubstancesunderthetestconditionsshouldbeknownfor
allstudies.
Asampleforanalyticalpurposesfromeachbatchoftestsubstancesshouldberetainedfor
studiesinwhichthetestsubstanceistestedlongerthan4weeks.
Ifthetestsubstanceisadministeredinavehicle,standardoperatingprocedure(SOP)should
beestablishedfortestingthehomogeneity&stabiltyofthetestsubstanceinthatvehicle.

Standard operating procedure (SOP)

AnytestfacilityshouldhavewrittenSOPsapprovedbythemanagementthat
areintendedtoensurethequality&integrityofthedata.
EachlaboratoryshouldhaveaseparateavailableSOP.Publishedtextbooks,
articles&manualsmaybeusedassupplements.
SOPshouldbeavailableforbutnotrestrictedtothefollowing
Test&referencesubstances:receipt,identification,labelling,handling,
sampling&storage.
Apparatus&reagents:use,maintenance,cleaning,calibrationofmeasuring
apparatus&environmentalcontrolequipment,reagentpreparation
Recordkeeping:reporting,storage&retrievalcodingofstudies,datacollection,
reportpreparation,indexingsystems,handlingofdata.
Testsystem:roompreparation&environmentalroomconditionsforthetest
system,testsystempreparation,observation&examinationsbefore,during&
attheterminationofthestudy.
Heath&safetyprecautions:Asrequiredbytheinstitutional,national/international
guidelines.