Basic Biochemical Lab Procedures

Basic Laboratory Procedures

CHNG 3804
Biological Systems

Biosafety
Risk assessment-Risk group
Disinfectants
Lab Safety Rules
Sterilisation
Agar Plates
Flasks

Fariba Dehghani

Overview

Exposure to Biohazards

Identify biohazards
Assess the risks of exposure
Control the risks

May cause infection of ourselves or others.

Biohazard agents may enter the body via

Facility, equipment, procedures

Decontamination
Hazardous waste disposal

Skin (direct contact, through broken skin)

Lung (inhalation) (>80% of infection)
Gut (ingestion)
Eyes (mucous membranes)
Blood stream (injection)
Across placenta during pregnancy

Biohazard

Factors Affecting Disease Progression

An agent of biological origin that has the capacity to

produce deleterious effects on human.
Biohazards include:

Mode of infection

microorganisms (bacteria, fungi, viruses, protozoa, amoeba

Animals (laboratory and farm animals)
Plants (crops or other species being researched)
Insects (flies, fleas, ticks, mites, etc.)
Biological fluids and tissues (blood, serum, culture media)
Toxins and allergens derived from those organisms, and
allergens and toxins derived from higher plants and animals

Dose received
Target organ affected
Susceptibility of the individual infected
Biological agent involved (risk group 14)

Risk Group

Risk Group Exercise

Group 1: (low individual & community risk)- micro-organism

that unlikely to cause human, plant or animal disease

Anthrax

Group 2: (moderate individual & limited community risk)- a

pathogen that can cause human, plant or animal disease,
unlikely to be serious hazard to laboratory workers, etc..

Ecoli
Salmonella SPP

Human tissue
HIV positive blood
Sewage
Cow

Group 3: (high individual & limited community risk)- A

pathogen that usually causes serious human or animal
disease and may present a serious hazard to laboratory
workers

Hep A

Cow poo

Group 4: (high individual & community risk)-A pathogen that

usually produces life threatening human or animal disease.
It is readily transmissible from one individual to another

HIV

Cow with salmonella

Lactobacillus spp

bat

Human blood

Risk Management

Physical Containment

Step 1- establish the context

PC 1- Suitable for containment of risk group 1

organisms

Step 2- Identify hazards

PC2 Suitable for containment of risk group 2

organisms

Step 3- Assess risks

PC3- Suitable for containment of risk group 3

organisms

Step 4- Rate risks

PC4- Suitable for containment of risk group 4

organisms

Step 5-Control risks

Properties of Cell Culture

Control of Biohazards

Deliberate infection with pathogens

Adventitious contaminating agents

Facility
Vaccination
Work Practice

Bacteria
Mycoplasma
Viruses
Parasites
prions

Vaccination
Vaccine

All people

Hep B

Biological Safety Cabinets

Work with
human
tissues

Vet science agriculture

Laminar flow
Product protection
Operator contamination

Class 1 Biosafety Cabinets

Operator protection
Product contamination

TB
Tetanus

Operator and product protection

Cytotoxic Drug Safety cabinets

Qfever

Class 2 Biosafety Cabinets

Operator, product and environment protection

Diphtheria

Clean Workstations

Class II cabinet

Self-Contained
Laminar Flow
horizontal
vertical

Decontamination in Biological
Laboratories

Biological Waste DisposalDisposalMicroorganisms

Autoclave
Disinfection

Risk Group 1 and 2:

Ethanol, isopropanol
Sodium hypochlorite
Formaldehyde, glutaraldehyde
Iodophors
Phenolics
Quaternary ammonium compounds
Paracetic acid

Thorough decontamination (autoclave or disinfection)

Seal in strong black bags
May be disposed of directly into industrial waste bins

Risk group 3 and 4

Thorough decontamination (autoclave or disinfection)
Seal in strong bags, appropriately labelled
Request disposal via university hazardous waste disposal
service
Wash hands

Biological Waste DisposalDisposal-Human/Animal

Tissues

Hazard Waste Program

Human tissues:

Fortnightly collection
Separate clinical/biological waste form otherwise
hazardous waste
Place sharps in approved sharps containers
Deposit clinical/biological wastes in dedicated
yellow bins

Tissue, organs, limbs and free-flowing or

expressible blood
The waste should be steam sterilised, placed into
yellow bags and placed into a clinical waste bin
for collection.

The management & disposal of these types

of wastes need to be conducted with public
perception and aesthetic consideration in
mind.
See: http://www.usyd.edu.au/ohs/forms/w-rform.pdf

Authoritative Bodies for Research

Project
Institutional biosafety committee (IBC)
Project involving recombinant DNA and genetically
manipulated material

Human Ethics Committee

Project involving humans (including questionaries)

Animal Care and Ethics Committee (ACEC)

General Laboratory Practices

Projects involving animals

Do
Dos & Don
Dont

Do not
Handle store or consume
food or drink in the
laboratory.
Smoke
Carry out hazardous work in
isolation
Wear open toed shoes in the
lab
Store mutually reactive
substances in the same
area.

Safety in the laboratory depends on personnel achieving a

recognized standard of behavior.
The following recommendation apply to all personnel working
within a laboratory
Never adopt a casual attitude in the laboratory
Ensure that personal clothing is suitable to laboratory
conditions (non-slip, closed in footwear).
Use protective clothing and devices appropriate to the type of
work.
Identify and control hazards associated with your work or the
lab.
Regard all substances as hazardous unless there is definite
information to the contrary.
Wash skin areas which come in contact with chemicals,
irrespective of concentration.

PC1

Always
Keep only the minimum required
quantities of hazardous
substances in the lab.
Use a fume cupboard, fume
cabinet or glove box when
working with highly toxic volatile
or odorous substances
Label all safety equipment and
maintain it in good operating
condition
Clean up spills immediately.
Dispose of specialized waste
Wear eye protection when in the
laboratory area.

Take care to minimize the production of aerosols

Take precaution to ensure that reading and writing
materials do not become contaminated.
Clean up all spills immediately and decontaminate
the area
Report significant spills and accidents immediately
to the laboratory supervisor.
A written record of accidents shall be prepared and
maintained.
Remove laboratory gowns and thoroughly wash
hands and finger nails before leaving the lab.

PC2

Do
Dos and Don
Donts

Restricted access.
Laboratory personnel shall receive instruction and training, with
regular updates in handling pathogens
Care shall be taken when using sharps
Maintenance and service personnel advised of the special
microbiological hazards in the laboratory.
Contaminated surfaces and equipment disinfected before
maintenance
Certain risk group 2 microorganisms requires special precautions
Take special care when handling human blood or body fluids.
You must notify the supervisor of any spills or accidents immediately
or ASAP.
Gloves should be worn and disposed of as biological waste
Remove both gowns and gloves before leaving the lab.
Wipe all work surfaces before leaving the laboratory
Wash hands before you exit laboratory.

Do not:
Store reference documents and papers other than worksheet on
lab bench.
Sniff bacterial culture for odors
Chew on pen-keep hand and pens away from your face. They could
be contaminated from bench surfaces or aerosols
Bring personal items such as clothing, mobile phones, etc. into the
PC2 lab.

Always:
Regarded clinical specimens as potentially hazardous.
Use double container for biological hazardous material for
transportation.
Autoclave contaminated re-usable glass wares
Dispose of biological wastes appropriately
Separate write up area & computers for lab areas

Lab Safety Rules

Gowns

Laboratory coats

Safety Glasses, Laboratory Coats and Enclosed

Shoes must be worn at all times, laboratory coats
should remain in laboratory.
No eating, drinking, smoking, shaving or applying
cosmetics in the laboratory.
No pipetting by mouth
No smelling of sniffing cultures for odors.
Use a separate clean area for paper work, computer
work, telephone calls etc.

Lab Safety Rules

Regard all samples as potentially infectious
Hands should be washed before leaving
laboratory.
Pay attention to personal hygiene hand
washing, touching face, hair, covering wounds.
Report accidents, possible exposures and any
ill-health which might relate to infection
immediately to the laboratory supervisor.
Minimize and contain aerosols

Lab Safety Rules

Know what to do in case of a spill.
Understand and practice disinfection and
sterilization methods.
Dispose of wastes correctly.
Label all samples with, the culture type, date and
your name.
Doors to the Biochemical Engineering Labs
should be kept closed.
Lab coats used in Biochemical Engineering labs
should remain in the labs.

Growing the Correct Microbe

An amazing variety of micro-organisms can be found
in any environment
These micro-organisms vary in the nutrient
requirements
Water + either carbon, nitrogen or phosphate source
and something will start to grow.
Necessary to sterilise/sanitise before starting
experiments

Sanitisation and Disinfection

Sanitisation
cleaning of pathogenic microorganisms
Disinfection
reducing the number of viable microorganisms
present in a sample
Not all disinfectants are capable of sterilizing

Autoclave

Sterilisation
Killing all micro-organisms on the surface of an
object
A surface is either sterile or non-sterile
Common methods include steam, ethylene oxide, rays

Sterilisation
Whether and how you sterilise, sanitise or disinfect
depends on
the organism you are trying to grow
The material of the container it is being grown in
The use of the organism/its product
Media stability

Different Type of Autoclaves

One of the most common ways to sterilise is through

the use of an autoclave.
In our Departments autoclaves steam at 121oC is
used.
Typical sterilisation times are 15-20 minutes
The large autoclave has a number of hazards
associated with it use
Hot
Heavy
Pressurised
Only materials which can withstand 121oC should be
autoclaved.

Procedure for Using Large Autoclave.

Enter your name, the date, time and material to be

autoclaved in the logbook.
Before opening, check that autoclave is not
pressurized, and side steam valve is open.
Undo wing nuts, open lid seek assistance if
necessary as the lid is heavy.
Add de-ionized water to cover the element by at
least 10 cm.
Place flasks and/or bottles in the autoclave. Cotton
bungs should be covered with aluminum foil and
the lids of bottles should be loosened one turn.
Carefully close lid, seek assistance if necessary.

Procedure for Using Large Autoclave.

Procedure for Using Large Autoclave.

Do not open the side steam valve as this will cause the
autoclave to rapidly depressurize resulting in your
media boiling, and spilling in the autoclave.
When the autoclave pressure has reduced to zero,
fully open the side steam valve.
Do not attempt to open the autoclave if it is still
pressurized.
Check that no steam is coming out of the rubber tubing
before loosening the wing nuts. If the wing nuts are too
tight to turn allow the autoclave to cool further and they
will become looser.
Open the lid of the autoclave, seek assistance if
necessary.
Remove items from autoclave.
Record any problems in the logbook.

Colony formation unit (CFU)

1.0 mL

Original
sample

1.0 mL

1/10
dilution

1/100
dilution

1.0 mL

1/1,000
dilution

1.0 mL

1/10,000
dilution

Tighten wing nuts in a cross pattern.

Switch on at wall and then at the front of the
autoclave, red light should come on.
When steam begins to flow through the rubber
hose connected to the side steam valve, close the
side steam valve. Where gloves for this step as the
valve will be hot. (This can happen between 10-30
minutes after starting the autoclave, depending on
initial temperature)
When pressure control valve begins to emit steam,
commence timing. (This typically happens 10-15
minutes after closing the side valve.)
When the required sterilization time has elapsed
turn off the autoclave at the front and at the wall.

Agar Plates

You will all have heard of agar plates

Agar is a gelling material derived from sea weed
Micro-organisms grown on agar plates can not move
Agar plates are used for a number of purposes

Uses of Agar Plates

Streaking to produce single colonies of microbes
from which pure cultures can be obtained.
Dilutions to determine the number of microbes in a
sample.
Identifying the type of microbe
Size, shape, appearance, growth rate
Specific Media

Working under flame

You may have seen Microbiologists working near a
lit bunsen burner.
This is to make sure there work is sterile.
The sterility is not due to the heat of the flame
The sterility is due to the convective flow

Flasks
To grow small volumes of microbes (100mL 1L)
conical flasks are often used.
The media is made up in the flasks and a cotton wool
bung is put in the top.
The cotton bung is covered with foil to stop it getting
wet.
The flasks are then autoclaved, allowed to cool and
inoculated.

Flasks
The cotton wool allows some air to pass in
and out of the flask.
Having a relatively small volume of liquid
and agitation maintains aeration in the
flask.

Summary

Risk management steps for biosafety

Lab Safety Rules
Sterilisation
Agar Plates
Flasks

References
Adapted from Biological safety course presented by
the Risk Management office training
Australian /NZ 2243.3:2002
Risk Management unit at University of Sydney and
the university of New South Wales
http://www.saiglobal.com/online/autologin.asp