Fundamentals of clinical

Microbiology
Lecture 11: Safety in Micro Lab
Martin Kalumbi
 L -- Label everything clearly
 A -- Appropriate containers in good condition
 B -- Be neat and orderly

 S -- Store only what you will use

 A -- Always wear protective clothing
 F -- Food allowed in eating areas only
 E -- Everything in its place on a shelf
 T -- Time to inventory & organize
 Y -- Your safety is important
 Why accidents happen?

Careless Inadequate Inadequate

Safety Training Instructions

Unsafe Over crowding

Experiments

Inadequate Facilities
Inadequate Equipment

Inadequate
Misbehavior Preparation
Ignorance

Poor Teaching
Laboratory Experiences
Management
Before Beginning Any Laboratory Exercise

Know the dangers associated with all materials

you will use in this laboratory exercise
Know the proper way of using all the materials
provided for the exercise
Know all the protective measures you must
use during the exercise
 Personal Protective Equipment
All users of the laboratory must be informed about the types
& use of PPE& how to access it.
Use personal protective equipment to prevent skin/mucous
membrane exposure during agent use, such as:
 Gloves
 Safety glasses/face shield
 Lab coat
 Closed toe shoes/foot covers
 Respiratory protection (BL-3 agents)
Personal Protection

Wear an apron or lab coat in the

proper manner

Wear gloves in the proper manner

Wear a face shield in the proper

manner
Personal Protection

Tie long hair neatly at the back of

the neck

Wear shoes that enclose your

entire feet
Emergency Procedures
Know where all exit doors are located

If a reagent splashes onto your face or eyes,

wash your face at the eyewash station for
several minutes
Emergency Procedures

If an irritating or harmful reagent splashes

onto your body, use the emergency shower to
wash your body for several minutes

Know where the fire extinguisher and fire

blanket are located
 Disinfectant Selection: Assignment

Disinfectants must be selected on a case by case basis to

ensure efficiency.
 Quaternary Ammonia Compounds: Brian
 Chloride Compounds (Bleach): Munthali
 Iodophores (Wescodyne): Silence madeya
 Phenolics (Amphyl)): Makondetsa
 Alcohols (70% Ethanol): Micheal
 Formaldehyde/Glutaraldehyde: Innocent
Describe the disinfectants, composition & mode of action?
 Biological Spill Kits
• Location
• Inside Tissue Culture Rooms
• Contents
 Gloves, Goggles/shield, N95 Respirator, Lab coat/gown
 Absorbent material
 Bleach or suitable disinfectant
 Tongs and dust pan
• Spill Procedures
• Place absorbent material over spill
• Pour disinfectant around outer edges of absorbent ending in the
middle
• Wait for inactivation of bio-hazardous material
• Properly dispose of all materials in biohazard box
Hazard Communication
Biohazard labels shall be placed on:
 the surface of all equipment (freezers, incubators,
refrigerators) which may be contaminated with
biohazardous materials.
 sample transport outer containers.
 medical waste bins

Biohazard signs shall be placed on:

 the outer door of BL 2 labs.
 medical waste storage areas
 CLASSIFICATION OF MICROORGANISMS
BASED ON RISK GROUP
 Infectious or pathogenic agents are those organisms capable of
causing disease in healthy humans, plants and animals.

These organisms are termed microorganisms bcoz they cant be seen

unaided

Based on the risks they pose on individuals / lab personnel or the

community they are grouped into four.
RISK GROUP I
These are organisms that present low risk to an individual or lab
worker and community
Unlikely to cause human disease

Not known to consistently cause disease in healthy human adults

CDC. Examples:
 E.Coli K12
 Saccharomyces cerevisae
 Adeno-associated virus
 Bacillus subtilus
 Bacillus sphericus
 -Lactobacillus spp.
Risk Group II
These organisms present moderate risk to an individual (lab worker) &
low risk to community
A pathogen that can cause human disease but is unlikely to be serious
hazard to laboratory workers & the community.

Laboratory exposures may cause serious infection, but effective

treatment and preventative measures are available & risk of spread of
infection is limited.

Biological safety cabinet needed Pathogens spread via ingestion,

inoculation and mucous membrane routes
Risk Group II
Examples:
 All 8 human herpesviruses - HSV,VSV, EBV, CMV, HHV6,7 & KSHV
 Adenovirus (all 50+ human serotypes)
 Cornybacteria diphtheria
 Neisseria meningitidis
 Stapylococcus ureus (including MRSA)
 Ringworm
 -Influenza virus
 -Herpes simplex
 -Hepatitis (A, B, C, D, E)
 -Tetanus
Risk Group III
Present high risk to an individual or lab worker but low risk to
community

A pathogen that usually causes serious human disease but does not
ordinarily spread from one infected individual to another, directly or
indirectly.
They do not ordinarily spread rapidly from one individual to another.

Effective treatment and preventative measures are

Risk Group III
Examples:
 Vibrio cholera (Senegal 05/05)
 Herpes B virus (upgraded to level 4 in some lists)
 HCV, HBV, HIV E coli O 157
 Shigella flexnerii
 Mycobacterium tuberculosis
 Hepatitis (some C’s)
 -Anthrax
 -TB
Risk Group IV
Organisms from this group present high risk to individual or lab
worker and community

A pathogen that usually causes serious human disease that can be

readily transmitted from one individual to another, directly or
indirectly.

Effective treatment and preventative measures are not usually

available.
Risk Group IV
Examples:
 Hemorrhagic fever viruses e.g. Rift Valley Fever (264 cases and 109
deaths in Tanzania 05/07)
 Marburg (423 cases and 357 deaths in Angola 06/05)
 Ebola (69 cases and 31 deaths in Republic of Congo 09/12)
 Yellow Fever (849 cases and 171 deaths, Dafur 10-12/12) actually
 RG3 with RG2 for vaccine strain 17D.
 Smallpox
 Influenza “1918” strain?
 Herpes B
 Examples of microorganisms by risk
group
Bacteria, Chlamydiae, Rickettsiae & Micoplasmas – Mostly risk group 2 &
some risk group 3

Parasites – Risk group 2

Fungi – Risk groups 2 & 3

Viruses – Risk groups 2, 3 & 4

Prions – Risk group 2

Containment levels
There are four physical containment levels or Biosafety levels
Assignment of an agent to a biosafety level for laboratory work must
be based on a risk assessment.
 Microbiological agents are typically, but not always, handled in
containment facilities at the same level as their risk group
 Containment Level 1 (CL1)
 Containment Level 2 (CL2)
 Containment Level 3 (CL3)
 Containment Level 4 (CL4)
Containment Level 1 (CL1)
This refers to the basic laboratory that handles low risk agents
CL1 does not require any special design features beyond those
suitable for a well-designed and functional laboratory

Work may be done on open bench tops

Containment is achieved by following basic microbiology laboratory

procedures / practices
Containment Level 2 (CL2)

CL2 labs will use ‘primary containment’ procedures, such as using

appropriate personal protective equipment (i.e., gloves, lab coats,
protective eyewear) and using containment devices such as Biological
Safety Cabinets (BSC’s)
Containment Level 3 (CL3)
CL3 labs use ‘primary and secondary’ containment barriers to
minimize the release of infectious organisms.

These secondary measures include using appropriate respiratory

protection, HEPA filtration of exhausted lab air and strictly controlled
laboratory access.
Containment Level 4 (CL4)
CL4 labs are isolated units, structurally independent of other areas.

Maximum containment of the agent is achieved by complete sealing

of the facility perimeter with confirmation by pressure decay testing

Isolation of the researcher from the agent by using a positive

pressure suit or the use of a Class III Biological Safety Cabinet.
CLASSIFICATION OF BIOSAFETY CABINETS
Safety cabinets are intended to protect a laboratory worker from
aerosols and airborne particles.

They will not protect the person from spillages and the consequences
of mishandling and poor technique.

There are three kinds of safety cabinet, Classes I, II, and III.
Class 1 Safety Cabinet
It has a front opening.
The operator sits at the cabinet, looks through the glass screen, and
works with the hands inside.
Any aerosols released from cultures or other infectious material are
retained because a current of air passes in at the front of the cabinet &
sweeps aerosols up through a HEPA filter which removes all or most of
the organisms.
Clean air then passes through the fan, which maintains the air flow, and
is exhausted (discharged) to atmosphere where any particles or
organisms that have not been retained on the filter are so diluted that
they are no longer likely to cause infection if inhaled.
Class II safety cabinet
In a Class II cabinet about 70% of the air is recirculated through filters
so that the working area is bathed in clean (almost sterile) air.
The air flow carries along any aerosols produced in the course of the
work and these are removed by the filters.
Some of the air (about 30%) is exhausted to atmosphere and is
replaced by a ‘curtain’ of room air which enters at the working face.
This prevents the escape of any particles or aerosols released in the
cabinet.
 Class III safety cabinet
 Class III safety cabinet for Risk Group 4
viruses
 This type of cabinet is totally enclosed
and is tested under pressure to ensure
that no particles can leak from it into
the room.
 The operator works with gloves which
form part of the cabinet.
 Air enters through a filter and is
exhausted to atmosphere through one
or two more filters.
 Waste
Disposal
 Universal Precautions

Treat ALL human blood and unfixed

tissue as if it contains HIV and HBV
(INFECTIOUS)
 Standard Microbiological Practices
NOT permitted in laboratories:
 Eating
 Drinking
 Smoking
 Handling contact lenses
 Pipetting by mouth
 Storing food and drink
Standard Microbiological Practices
ALWAYS wash hands:

 After handling microorganisms and animals

 After removing gloves

 Before leaving laboratory

Standard Microbiological Practices
 Discard needles, razor blades, and scalpel blades
into red, puncture resistant sharps containers

 Dispose of broken glass into “broken glass”

containers, never regular trash
Standard Microbiological Practices
NEVER
 recap, bend, or break needles
 discard needles or sharps into biological waste
bags
 discard needles into regular trash
Standard Microbiological Practices
Decontaminate all biological waste (including BL1)
before disposal

 Solid waste (Petri dishes, cultures): autoclave and put

in dumpster

 Liquid waste: add disinfectant (bleach to 10%) and

pour down drain
How to do a Risk Assessment?
1. Determine hazards and evaluate
risks
2. Use all relevant available data
3. Determine controls needed to
minimise those risks
4. Document the assessment
5. Agree it with your supervisor
6. Use those control measures
Before You Leave The Laboratory
 Clean your workbench with
disinfectant at the beginning
and at the end of the
laboratory exercise
 Leave all equipment, samples
and reagents in the lab
 Leave your lab coat (apron) in
the lab
 Wash your hands with soap
and water
 THE END
ANY QUESTION???