Safety and Laboratory Guidelines

MICROORGANISMS
•belong to the Protista biologic kingdom

•include some eukariotes and prokaryotes, viruses

and prions
•are classified according to their:
structure
chemical composition,
biosyntetic organization
genetic organization
 In microbiology- study those
organisms that can only be seen
with a microscope
 Bacteria:
• unicellular organisms whose genetic material is not
enclosed inside a nucleus

•are called prokaryotes, from the Greek words meaning

prenucleus

•cell wall- is largely composed of a molecule called

peptidoglycan

•bacterial shapes: the bacillus – or rodlike-cell, the

coccus – or spherical-cell, and the spiral, which is a
corkscrew-shaped or curved cell

•bacterial disposal : pairs, chains

•reproduce by binary fission, with one cell dividing into

two equal cells.
•nutrition: some use organic chemicals and others use
photosynthesis or inorganic substances
 PROCARYOTIC CELLS EUCARYOTIC CELLS
Bacteria Fungi, Plants , Protozoans , Animals
(including Humans)

Size : 1-10m Size : 10-100m

No nuclear membrane Nuclear membrane
Single chromosome Multiple chromosomes
No DNA-associated histones Histones associated with DNA
Binary fission Mitotic division
Lack membranous compartments Have membranous compartments
Peptidoglycan cell wall(except for Chitin or cellulose wall( when present)
mycoplasmas)
Steroids absents (except in Steroids may be present
Mycoplasma membranes)
70S ribosomes 80S ribosomes
Anaerobic respiration possible No anaerobic respiration
No tissue differentiation Ussualy differentiated in tissues
Nitrogen fixation possible Nitogen fixation not possible
Bacterial shapes
The nature of bacterial interaction with humans
•Commensal organisms
•routinely colonize body surfaces without doing harm
•often referred to as normal microbial flora (Escherichia coli)

•Pathogens
•damage the human host
•by direct invasion and injury (Shigella sp)
• by the production of harmful toxic products(Clostridium sp)

•Opportunistic organisms
•usually found in the environment or as part of the normal flora.
•in normal individuals , they are harmless
•they may cause severe disease in imunocompromise patients or if they
penetrate a territory from which they are ussualy excluded (as a result of
trauma or surgery).

•Zoonotic organisms
• usually cause disease in vertebrates other than humans
• may be acquired through contact with infected animals or animal products
 Microbiology laboratory

• intersting, exciting experience

• you sould be aware of some potential hazards
• improper handling of chemicals, equipment and microbial cultures
– is dangerous; can result in injury or infection

Microorganisms present varying degree of risk to :

• laboratory personnel (students, technicians , faculty )
• people outside the laboratory
• the environment
 Student conduct
to reduce the risk of infection:

• do not smoke, eat, drink, bring food or drinks into the laboratory
room-even if lab work is not being done at the time
• do not apply cosmetics or handle contact lenses in the laboratory
• wash your hands thoroughly with soap and water after handling
living microbes and before leaving the laboratory each day
• wash your hands after removing gloves
• come to lab prepared for that day’s work
• do not remove any organisms or chemicals from the laboratory
• work carefully and methodically
• do not hurry through any laboratory procedure
 Basic Laboratory Safety
• Wear protective clothing (lab. coat) in the laboratory when
handling microbes
• Remove the coat prior to leaving the lab
• Do not wear sandals or open –toed shoes in the laboratory
• Wear eye protection whenever you are heating chemicals , even
if you wear glasses or contacts
• Turn off your Bunsen burner when it is not in use
• Tie back long hair, as it is a potential source of contamination as
well as a likely target for fire
• Not all procedures require gloves and eye protection
• If you are feeling ill, go home
• Wear disposable gloves while staining microbes
• Use an antiseptic on your skin if it is exposed to a spill
containing microorganisms
• Never pipette by mouth; always use mechanical pipettors
Reducing Contamination of self, others, cultures
and the environment
• Place all nonessential books and papers under the desk
• Never lay down culture tubes on the table ; they always should remain
upright in a tube holder
• When pipetting microbial cultures, place a disinfectant-soaked towel
on the work area
 microbial cultures must be handled safely
Classification of microbes into 4 biosafety levels (BSL)- provides a

Classification of microbes into 4 biosafety levels (BSL)- provides

a set of minimum standards for laboratory practices

BSL -1
• Agents: not know to consistently cause disease in healthy
individuals, present a minimal threat to the environment and lab
perssonel
• Practices:
• Primary barriers: microbes may be handled in the open, no special
containment equipment is required
• Secondary barriers: open benchtop sink required

CLASS I BIOLOGICAL SAFETY

CABINET
(Used in conjunction with the
building system. Glove ports are
optional.)
CLASS I BIOLOGICAL SAFETY CABINET
(Complete with internal motor/blower assembly. HEPA filtered
exhaust air vented to the atmosphere. Glove ports are optional.)
BSL-2
Agents : associated with human disease, hazard is through
percutaneous injury, ingestion, exposure to mucous membrane, present
a moderate environmental or health hazard
Practices:
Primary barriers: class I/ II BSCs , physical containment devices used
for all manipulations of the agents that cause splashes or aerosols of
infectious materials
PPS: Personal Protective Equipment: laboratory coats, gloves, face
protection
Secondary barriers: BSL1 Plus: autoclave available

CLASS II TYPE A1 BIOLOGICAL

SAFETY CABINET
(Can be room re-circulated or air
gap type (thimble)
CLASS II TYPE A2 BIOLOGICAL SAFETY CABINET
(Can be room re-circulated or air gap type (thimble).
 CLASS II TYPE B1 BIOLOGICAL SAFETY CABINET
(Hard-ducted.)
BSL-3
• Agents: are local or exotic origin, are associated with
respiratory transmission and serious or lethal diseases
• Practices:
• Primary barriers: class I/ II BSCs, physical containment devices
used for all open manipulations of the agents; PPS + respiratory
protection
• Secondary barriers: BSL-2 plus: physical separation from acces
corridors; access to self closing, double door, exhausted air not
recirculated, negative airflow into laboratory

BSL-4
• Agents: have a great potential for lethal infection
• Primary barriers: class III BSC
• Secondary barriers: BSL-3 plus: separate building or isolate
zona, dedicated supply and exhaust, vacuum, and decon systems,
other requirements
CLASS III BIOLOGICAL SAFETY CABINET
(Hard-ducted.)
 Lab tools- collection, inoculation

Graduated pipettes Collection Swabs Steel Forceps

Inoculation Loop

Disposable polystyrene
Standard sized sterile Petri dishes
used for culturing and examination
Specimen Container of microorganisms.
 
 Practices Goal
LABORATORY DIAGNOSIS OF BACTERIAL INFECTIONS
In order to properly execute antibacterial therapy, it is necessary to
definitively identify the causative organism.
•Specimen collection
•Microscopic examination of patient specimens, glas slides:
staining –common staining include Gram
stain, acid fast stain, iodine stain, Giemsa stain
- fluorescent dyes
darkfield examination
direct immunofluorescence
•Isolation of microorganisms in bacterial pure culture
•Identification by growth characteristics on media and the results of
biochemical tests.

•Antimicrobial susceptibility testing

SEROLOGIC TESTING
In general, the diagnosis of an infectious disease based on the detection of
specific antibody by serological tests.
STERILITY- the total absence of viable microorganisms as assessed by
no growth on any medium

STERILIZATION
• the process of killing or removing of
all the forms of microbial life (including
bacterial spores, fungi and
nonenveloped viruses)
•can be accomplished using physical,
chemical and gas vapor sterilants

-
DISINFECTION
the process of reducing the bacteria
number or inhibiting the growth of
bacteria, to the point where they don't
pose a threat of disease
 Sepsis
infection
Bactericidal
Kills bacteria

Antisepsis
any procedure that inhibits
the growth and multiplication
of microorganisms

Bacteriostatic
inhibits growth of
Antiseptics bacteria
are applied topically to human
tissue, inhibit the growth of
microorganims / they kill them,
without sporicidal action
cannot be taken internally
 Study case

Simptoms:
•cough
66 year old man, • fever
homeless, drinking •night sweats
2 quarts of vodka •chest pain,
per day • in the last several days he
had produced abundant, thick,
tenacious, blood –tinger
(currant –jelly) sputum

History: he was cough free a month ago, his

cough had become progressively worse since
then
Imaging
A chest x-ray revealed right lobe infiltrate with cavitary lesion

A clinical diagnosis of pneumonia should be considered

Diagnostic work-up
Blood cultures
Sputum Acid –fast and Gram stain examination + culture

Gram stain – PMN + Gram negative rods ,

Differential diagnosis
• Haemophilus influenzae

•Klebsiella pneumoniae- one of the more common cause of pneumonia

in the homeless

•Legionella pneumophila

•Staphylococcus aureus

•Streptococcus pneumoniae

•Homelessness should prompt ruling out tuberculosis ( the presence

of cavitary lesions)