Practical Microbiology

Lab Safety
Hazards of Clinical Laboratory Work
Clinical laboratory personnel and employees are
subjected to the risk of the followings:
1. Biological hazards (infection).
2. Chemical hazards.
3. Physical hazards.

Such risks can be prevented or minimized by:-

1.laboratory safety program.
2.Aseptic technique.
 Lab safety
laboratory Classification and Biosafety

Group 1 : agents (bacteria , viruses, and

parasites that are unlikely to cause disease in
healthy workers

.
 Lab safety
Group 2 agents (those that produce moderate
individual risk and limited community risk),
Example : microbiology laboratory Teaching medical
microbiology and using pathogenic microorganisms.
 Lab safety

Group 3: Containment level 3

usually is recommended for work
with cultures of M. tuberculosis
rickettsiae, brucellae, Y pestis, and a
wide variety of viruses, including
human immunodeficiency viruses.
 Lab Safety

Group 4 Agents
is found in only a few reference and
research laboratories.
a few viruses (including Lassa,
Ebola, and Marburg viruses) are classified
in biosafety level 4.
What needs to be sterilized?
1. Culture media
2. Fluids used in labs
3. Reagents
4. Laboratory containers
5. Laboratory equipments
6. Syringes
7. Complete operating theater
8. Nursing equipments
9. Industrial equipments
 Nursing equipment Lab reagent &containers

Operating theater
Canned food
Types of sterilization
The methods of sterilization chosen depend on:-
• Object to be sterilized.
• Number and kind of microorganisms that
could be present.
A- Physical methods:
B- Chemical methods:
 Physical methods
1- Sun light
2- Heat
3- Radiation
4- Filtration
 Physical methods

Heat :-
We either use dry heat or moist heat.
• A- Dry heat :- Kills by oxidation, protein denaturation & toxic effect of elevated
levels of electrolyte
• Types of processes
1- Red hot
2-Flaming
3-Incineration
4-Hot air oven
- Red heat-, e.g. inoculating loops, needles, forceps
are sterilized in flame until they become red hot
Dry heat
Flaming :-is used to sterile, glass slides, mouths of
test tubes
 Moist heat
• Moist heat Lethal effect due to denaturation & coagulation of
proteins
– Temp below 1000C (Pasteurization)
– Temp at 1000C (Boiling)
– Temp above 1000C (Autoclave)
 Temperature above 100°C.(Autoclave)
• . In this apparatus material for
sterilization is exposed to
121°C for 15-20 minutes at 15
Ib pressure per square inch.
 Radiation

Ionizing radiation- U.V. light-

 Filtration:-
. Used to sterilize heat sensitive materials like
vaccines, enzymes, antibiotics, serum, sugars
and toxins
 Chemical methods
• Chemical methods:
1. Alcohols
2. Aldehydes
3. Gases
4- Halogens
5- Phenols
6- Salts
7- Dyes
 Culture media
A microbiological medium is the food that we use for
culturing bacteria, molds, and other microorganisms
The media are contained either in test tubes, plates (Petri
Dishes, flasks or screw capped bottles etc…..,
 Advantages of culture media
1- to identify the cause of infection from the clinical sample, so that proper
treatment can be given.

2- to study the characteristics or properties of microorganisms.

3- to prepare biological products like vaccines,, antigens…etc.

4- For storage of stock cultures

5- As transport media to preserve bacteria during transportation to the laboratory

 Types of Culture Media
A- According to physical consistencies

culture media

Liquid media -1 Solid media -2 Semi solid media -3

Broth Agar Gelatin

B- According to the purpose of application

Simple-1
media

Transport -5 Differential -2
media media
Culture media

Enriched -4 Selective -3
media media
 Basic media

Nutrient broth Nutrient agar

 Enriched media

Blood agar Chocolate agar

MacConkey Agar

left: no lactose fermentation

right: lactose fermentation
Mannitol salt agar inoculated with Staphylococcus auraes and
Staphylococcus epidermidis
Anaerobic media
• These media are used to grow anaerobic organisms.
• Eg: Robertson’s cooked meat medium, Thioglycolate
medium.
Thioglycolate
medium
Robertsons cooked medium
Transport media e.g. Stuarts transport medium
Anaerobic, aerobic, and pediatric blood
culture bottles
 Antibiotic Sensitivity Test

Kirby-Bauer Test (disc diffusion test)

Coagulase test
Catalase test
API 20E test
Macro examination of Urine sample
 Cultivation of
microorganisms
 Method of culture

Two methods are widely used for the preparation of pure

cultures from laboratory specimens containing mixed
:microorganisms
.A. Streak plate method (Quadrant method) •
.B. Pour plate method •
 1

1
2
4

3
4
Streak pattern
3

Bacterial growth pattern

All labeling is done on the bottom of the agar plate •

1. Initials
2. Date (mm/dd/yy)
3. Code # or letter
Streak method
Physical condition required for growth
Oxygen

- Obligate Aerobic like Bacillus.

- Obligate Anaerobic like Clostridium.
- Microaerophilic Like Helicobacter pylori
- Facultative anaerobic like Salmonella,.
- CO2 like Neisseria gonorrhoeae
Physical condition required for growth
Oxygen
 Preparation of Smear
From Liquid medium

1 2
Loop sterilization Thin smear

3 4
Dry Fixation
 Preparation of smear from solid medium

1 2
Drop of water Loop Sterilization

3 4
mix colony with water Thin smear
5 6
Dry Fixation

Aim of smear fixation

1- Kill the m.o
2- Make the m.o stuck to the surface of the slide.
3- Make the m.o permeable to the stain.
4- prevent the m.o from going autolytic change
Simple stain : all the bacterial cells on the slide take the color of
dye.

Blue color (methylene blue) Red color (safranin)

Special stain: flagella staining (carbolfuchsin and tannic acid)
Special stain: negative staining for capsule with India ink
Special stain: endospore staining (malachite green)
Arrangements of the bacteria microscopically

Cocci

Clusters
Tetrads Chains

Staphylococci Micrococci Streptococci

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 1- Gram stain
Gram stain: bacteria fall into two fundamentally different
categories:
Gram positive bacteria appear in violet color.
Gram negative bacteria appear in red color
Gram stain: can be used to determine the relative number of
bacteria and the morphological features of bacteria which include:
Staining type: whether Gram negative or Gram positive.
Arrangement: whether in pairs, tetrads, chains or clusters.
Shape: cocci, bacilli , vibrio, coccobacilli.
 Gram staining procedure

1-Crystal violet 2- iodine 3- Alcohole 4- Sfranin

Direct Gram stain Secondary Gram stain

Bacillus anthracis
Gram +bacilli in chains
Direct Gram stain Secondary Gram stain

Staphylococcus spp
Gram +cocci in pairs, tetrads & clusters
Direct Gram stain Secondary Gram stain

Neisseria spp.
Gram –ve , diplococci
Direct Gram stain Secondary Gram stain

Escherichia coli

Escherichia coli
Gram –ve single bacilli
Gram stain of a mixture of Staphylococcus aureus and
Escherichia coli
SPECIMEN COLLECTION
 Serum
Collection
Venous blood in sterile tube
• let clot for 30 minutes at ambient
temperature (37°C)
• glass better than plastic
 Throat swab
 Are performed when tonsillitis, sore throat, &diphtheria
is suspected.
Swabs for Bacterial (red) and Viral (green)
Cultures
 Nasal Cultures
• Virus
– Use wire swab
– Place in nose 1-3 cm, rotate, 10-15 sec
– Obtain viral transport medium from lab
• Bacterial
– Cultrate with rigid or wire swab
 Ear Swab
• Take a swab from ear pus by inserting a swab in ear, be careful
not touch a skin.
• Rarely, middle ear fluid my be aspirated through the ear drum
by tympanocentesis.
Eye Swab
Gently clean excess debris from the outside of the eye with a gauze
pad moistened with sterile normal saline solution.
 Retract the lower eyelid and gently rub a sterile swab over the
conjunctiva. Sample each eye with separate swabs.
.
 Urine

 Are performed when UTI (Urinary Tract Infection)

is suspected.
Macro examination of Urine sample
Micro examination of Urine
Stool Sample
 Sputum
Sputum collected when pneumonia, tuberculosis, or
lung abscess is suspected.

Be sure that the specimen is sputum not saliva.

 Wound and Abscess (pus)

 Pus can be collected

in a syringe or it may
be taken by swab
from surface of the
lesion.