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Dr. Salma Elamin
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A safety updated manual should be available
in the laboratory, to regulate proper handling
of infectious and hazardous materials, as well
as proper using and handling of equipments.
Emergency procedures should be known to
all laboratory personnel.
Lab-coats should be worn in the laboratory
at all times.
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Learn the location of first aid boxes, and fire
fighting equipments.
To avoid the risk of transmitting infectious
pathogens when working with blood
specimens, precautions must be taken.
Proper hand wash procedure to be followed
before and after any procedure or wearing
gloves:
1. Wet hands and wrists with water
2. Apply soap to the palms from a
dispenser.
3. Rub both hands, wrists, fingernails, and
between fingers, well with soap, for a
minimum of 5 seconds.
4. Rinse well with water and dry.
5. Sinks with foot – operated controls are
ideal, when not available try to use a paper
towel to turn off water so that clean hands
would not be contaminated again.
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When working with a potentially infectious
biological material, procedures to follow:
1. Never mouth- pipette.
2. Handle infectious fluids carefully to avoid
spilling and avoid occurring of aerosols.
3. Avoid using needles and syringes unless
necessary. Dispose of sharps in the
designated container.
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4 Use protective gloves and laboratory.
5. wash hands frequently, after taking of the
gloves, after laboratory activities, and after
coming in contact with an infectious material.
6. Decontaminate surfaces before and after
use. Wipe any spills immediately.
7. Never eat , drink, or smoke in the
laboraty.
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Infectious waste: e.g. Blood, blood products,
sharps, microbiological waste, should be
packed in for disposal in color coded
containers labeled with the universal symbol
for biohazards .
Containers for waste must be easily
accessible and not to be over filled.
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Incubators
Temperature is set as required e.g.:
37˚C
CO² Incubator
Are device for controlling the
temperature, humidity, and other
Cabinet Purpose
In varying degrees, a laminar flow
biological safety cabinet is designed to
provide three basic types of protection.
Personnel protection from harmful
agents inside the cabinet.
Product protection to avoid
contamination of the work,
experiment or process.
Environmental protection from
contaminants contained within
the cabinet.
How Biological Safety Cabinets
Are Classified”
Classification Biosafety Level
ApplicationClass I1,2,3low to
moderate risk biological
agentsClass II1,2,3low to
moderate risk biological
agentsClass III4high risk
biological agents
•Hot Air Oven: Sterilization by Dry Heat
•General Biohazard Sign - equipment
Biohazard sticker/label, that is red or orange in
colour with a biohazard symbol and lettering in
black as illustrated.
It identifies equipment/instrument containing
biological materials: refrigerators, sharps
containers, storage areas, freezers, centrifuges and
wastes containers. DO NOT TOUCH.
Safety Goggles
•Disposable Mask:
•Inoculating Loops and Needles
•Petri Plates or Petri Dishes:
•Compound light microscope:
Forms of Culture Media:
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Examples of simple stains are:
a- Loffler’s methylene blue: the most
valuable reagent available for staining
bacteria.
It is excellent for the genus
Corynebacterium, it can demonstrate
beading, barring, and granules.
With sporing organisms stained with
methylene blue, spores appear as unstained
bodies within the cell.
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To stain a slide with methylene blue:
1. Apply stain for 1 minute.
2. Rinse with water.
3. Drain or blot dry.
Methylene blue staining
demonstrating the typical
morphology of
Corynebacterium
diphtheriae
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The color of the bacteria at the end of
the gram-staining procedure (either purple
or pink) depends on the chemical
composition of the cell wall.
Gram-positive bacteria retain the purple
color of the crystal violet.
In Gram-negative cells, the crystal violet
is removed during the decolorization step
and the cells are subsequently stained pink
by the safranin.
Some strains of bacteria are neither
consistently purple nor pink, following this
procedure. They are referred to as Gramvariable bacteria.
Components of Gram Stain:1. Crystal violet (The primary stain).
2. Iodine (fixative or mordant).
3. Acetone or Alcohol (the decolorizer).
4. Safranin ( the counter stain).
1.The heat-fixed smear is covered with a
solution of the basic dye: crystal violet (a
purple dye) for 1 minute.
2. Rinse gently with water and cover the
smear with Gram’s iodine solution (fixative or
mordant) for 1 minute.
3. Wash off the iodine with water and
decolorize with acetone (Decolorizer) for 1-5
seconds, if the decolorizer is left longer on
the slide it can wash of the primary stain off.
4. The slide is counterstained with safranin
(a bright red dye) for I minute, rinse, dry, and
examine using the oil immersion objective.
Gram
stain of
Lactobacil
lus
Illustrating
species
gram
positive
bacilli, single
and in chains
Gram stain of Escherichia coli
Illustrating short gram
negative bacilli
Endospores are formed by a few groups of
Bacteria as intracellular structures, but
ultimately they are released as free
endospores.
 Biologically: endospores show no signs of
life.
 They are highly resistant to environmental
stresses such as high temperature (some
endospores can be boiled for hours and retain
their viability), irradiation, strong acids,
disinfectants, etc.
 They are probably the most durable cell
produced in nature.
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It is difficult to get a dye into an endospore
because of its low penetrability and high
degree of resistance due to multiple coats
surrounding the spore.
After applying the primary stain, the slide is
heated over a steam bath to soften the hard
outer layer of the cell, and allow the Malchite
green to bind to within the spore.
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This stain cannot be decolorized from the
spore once they are removed from the heat ;
however water can remove the Malachite
Green from the vegitative cells making them
colorless again.
When we apply Safranin as the counter stain,
the vegetative cells take up this stain and
appears red/pink.
Spore Stain Method:
- Flood the slide with 5% aqueous
malachite green and steam for 1 min.
- Wash under running water.
- Counterstain with 0.5% aqueous
safranin for 15 seconds.
- Rinse with water and drain or blot
dry.
Bacterial bodies stain red, spores green.
Capsules are not usually stainable by basic
stains, because of their chemical
characteristics.
 They are best observed by a procedure called
negative staining.
 Two forms of negative staining are used for
capsule detection: a. Uses India Ink and wet
preparation.
b. Uses India Ink followed by smearing and
drying on the slide.
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Procedure:
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India ink wet-film staining method for capsules:
- This is a negative stain: colouring the background so
that the cells are shown as clear objects. Performed as
follows:
1. Place a large loopful of undiluted India ink on a
slide.
2. Mix this with a small portion of the bacterial
colony or a small loopful of the deposit from a centrifuged
liquid culture.
3. Place a coverslip on top and press down under a
pad of blotting paper.
The capsule appears as a clear light zone between the
refractile cell outline and the dark background.
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India ink is composed of fine carbon particles
that are suspended in water and that form a true
colloid(do not settle out of suspension).
These particles are too large to penetrate the
gel-like matrix of the capsule.
When a drop of India Ink is mixed with a drop of
broth culture (when seen by light microscope),
the capsule and cells are seen as transparent
zones of different density, surrounded by a dark
(India Ink) background.
So the capsule and cell are observed indirectly
because they exclude the carbon particles:
Negative Stain.
Bacterial capsules outlined by
India ink viewed by light
microscopy.
This is a true capsule, a
discrete layer of
polysaccharide
surrounding the cells.
Sometimes bacterial cells
are embedded more
randomly in a
polysaccharide matrix
called a slime layer or
biofilm.
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The most obvious characteristic of the
mycobacteria is the large amount of lipid
presents in their cell walls−approximately
40% of the total cell dry weight−causing them
to grow as extremely rough, hydrophobic
colonies.
Mycobacteria are also difficult to stain,
but once stained, they resist
discoloration.
 Organisms with the ability to retain a
stain in spite of washing with acid
alcohol are referred to as acid-fast.
 Only the members of the genus
Mycobacterium and a few species of
Nocardia possess this property and
this characteristic helps in detecting
mycobacteria in body fluids such as
sputum.
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Differential staining procedures have two
components: a differential stain, followed by
a simple stain.
The dyes used in each are different colors, so
that a positive cell will be one color and a
negative cell another.
Acid-fast stain is a differential stain
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To get the dyes into these cells, it is
necessary to employ methods such as heating
the organisms in the stain or including
detergents in the stain.
However, once these cells are stained by
carbolfuchsin (a mixture of phenol and the
dye fuchsin), their cell-wall structure allows
them to retain the stain even when washed
with 95% alcohol containing 3% HCl−hence,
the term acid-fast. All other bacteria are
decolorized by this procedure.
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Ziehl- Neelsen’s method (Acid- fast stain):
1. Flood the slide with strong carbol fuschin
and heat until steam rises (but do not boil).
2. After 3-4 min apply more heat until steam
rises again; do not let the stains dry on the slide.
3.About 5-7min after first application of heat,
wash the slide thouroughly under running water.
4. Decolorize with acid-alcohol until all traces of
red have disappeared from the film.
5. Wash well in water when decolorization is
complete.
6. Counterstain with Loeffler’s methylene blue or
0.5% malachite green for 1 min.
7. Wash and stand on end to drain : DO NOT
BLOT.