Transcript Gram stain

416PHT
Lab#2
Gram’s stain
Acid fast stain
Spore stain
Identification of Bacteria
 Microscopical Examination:
• Examination of wet mount preparation.
• Examination of stained preparation.
 Macroscopical Examination:
• Characters of colonies.
• Hemolysis on blood agar.
• Pigment production.
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Identification of Bacteria
Biochemical Tests.
Additional Tests:
• such as serological tests
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Staining of Bacteria
• Bacteria cells are almost colorless,
and for this reason a staining technique
is often applied to the cells to color them
so that their shape and size can be easily
determined under the microscope.
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Staining of Bacteria
• Types of staining technique:Simple staining
(use of a single stain)
For visualization of
morphological
shape &
arrangement.
Differential staining
(use of two contrasting stain)
Identification
Gram
stain
Visualization
of structure
Acid fast
stain Spore
stain
Capsule
stain
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Staining of Bacteria
• Principle of staining:Dye are generally salts in which one
of the ions is colored.
Example: methylene blue (simple
dye) is the salt of methylene blue
chloride (MBC)
+
MBC
MB + C
Dyes may be either:
Acidic dyes [ -ve]
Basic dyes [ +ve]
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Preparation and Fixation of
Bacteria for Staining
(Preparation of Smear)
• Objective:To kill the microorganism &fix them to the
slide to prevent them from being washed
out during the process of staining.
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Preparing a smear for staining.
(The following procedure is used for all of our staining)
1. Flame (sterilize)
your inoculating
loop/needle before and
after use. Heat from
base to tip. Be sure
to get the entire wire
red hot.
Make sure
that you are
collecting
your hair
.
2. Prepare the smear
a. With solid culture
(agar colony), place a small drop
of distilled water on a clean
slide. Drag the sterile
inoculating needle tip through
the edge of an isolated colony.
Gently spread the mixture into
a circle the size of a quarter.
b. With liquid culture
3.loop
Letofthe
smear
(A
liquid
cultureair
candry
be completely. Do not
apply directly
heat while
because this can lyse
placed
on thedrying
slide and
spread
out.)
the cells
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Smear preparation
S
Fixation
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Simple Staining
• Objective:To show the morphological shapes and
arrangement of bacterial cells.
a) direct staining
b) Indirect staining with acidic dye
(Negative staining)
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Simple Staining
 Materials: Cultures of S. aureus, B. subtilis
 Stain:


Methylene blue stain
Crystal violet
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Simple Staining
• Procedure:-
MB
1-2 min
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Basic Shapes of Bacteria
Cocci
Bacilli
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Arrangements
Cocci
Irregular Clusters
Tetrads
Staphylococci
Micrococci
Chains or Pairs
Streptococci
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Results
Name of stain:
Name of dye:
Shape of cells:
Arrangement of cells:
Color:
Name of m.o:
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Simple Staining
• Name of stain:- Simple
•
•
•
•
•
Stain
Name of dye:Methylene blue
Shape of cells:- bacilli
Arrangement of cells:Chinese letter
Color:- Blue
Name of m.o:-
Coryebacterium
diphtheria
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Simple Staining
•Name of stain:- simple
stain
•Name of stain:-
Methylene blue
•Shape of cells:- cocci
•Arrangement of cells:clusters
•Color:- Blue
•Name of m.o:-
Staphylococcus aureus
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Simple Staining
•Name of stain:- simple
stain
•Name of stain:- Crystal
violet
•Shape of cells:- cocci
•Arrangement of cells:clusters
•Color:- purple
•Name of m.o:-
Staphylococcus aureus
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Indirect staining with acidic
dye (Negative staining)
• The negative stain technique does not
•
•
•
stain the bacteria but stain the
background.
The bacteria will appear clear against a
dark background.
No heat fixation or strong chemicals are
used, so the bacteria less distorted than in
other staining procedure.
Example: Nigrosine are acidic stain
(negatively charged), so the –ve stain
doesn’t stain the bacteria due ionic
repulsion of bacterial cell wall
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Negative staining
Candida albicans
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Negative staining
S. aureus
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Negative staining
B. subtilis
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Bacterial Morphology
Shape
Special arrangement
Spore formation
Capsule formation
Motility
Staining affinity
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Bacterial Shapes
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Bacterial
Arrangement
- Clusters (group).
- Chains.
- Pairs (diploids).
- No special
arrangement.
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Clusters
Chains
Pairs
Gram-Stained Cocci
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Gram-Stained Bacilli
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Bacterial Spores
- Morphological characters of bacterial
spores:
* Shape.
* Position.
* Staining.
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Staining of Bacteria
• Types of staining technique:Simple staining
(use of a single stain)
For visualization of
morphological
shape & arrangement.
Differential staining
(use of two contrasting stain)
Identification
Gram
stain
Visualization
of structure
Acid fast
stain Spore
stain
Capsule
stain
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Smearing out of the sample
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Smear Fixation
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Principle of Differential Stains
* Application of the primary
stain.
* Decolourization.
*Application of the counterstain.
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Gram Staining
“One of the
most common
mistakes is to
decolorize a
smear for too
long a time
period. Even
Gram-positive
cells can lose
the crystal
violet-iodine
complex
during
prolonged
decolorization.
Gm+ve cocci
G-ve bacilli
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Gram Stain
• It is the most
important differential
stain used in
bacteriology because
it classified bacteria
into two major
groups:
a) Gram positive:
Appears violet after
Gram’s stain
b) Gram negative:
Appears red after Gram’s
stain
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Gram’s +ve Bacteria
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Gram’s -ve Bacteria
Gram’s +ve Bacteria
Gram’s -ve Bacteria
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Gram Stain
Gram-positive bacteria
• Have a thick peptidoglycan layer surrounds the
cell.
• The stain gets trapped into this layer and the
bacteria turned purple.
• Retain the color of the primary stain (crystal
violet) after decolorization with alcohol
Gram-negative bacteria
• have a thin peptidoglycan layer that does not
retain crystal violet stain.
• Instead, it has a thick lipid layer which dissolved
easily upon decoulorization with Acetone-Alcohol.
• Therefore, cells will be counterstained with
safranin and turned red.
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Gram Stain
• Materials:-
• Cultures of S.aureus, C.albican, B.subtilis,
E.coli
• Crystal violet (primary stain)
• Gram’s iodine (mordant)
• Acetone-alcohol (decolorizing agent)
• Safranin (counter stain)
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Gram Staining Technique
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Gram Stain [single]
• Procedure:
safranin
CV
iodine
s
30
sec
30-60
sec
10
sec
2 min
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Gram +ve
S.aureus
Gram –ve
E.coli
Step 1: Crystal Violet
Step 2: Gram’s Iodine
Step 3: Decolorization
(Aceton-Alcohol)
Step 4: Safranin Red
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Step 1: Crystal Violet
Step 2: Gram’s Iodine
Step 3: Decolorization
(Aceton-Alcohol)
Step 4: Safranin Red
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Results:
Shape: Cocci
Arrangement: clusters
Colour: Violet
Gram’s reaction: Gram’s +ve
Name of microorganism:
Staphylococcus aureus (S. aureus)
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Results:
Shape: Oval
Arrangement: Single
Colour: Violet
Gram’s reaction: Gram’s +ve
Name of microorganism:
Candida albicans (C. albicans)
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Results:
Shape: Bacilli
Arrangement: Chains
Colour: Violet
Gram’s reaction: Gram +ve
Name of microorganism:
Bacillus subtilis (B. subtilis)
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Results:
Shape: Rods
Arrangement: Single
Colour: red
Gram’s reaction: Gram -ve
Name of microorganism:
Escherichia coli (E. coli)
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Staining of Bacteria
• Types of staining technique:Simple staining
(use of
a single basic stain)
Differential staining
(use of two contrasting stains
separated by a decolorizing agent)
Identification
For visualization of
morphological
shape & arrangement.
Gram
stain
Visualization
of structure
Acid fast
stain Spore
stain
Capsule
stain
Acid Fast Staining
• Mycobacteria have a 3rd type of
cell envelope (rather than the basic
gram- related properties).
• The cell wall of bacteria in this genus
contain → considerable amounts of
lipids → form an extremely
hydrophobic external layer.
Acid Fast Staining
• These organisms are not readily stainable
with ordinary stains.
• Staining of these bacteria needs exposure
to a strong stain e.g., concentrated
carbol fucsin
With application of heat.
• Once they are properly stained, they
resist decolorization by strong
mineral acids or acid-alcohol→ so they are
said to be
Acid-fast.
Acid Fast Staining
AFS is an important diagnostic value in
identifying pathogenic members of
genus Mycobacterium such as M.
tuberculosis and M. leprae.
Acid Fast Staining
Materials:Culture of M. phelei
Acid-fast staining kit:
Conc. carbol fuchsin (primary
dye)
Acid-alcohol (decolorizing
agent)
Methylene blue (counter
stain)
Acid Fast Stain
• Procedure
:-
5 min
Carbol
acid
MB
fuchsin
\\\\
30-60
sec
1 min
Results
Type of Staining: Acid fast stain
Shape: beaded bacilli
Arrangement: Tree shaped
Colour: red
Name of microorganism:
M.phelei
The Spore Stain
• Some bacteria (e.g., Bacillus and
•
•
•
•
Clostridia) form resistant bodies in the cell
known as endospors.
Bacterial spores are highly resistant to
physical & chemical agents (primarily due
to a thick tough spore coat).
They are not easily stained by routine
staining.
Heat is required in spore staining to
promote the penetration of the dye into
the spore.
Once the spores stained they resist
decolorization.
The Spore Stain
Materials :• Culture of B. subtilis
• Spore-staining kit:
•Malachite green (primary stain)
•Safranine (counter stain)
Spore Stain of
Bacillus subtilis
Type of Staining: Spore stain
Shape: bacilli
Arrangement: Chains
Colour of spores: green
Colour of vegetative cells: red
Name of microorganism:
B. subtilis