226 PHT Lab#2 Staining techniques
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Transcript of 226 PHT Lab#2 Staining techniques
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Microscopical Examination:
• Examination of wet mount preparation.
• Examination of stained preparation.
Identification of Bacteria
Macroscopical Examination:
• Characters of colonies.• Hemolysis on blood agar.• Pigment production.
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Biochemical Tests.
Identification of Bacteria
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)
Differential staining (use of two contrasting stain)
For visualization of morphological
shape & arrangement.
Identification Visualization of structure
Gram stain
Acid fast stain Spore
stainCapsule
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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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 to ionic repulsion of bacterial cell wall
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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
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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(A loop of liquid culture can be placed directly on the slide and spread out.)
10
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3. Let the smear air dry completely. Do not apply heat while drying because this can lyse 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 with basic dye: Materials:-
Cultures of Staphylococci, Bacillus Methylene blue stain
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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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ArrangementsCocci
Irregular Clusters Chains or PairsTetrads
Staphylococci Micrococci Streptococci
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Bacterial Arrangement- Clusters (group).
- Chains.
- Pairs (diploids).
- No special arrangement.
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ResultsName of staining technique: Name of dye: Shape of cells:Arrangement of cells: Color:Name of m.o:
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Simple Staining
• Name of stain. tech.:- Simple Stain
• Name of dye:- Methylene blue
• Shape of cells:- bacilli
• Arrangement of cells:- Chinese letter
• Color:- Blue• Name of m.o:-
Coryebacteria
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Simple Staining
• Name of stain. tech. :- simple stain
• Name of stain:- Methylene blue
• Shape of cells:- cocci• Arrangement of cells:-
clusters• Color:- Blue • Name of m.o:-
Staphylococci
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Simple Staining
• Name of stain. tech. :- simple stain
• Name of stain:- Crystal violet
• Shape of cells:- cocci• Arrangement of
cells:- clusters• Color:- purple• Name of m.o:-
Staphylococci
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Negative staining
Candida
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Negative staining
Staphylococci
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Negative staining
Bacillus
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Principle of Differential Stains
* Application of the primary stain.
* Decolourization.
*Application of the counter-stain.
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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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Flaming of Loop
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Smearing out of the sample
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Smear Fixation
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Gra
m S
tain
ing
“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.
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Gram-positive bacteria• Have a thick peptidoglycan layer surrounds
the cell. • The stain gets trapped into this layer and
the bacteria turned violet.• 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.
Gram Stain
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Gram’s +ve Bacteria
Gram’s -ve Bacteria
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Gram Stain• Materials:-
• Cultures of Staphylococci, Candida, Bacillus, gram –ve bacteria
• 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 +veStaphylococci
Gram –ve bacteria
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: CocciArrangement: clustersColour: VioletGram’s reaction: Gram’s +veName of microorganism: Staphylococci
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Results:Shape: OvalArrangement: SingleColour: VioletGram’s reaction: Gram’s +veName of microorganism: Candida
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Results:Shape: BacilliArrangement: ChainsColour: VioletGram’s reaction: Gram +veName of microorganism: Bacillus
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Results:Shape: RodsArrangement: SingleColour: redGram’s reaction: Gram -veName of microorganism: Gram –ve bacteria
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