226pht Lab#2 Gram staining. 2 Microscopical Examination: Examination of wet mount preparation....

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226phtLab#2

Gram staining

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Microscopical Microscopical Examination:Examination:

• Examination of wet mount Examination of wet mount preparation.preparation.

• Examination of stained preparation.Examination of stained preparation.

Identification of BacteriaIdentification of Bacteria

Macroscopical Macroscopical Examination:Examination:

• Characters of colonies.Characters of colonies.• Hemolysis on blood agar.Hemolysis on blood agar.• Pigment production.Pigment production.

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Biochemical Tests.Biochemical Tests.

Identification of BacteriaIdentification of Bacteria

Additional Tests:Additional Tests:• such as serological testssuch as serological tests

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Staining of BacteriaStaining of Bacteria

• Bacteria cells are almost Bacteria cells are almost colorlesscolorless, and for this reason , and for this reason a a staining techniquestaining technique is often applied is often applied to the cells to color them so that to the cells to color them so that their shape and size can be easily their shape and size can be easily determined under the microscope. determined under the microscope.

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Staining of BacteriaStaining of Bacteria

•Types of staining technique:-Types of staining technique:-

Simple stainingSimple staining (use of a single stain)(use of a single stain)

Differential stainingDifferential staining (use of two contrasting stain)(use of two contrasting stain)

For visualization of For visualization of morphologicalmorphological

shape & shape & arrangement.arrangement.

IdentificationIdentification Visualization Visualization of structureof structure

Gram Gram stainstain

Acid fastAcid fast stainstain Spore Spore

stainstainCapsuleCapsule

stainstain

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StainingStaining ofof BacteriaBacteria• Principle of staining:-Principle of staining:- Dye are generally salts in which Dye are generally salts in which

one of the ions is colored.one of the ions is colored.Example: methylene blue Example: methylene blue

(simple dye) is the salt of (simple dye) is the salt of methylene blue chloride (MBC)methylene blue chloride (MBC)

MBC MB + MBC MB + CC

Dyes may be either:Dyes may be either:Acidic dyes [ -ve]Acidic dyes [ -ve]Basic dyes [ +ve]Basic dyes [ +ve]

++ --

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Preparation and Fixation of Preparation and Fixation of Bacteria for StainingBacteria for Staining(Preparation of Smear)(Preparation of Smear)

• Objective:-Objective:-

To kill the microorganism &fix them to To kill the microorganism &fix them to the slide to prevent them from being the slide to prevent them from being washed out during the process of washed out during the process of staining.staining.

Preparing a smear for staining.Preparing a smear for staining.

(The following procedure is used for all of our (The following procedure is used for all of our staining)staining)

1. Flame (sterilize) 1. Flame (sterilize) your inoculating your inoculating loop/needle before loop/needle before andand after use. Heat after use. Heat from base to tip. Be from base to tip. Be sure to get the sure to get the entire wire red hot.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(A loop of liquid culture can be

placed directly on the slide and spread out.)

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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 preparationSmear preparation

SS Fixation Fixation

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Simple StainingSimple Staining

• Objective:-Objective:-

To show the morphological shapes and To show the morphological shapes and arrangement of bacterial cells.arrangement of bacterial cells.

a)a)Direct staining with basic dye:Direct staining with basic dye: Materials:-Materials:-

Cultures of S. aureus, B. subtilisCultures of S. aureus, B. subtilis Methylene blue stainMethylene blue stain

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Simple StainingSimple Staining• Procedure:-Procedure:-

MBMB

1-2 min1-2 min

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Basic Shapes of Bacteria Basic Shapes of Bacteria

CocciCocci BacilliBacilli

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ArrangementsArrangements

CocciCocci

Irregular ClustersIrregular Clusters Chains or PairsChains or PairsTetradsTetrads

StaphylococciStaphylococci MicrococciMicrococci StreptococciStreptococci

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ResultsResults

Name of stain:Name of stain: Name of dye:Name of dye: Shape of cells:Shape of cells:Arrangement of cells: Arrangement of cells: Color:Color:Name of m.o:Name of m.o:

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Simple StainingSimple 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 StainingSimple Staining

•Name of stain:- Name of stain:- simple simple stainstain•Name of stain:- Name of stain:- Methylene blueMethylene blue•Shape of cells:- Shape of cells:- coccicocci•Arrangement of cells:- Arrangement of cells:- clustersclusters•Color:- Color:- BlueBlue •Name of m.o:- Name of m.o:- Staphylococcus aureusStaphylococcus aureus

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Simple StainingSimple Staining

•Name of stain:- Name of stain:- simple stainsimple stain•Name of stain:- Name of stain:- Crystal violetCrystal violet•Shape of cells:- Shape of cells:- coccicocci•Arrangement of Arrangement of cells:- cells:- clustersclusters•Color:- Color:- purplepurple•Name of m.o:- Name of m.o:- Staphylococcus aureusStaphylococcus aureus

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Indirect staining with acidic Indirect staining with acidic dye (Negative staining)dye (Negative staining)• The negative stain technique does The negative stain technique does

not stain the bacteria but stain the not stain the bacteria but stain the background.background.

• The bacteria will appear clear against The bacteria will appear clear against a dark background.a dark background.

• No heat fixation or strong chemicals No heat fixation or strong chemicals are used, so the bacteria less are used, so the bacteria less distorted than in other staining distorted than in other staining procedure.procedure.

• Example:Example: NigrosineNigrosine are acidic stain are acidic stain (negatively charged), so the –ve stain (negatively charged), so the –ve stain doesn’t stain the bacteria due ionic doesn’t stain the bacteria due ionic repulsion of bacterial cell wallrepulsion of bacterial cell wall

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Negative stainingNegative staining

Candida albicansCandida albicans

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Negative stainingNegative staining

S. aureusS. aureus

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Negative stainingNegative staining

B. subtilisB. subtilis

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Bacterial MorphologyBacterial Morphology

SShape hape

SSpecial arrangementpecial arrangement

SSpore formationpore formation

CCapsule formationapsule formation

MMotilityotility

SStaining affinitytaining affinity

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Bacterial Shapes Bacterial Shapes

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Bacterial ShapesBacterial Shapes

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Bacterial Bacterial

ArrangementArrangement

- Clusters (group).Clusters (group).

- Chains.Chains.

- Pairs (diploids). Pairs (diploids).

- No special No special arrangement. arrangement.

2727Gram-Stained CocciGram-Stained Cocci

Clusters

Chains

Pairs

2828Gram-Stained BacilliGram-Stained Bacilli

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Bacterial SporesBacterial Spores

- Morphological characters of Morphological characters of bacterial spores:bacterial spores:

* Shape.* Shape.

* Position.* Position.

* Staining.* Staining.

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Bacterial SporesBacterial Spores

Unstained Spores

Spore Stain

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Bacterial CapsuleBacterial Capsule

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Flaming of LoopFlaming of Loop

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StainingStaining

Types of Stains:Types of Stains:

1- 1- Simple stains:Simple stains: e.g. Methylene blue e.g. Methylene blue stain.stain.

2- 2- Differential stains:Differential stains: e.g. Gram stain e.g. Gram stain & Ziehl-Neelsen stain (acid fast & Ziehl-Neelsen stain (acid fast stain). stain).

3- 3- Special stains:Special stains: e.g. Fontana stain, e.g. Fontana stain, auramine O stain or seeing a auramine O stain or seeing a structure. structure.

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Staining of BacteriaStaining of Bacteria

•Types of staining technique:-Types of staining technique:-

Simple stainingSimple staining (use of a single stain)(use of a single stain)

Differential stainingDifferential staining (use of two contrasting stain)(use of two contrasting stain)

For visualization of For visualization of morphologicalmorphological

shape & arrangement.shape & arrangement.

IdentificationIdentification Visualization Visualization of structureof structure

Gram Gram stainstain

Acid fastAcid fast stainstain Spore Spore

stainstainCapsuleCapsule

stainstain

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Smearing out of the Smearing out of the samplesample

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Smear FixationSmear Fixation

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Principle of Differential StainsPrinciple of Differential Stains

* Application of the primary * Application of the primary stain.stain.

* Decolourization.* Decolourization.

*Application of the counter-*Application of the counter-stain.stain.

Gra

m S

tain

ing

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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G-ve bacilliG-ve bacilli

Gm+ve cocci

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Gram StainGram Stain• It is the most It is the most

important important differential stain differential stain used in bacteriology used in bacteriology because it classified because it classified bacteria into two bacteria into two major groups:major groups:

a)a) Gram Gram positive:positive:

Appears violet after Gram’s stain

b)b) Gram Gram negative:negative:

Appears red after Gram’s stain

4141Gram’s +ve Bacteria

Gram’s -ve Bacteria

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Gram’s +ve Bacteria

Gram’s -ve Bacteria

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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 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.

Gram StainGram Stain

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Gram StainGram Stain

• Materials:-Materials:- •Cultures of Cultures of S.aureus, C.albican, S.aureus, C.albican,

B.subtilis, E.coli B.subtilis, E.coli

•Crystal violet (primary stain)Crystal violet (primary stain)

•Gram’s iodine (mordant)Gram’s iodine (mordant)

•Acetone-alcohol (decolorizing agent)Acetone-alcohol (decolorizing agent)

•Safranin (counter stain) Safranin (counter stain)

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Gram Staining Gram Staining TechniqueTechnique

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Gram Stain Gram Stain [single][single]

• Procedure:Procedure:

ss

CVCViodineiodine

30-60 sec30-60 sec2 min2 min10 sec10 sec

safraninsafranin

30 sec30 sec

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Gram +veGram +veS.aureusS.aureus

Gram –veGram –ve E.coliE.coli

Step 1:Step 1: Crystal Violet

Step 2:Step 2: Gram’s IodineGram’s Iodine

Step 3: Decolorization Step 3: Decolorization (Aceton-Alcohol)(Aceton-Alcohol)

Step 4:Step 4: Safranin RedSafranin Red

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Step 1:Step 1: Crystal Violet

Step 2:Step 2: Gram’s IodineGram’s Iodine

Step 3:Step 3: Decolorization Decolorization (Aceton-Alcohol)(Aceton-Alcohol)

Step 4:Step 4: Safranin RedSafranin Red

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Results:Results:

Shape: Shape: CocciCocci

Arrangement: Arrangement: clustersclusters

Colour: Colour: VioletViolet

Gram’s reaction: Gram’s reaction: Gram’sGram’s +ve+ve

Name of microorganism: Name of microorganism: Staphylococcus aureusStaphylococcus aureus ( (S. S. aureusaureus))

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Results:Results:

Shape: Shape: OvalOval

Arrangement: Arrangement: SingleSingle

Colour: Colour: VioletViolet

Gram’s reaction: Gram’s reaction: Gram’s +veGram’s +ve

Name of microorganism: Name of microorganism:

Candida albicansCandida albicans ( (C. albicansC. albicans))

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Results:Results:

Shape: Shape: BacilliBacilli

Arrangement: Arrangement: ChainsChains

Colour: Colour: VioletViolet

Gram’s reaction: Gram’s reaction: Gram +veGram +ve

Name of microorganism: Name of microorganism:

Bacillus subtilisBacillus subtilis ( (B. subtilisB. subtilis))

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Results:Results:

Shape: Shape: RodsRods

Arrangement: Arrangement: SingleSingle

Colour: Colour: redred

Gram’s reaction: Gram’s reaction: Gram -veGram -ve

Name of microorganism: Name of microorganism:

Escherichia coliEscherichia coli ( (E. coliE. coli))

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