381 PHT Lab #2

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381 PHT381 PHTLab #2Lab #2

Staining of BacteriaStaining of Bacteria

Bacteria cells Bacteria cells are are almost almost colorlesscolorless and and

transparenttransparent

A staining technique is often applied to the A staining technique is often applied to the

cells to cells to color themcolor them → →

Their Their shapeshape and and size size can be easily can be easily determined under the microscope. determined under the microscope.

Principle of stainingPrinciple of staining

StainsStains → combine → combine chemicallychemically with the with the bacterial protoplasm.bacterial protoplasm.

Commonly used stains are Commonly used stains are saltssalts::Basic dyesBasic dyes:: colored cationcolored cation + colorless + colorless

anionanion e.g. e.g. methylene blue methylene blue (methylene blue (methylene blue

chloride)chloride) MBMB++ + Cl + Cl- -

Acidic dyes:Acidic dyes: colored anioncolored anion + colorless + colorless cationcation

e.g. e.g. eosin ( Naeosin ( Na++ + eosin + eosin--).).

Bacterial cells areBacterial cells are slightly negatively slightly negatively chargedcharged ( rich in nucleic acids bearing ( rich in nucleic acids bearing negative charges as phosphate groups)negative charges as phosphate groups)

→ → combine withcombine with positively charged positively charged basic dyesbasic dyes

Acidic dyesAcidic dyes do do not stain the bacterial not stain the bacterial cellcell → → can can stain the background stain the background materialmaterial with a contrasting color.with a contrasting color.

Types of staining techniquesTypes of staining techniques

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

Differential stainingDifferential staining (use of (use of two contrasting stainstwo contrasting stains

separated by separated by a decolorizing agent)a decolorizing agent)

For visualization ofFor visualization of morphologicalmorphological

shape & arrangement.shape & arrangement.IdentificatioIdentificationn

Visualization Visualization of structureof structure

Gram Gram stainstain

Acid fastAcid fast stainstain Spore Spore

stainstainCapsuleCapsule

stainstain

Smear Preparation:Smear Preparation: Preparation and Preparation and FixationFixation of Bacteria for of Bacteria for Staining.Staining.

Objective:Objective:

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

Smear preparationSmear preparation

S Fixation

Definition:Definition:

It is the use of It is the use of single basic dyesingle basic dye to to color the bacterial organism.color the bacterial organism.

e.g. methylene blue, e.g. methylene blue, crystal violet, crystal violet, safranin.safranin. All bacteriaAll bacteria take take the color of the dyethe color of the dye..

Objective:-Objective:- To show the To show the morphological shapesmorphological shapes and and

arrangementarrangement of bacterial cells. of bacterial cells.

Simple StainingSimple StainingProcedure:-Procedure:-

1-2 min

Basic Shapes of Bacteria Basic Shapes of Bacteria

Cocci Bacilli

ArrangementsArrangements

Irregular Clusters Chains or PairsTetrads

Staphylococci Micrococci Streptococci

ResultsResults

Type of staining:Type of staining: Name of stain:Name of stain:

Shape of cells:Shape of cells:Arrangement of cells: Arrangement of cells: Color:Color:

Name of m.o:Name of m.o:

Simple StainingSimple Staining

Type of staining:-Type of staining:- Simple Simple StainStainName of dye:-Name of dye:- Methylene Methylene blueblue

Shape of cells:-Shape of cells:- bacillibacilliArrangement of cells:-Arrangement of cells:- strainstrainColor:-Color:- BlueBlue

Name of m.o:-Name of m.o:- Bacillus Bacillus

Simple StainingSimple StainingType of staining:-Type of staining:- Simple StainSimple Stain

Name of dye:-Name of dye:- 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:-

StaphylococciStaphylococci

Simple StainingSimple StainingType of staining:-Type of staining:- Simple Simple StainStain

Name of dye:-Name of dye:- Crystal violet.Crystal violet.

Shape of cells:-Shape of cells:- coccicocci

Arrangement of cells:Arrangement of cells:clustersclusters

Color:-Color:- PurplePurple

Name of m.o:-Name of m.o:-

Gram Stain:Gram Stain:It is the It is the most important most important differential staindifferential stain used in used in bacteriology because bacteriology because it classified bacteria it classified bacteria into into two major groups:two 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

Crystal violetCrystal violet↓↓

IodineIodine

↓↓AlcoholAlcohol

↓↓

SafraninSafranin

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

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

2424Gram’s +ve

BacteriaGram’s -ve Bacteria

Gram’s +ve Bacteria

Gram’s -ve Bacteria

Gram-positive bacteriaGram-positive bacteria Have Have a thick peptidoglycan layera thick peptidoglycan layer surrounds the cell. surrounds the cell. The stain gets trappedThe stain gets trapped into this layer and the into this layer and the bacteria turned bacteria turned purple.purple.Retain the color of the primary stain (crystal violet) Retain the color of the primary stain (crystal violet) after decolorization with alcoholafter decolorization with alcohol

Gram-negative bacteriaGram-negative bacteria have have a thin peptidoglycan layera thin peptidoglycan layer that does not retain that does not retain crystal violet stain.crystal violet stain.Instead, it has Instead, it has a thick lipid layera thick lipid layer which dissolved which dissolved easily upon decoulorization with Aceton-Alcohol. easily upon decoulorization with Aceton-Alcohol. Therefore, cells will be counterstained with safranin Therefore, cells will be counterstained with safranin and turnedand turned redred. .

Gram StainGram Stain

Materials:-Materials:- Cultures of Cultures of Staphylococcus aureus, Staphylococcus aureus,

Candida albican, Candida albican,

Bacillus subtilis, Bacillus subtilis,

E.coli E.coli

Gram stain:Gram stain:

Crystal violetCrystal violet (primary stain) (primary stain)

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

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

Safranin Safranin (counter stain) (counter stain)

Gram Stain Gram Stain

Procedure:Procedure:

CVCViodineiodine

30-60 sec30-60 sec2 min2 min10 sec10 sec

safraninsafranin

30 sec30 sec

Results:Results:Shape: Shape: CocciCocci

Arrangment: Arrangment: irregularirregular clustersclusters

Colour: Colour: VioletViolet

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

Name of microorganism: Name of microorganism:

Results:Results:Shape: Shape: OvalOval

Arrangment: Arrangment: SingleSingle

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

Candida Candida

Results:Results:Shape: Shape: BacilliBacilli

Arrangment: Arrangment: ChainsChains

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

Bacillus Bacillus

Results:Results:

Shape: Shape: RodsRods

Arrangment: Arrangment: SingleSingle

Colour: Colour: redred

Gram’s reaction: Gram’s reaction: Gram’s –veGram’s –ve

Gram negative Gram negative bacillibacilli

Negative stainingNegative staining (Indirect staining with acidic (Indirect staining with acidic

dye)dye)

The negative staining technique The negative staining technique does not does not stain the bacteriastain the bacteria due ionic repulsion. due ionic repulsion.

but but stain the backgroundstain the background..The bacteria will appear The bacteria will appear colorless colorless against against a dark background.a dark background.No heat fixation or strong chemicalsNo heat fixation or strong chemicals are are used→ used→ the bacteria are less distortedthe bacteria are less distorted than in other staining procedure.than in other staining procedure.

Example: Example: Nigrosine Nigrosine

Negative stainingNegative staining

Candida

Staphylococci

Bacillus

381 PHT Lab #2

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