381 PHT Lab #2
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381 PHT381 PHTLab #2Lab #2
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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.
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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--).).
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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.
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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
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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.
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Smear preparationSmear preparation
S Fixation
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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.
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Simple StainingSimple StainingProcedure:-Procedure:-
MB
1-2 min
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Basic Shapes of Bacteria Basic Shapes of Bacteria
Cocci Bacilli
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ArrangementsArrangements
Cocci
Irregular Clusters Chains or PairsTetrads
Staphylococci Micrococci Streptococci
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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:
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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
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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
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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:-
StaphylococciStaphylococci
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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
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Crystal violetCrystal violet↓↓
IodineIodine
↓↓AlcoholAlcohol
↓↓
SafraninSafranin
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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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BacteriaGram’s -ve Bacteria
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Gram’s +ve Bacteria
Gram’s -ve Bacteria
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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. .
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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)
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Gram Stain Gram Stain
Procedure:Procedure:
ss
CVCViodineiodine
30-60 sec30-60 sec2 min2 min10 sec10 sec
safraninsafranin
30 sec30 sec
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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:
StaphylococciStaphylococci
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Results:Results:Shape: Shape: OvalOval
Arrangment: Arrangment: SingleSingle
Colour: Colour: VioletViolet
Gram’s reaction: Gram’s reaction: Gram’s +veGram’s +ve
Name of microorganism: Name of microorganism:
Candida Candida
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Results:Results:Shape: Shape: BacilliBacilli
Arrangment: Arrangment: ChainsChains
Colour: Colour: VioletViolet
Gram’s reaction: Gram’s reaction: Gram’s +veGram’s +ve
Name of microorganism: Name of microorganism:
Bacillus Bacillus
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Results:Results:
Shape: Shape: RodsRods
Arrangment: Arrangment: SingleSingle
Colour: Colour: redred
Gram’s reaction: Gram’s reaction: Gram’s –veGram’s –ve
Name of microorganism: Name of microorganism:
Gram negative Gram negative bacillibacilli
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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
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Negative stainingNegative staining
Candida
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Negative stainingNegative staining
Staphylococci
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Negative stainingNegative staining
Bacillus
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