22
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.
33
Biochemical Tests.Biochemical Tests.
Identification of BacteriaIdentification of Bacteria
Additional Tests:Additional Tests:• such as serological testssuch as serological tests
44
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.
55
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
66
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]
++ --
77
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.)
99
.
3. Let the smear air dry completely. Do not apply heat while drying because this can lyse the cells
1010
Smear preparationSmear preparation
SS Fixation Fixation
1111
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 direct staining
b)b) Indirect staining with acidic dye Indirect staining with acidic dye (Negative staining)(Negative staining)
Simple StainingSimple Staining
Materials:-Materials:- Cultures of S. aureus, B. subtilisCultures of S. aureus, B. subtilis Stain:Stain:
Methylene blue stainMethylene blue stain Crystal violetCrystal violet
1212
1313
Simple StainingSimple Staining• Procedure:-Procedure:-
MBMB
1-2 min1-2 min
1414
Basic Shapes of Bacteria Basic Shapes of Bacteria
CocciCocci BacilliBacilli
1515
ArrangementsArrangements
CocciCocci
Irregular ClustersIrregular Clusters Chains or PairsChains or PairsTetradsTetrads
StaphylococciStaphylococci MicrococciMicrococci StreptococciStreptococci
1616
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:
1717
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
1818
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
1919
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
2020
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
2121
Negative stainingNegative staining
Candida albicansCandida albicans
2222
Negative stainingNegative staining
S. aureusS. aureus
2323
Negative stainingNegative staining
B. subtilisB. subtilis
2424
Bacterial MorphologyBacterial Morphology
SShape hape
SSpecial arrangementpecial arrangement
SSpore formationpore formation
CCapsule formationapsule formation
MMotilityotility
SStaining affinitytaining affinity
2525
Bacterial Shapes Bacterial Shapes
2626
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
2929
Bacterial SporesBacterial Spores
- Morphological characters of Morphological characters of bacterial spores:bacterial spores:
* Shape.* Shape.
* Position.* Position.
* Staining.* Staining.
3030
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
3131
Smearing out of the Smearing out of the samplesample
3232
Smear FixationSmear Fixation
3333
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.
3535
G-ve bacilliG-ve bacilli
Gm+ve cocci
3636
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
3737Gram’s +ve Bacteria
Gram’s -ve Bacteria
3838
Gram’s +ve Bacteria
Gram’s -ve Bacteria
3939
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
4040
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)
4141
Gram Staining Gram Staining TechniqueTechnique
4242
Gram Stain Gram Stain [single][single]
• Procedure:Procedure:
ss
CVCViodineiodine
30-60 sec30-60 sec2 min2 min10 sec10 sec
safraninsafranin
30 sec30 sec
4343
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
4444
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
4545
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))
4646
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))
4747
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))
4848
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))
4949
Staining of BacteriaStaining of Bacteria•Types of staining technique:-Types of staining technique:-
Simple stainingSimple staining (use of (use of
a single basic stain)a single basic stain)
Differential stainingDifferential staining (use of (use of two contrasting stainstwo contrasting stains
separated by separated by a decolorizing agenta decolorizing agent))
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
Acid Fast StainingAcid Fast Staining
•MycobacteriaMycobacteria have have a 3a 3rdrd type type of cell envelopeof cell envelope (rather than the (rather than the basic gram- related properties).basic gram- related properties).
• The cell wall of bacteria in this The cell wall of bacteria in this genus contain → genus contain → considerable considerable amounts of lipidsamounts of lipids → form an → form an extremely hydrophobic external extremely hydrophobic external layer.layer.
Acid Fast StainingAcid Fast Staining
• These organisms are These organisms are not readily stainable not readily stainable with ordinary stains.with ordinary stains.
• Staining of these bacteria needs exposure Staining of these bacteria needs exposure to to a strong staina strong stain e.g., e.g., concentrated concentrated carbol fucsincarbol fucsin
With application of With application of heat.heat.• Once they are properly stained, they Once they are properly stained, they
resist decolorizationresist decolorization by by strong strong mineral acidsmineral acids or or acid-alcoholacid-alcohol→ so they are → so they are said to besaid to be
Acid-fast.Acid-fast.
AFS is an AFS is an important diagnostic important diagnostic valuevalue in identifying in identifying pathogenic pathogenic membersmembers of genus of genus Mycobacterium Mycobacterium such as such as M.M. tuberculosistuberculosis and and M. leprae.M. leprae.
Acid Fast StainingAcid Fast Staining
Acid Fast StainingAcid Fast Staining
Materials:- Materials:- Culture of Culture of M. phelei M. phelei
Acid-fast staining kit: Acid-fast staining kit:
Conc. carbol fuchsinConc. carbol fuchsin (primary dye)(primary dye)
Acid-alcoholAcid-alcohol (decolorizing (decolorizing agent)agent)
Methylene blueMethylene blue (counter (counter stain)stain)
Acid Fast StainAcid Fast Stain• Procedure:-Procedure:-
Carbol fuchsin\\\\
5 min
acid
30-60 sec
MB
1 min
ResultsResultsType of Staining: Acid fast stain
Shape: beaded bacilli
Arrangement: Tree shaped
Colour: red
Name of microorganism: M.phelei
The Spore StainThe Spore Stain • Some bacteria (e.g., Bacillus and Some bacteria (e.g., Bacillus and
Clostridia) form resistant bodies in Clostridia) form resistant bodies in the cell known as the cell known as endosporsendospors..
• Bacterial spores are Bacterial spores are highly resistanthighly resistant to to physical & chemical agents physical & chemical agents (primarily due to a thick tough spore (primarily due to a thick tough spore coat).coat).
• They are They are not easily stainednot easily stained by routine by routine staining.staining.
• Heat Heat is required in spore staining to is required in spore staining to promote the penetration of the dye promote the penetration of the dye into the spore.into the spore.
• Once the spores stained they Once the spores stained they resist resist decolorizationdecolorization..
The Spore StainThe Spore Stain
Materials :-Materials :-•Culture of Culture of B. subtilisB. subtilis
•Spore-staining kit:Spore-staining kit:•Malachite greenMalachite green (primary (primary stain)stain)
•SafranineSafranine (counter stain (counter stain))
Spore Stain ofSpore Stain ofBacillus subtilisBacillus subtilis
Type of Staining: Spore stain
Shape: bacilli
Arrangement: Chains
Colour of spores: green
Colour of vegetative cells: red
Name of microorganism:
B. subtilis