Embed Size (px)
Citation preview
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 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
1212
Simple StainingSimple Staining• Procedure:-Procedure:-
MBMB
1-2 min1-2 min
1313
Basic Shapes of Bacteria Basic Shapes of Bacteria
CocciCocci BacilliBacilli
1414
ArrangementsArrangements
CocciCocci
Irregular ClustersIrregular Clusters Chains or PairsChains or PairsTetradsTetrads
StaphylococciStaphylococci MicrococciMicrococci StreptococciStreptococci
1515
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:
1616
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
1717
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
1818
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
1919
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
2020
Negative stainingNegative staining
Candida albicansCandida albicans
2121
Negative stainingNegative staining
S. aureusS. aureus
2222
Negative stainingNegative staining
B. subtilisB. subtilis
2323
Bacterial MorphologyBacterial Morphology
SShape hape
SSpecial arrangementpecial arrangement
SSpore formationpore formation
CCapsule formationapsule formation
MMotilityotility
SStaining affinitytaining affinity
2424
Bacterial Shapes Bacterial Shapes
2525
Bacterial ShapesBacterial 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
Bacterial SporesBacterial Spores
Unstained Spores
Spore Stain
3131
Bacterial CapsuleBacterial Capsule
3232
Flaming of LoopFlaming of Loop
3333
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.
3434
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
3535
Smearing out of the Smearing out of the samplesample
3636
Smear FixationSmear Fixation
3737
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.
3939
G-ve bacilliG-ve bacilli
Gm+ve cocci
4040
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
4242
Gram’s +ve Bacteria
Gram’s -ve Bacteria
4343
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
4444
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)
4545
Gram Staining Gram Staining TechniqueTechnique
4646
Gram Stain Gram Stain [single][single]
• Procedure:Procedure:
ss
CVCViodineiodine
30-60 sec30-60 sec2 min2 min10 sec10 sec
safraninsafranin
30 sec30 sec
4747
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
4848
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
4949
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))
5050
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))
5151
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))
5252
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))
5353
