Ex. 8 : Aseptic Technique – Inoculating Different Types of Media
313 PHT LAB#1 ☠ Lab coat & marker. ☠ No eating, drinking, ☠ Benches disinfection. ☠...
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Transcript of 313 PHT LAB#1 ☠ Lab coat & marker. ☠ No eating, drinking, ☠ Benches disinfection. ☠...
313PHT
LAB#1
☠ Lab coat & marker.☠ No eating,
drinking, ☠ Benches
disinfection.☠ Inoculating loop
sterilization.☠ Aseptic technique. ☠ Discarded cultures
& infectious materials.
☠ Broken or spilled living cultures.
☠ Microscope.☠ At the end of each
lab check:•Gas tap is turned off.
•Water tap is closed properly.
•Microscope lamp is turned off.
☠ Finally wash your hands thoroughly.
• Discard cultures and other infectious materials:
Petri dishes→ Plastic bag → Autoclave.Test tube cultures → wire basket →
Autoclave.Used pipettes → Jar containing a
disinfectant → Plastic bag → Autoclave
Used slides, covers → Jar containing a disinfectant
Broken glass → swept in a dustpan → container for broken glass.
NEVER place contaminated material in waste basket.
• Broken or spilled living cultures:
Clothing → Autoclave plastic bag → Autoclave.
Flood the area with a disinfectant ( or paper towels are placed over the spills).
After 20- 30min→ wipe up & discard the waste in autoclavable dustpan→ Autoclave.
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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
66
Flaming of LoopFlaming of Loop
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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-stain.*Application of the counter-stain.
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Gram’s StainGram’s Stain
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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
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Gram StainGram Stain
• Materials:-Materials:- • Cultures of Cultures of Staphylococci, Candida, Staphylococci, Candida,
Bacillus, gram –ve bacteria.Bacillus, gram –ve bacteria.• 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 30-60 secsec
2 min2 min10 sec10 sec
safraninsafranin
30 30 secsec
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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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Results:Results:
Shape: Shape: CocciCocci
Arrangement: Arrangement: clustersclusters
Colour: Colour: VioletViolet
Gram’s reaction: Gram’s reaction: Gram’s +veGram’s +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))
2020
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:
Gram –ve bacilliGram –ve bacilli
Culture MediaCulture Media• The culture media is an artificial
preparation contains the essential element and nutrient in a proper concentration needed by the microorganism to grow.
• It may be:-•Liquid (broth)•Solid (containing agar)•Semisolid (containing low conc of agar)
Culture MediaCulture Media• Most common ingredients:-
1. Essential elements and nutrients.
2. Solidifying agents.
Types of culture media:Types of culture media:•Simple media•Enriched media•Selective media• Indicator media•Selective and indicator media
Isolation of Pure Colonies Isolation of Pure Colonies of Microorganism of Microorganism
“Streak Plate Method”“Streak Plate Method”
In natural environments, bacteria & other m.o exist in mixed population.
“Streak Plate Technique”The individual cells are separated from each other by certain distance on the surface of the agar.After incubation, each single deposited cell divide many times and finally form visible mass of growth “COLONY”.
The streak Plate MethodThe streak Plate Method
• The culture prepared from a single type of colony is regarded as a pure culture.
• The streak Plate Method is used for:Checking the purity of a bacterial
culture.Isolating individual species from a
mixture culture.
The streak Plate MethodThe streak Plate Method• Objective:- for isolation of individual
species of a mixed broth culture.• Materials:-
Nutrient agar plate. Mixed broth culture of Serratia
marcescens and staph. aureus.
The streak Plate MethodThe streak Plate Method
• Procedure:
S & SS & S
Aseptic technique Aseptic technique
Invert the plate and Invert the plate and Incubate for 24h at 37Incubate for 24h at 37℃℃
Drop of the cultureDrop of the cultureFlame & Cool
Flame & Cool
Flame & Cool
The streak Plate MethodThe streak Plate Method
The streak Plate MethodThe streak Plate Method
Antibiotic Sensitivity Antibiotic Sensitivity TestingTesting
• Antibiotic sensitivity testing is used to determine the susceptibility of the microorganism to various antimicrobial agents.
•Sensitivity testing techniques:
– Disc Diffusion Method.
– Serial Dilution Method (Minimum inhibitory concentration).
Disc Diffusion Disc Diffusion MethodMethod
The effectiveness of an antibiotic in this technique is based on the size of the zone of inhibition that surrounds a disc that has been impregnated with a specific concentration of the agent.
Advantages:Rapid Accurate Inexpensive
Disc Diffusion methodDisc Diffusion method• Materials:
– Cultures of Staph. aureus, E. coli Pseudomonas aeruginosa– Filter paper disc – Antibiotic solutions (Vancomycin,
Augmentin, Ceftazidime, Azactam)– 20 ml melted nutrient agar– Petri-dish– Sterile 1ml pipette
ProcedureProcedure
45°c
0.1ml
m.o
inoculate Aug
Az Van
Cef
Az
24 h
at 37°c
incubate
Don’t invertDon’t invert
Results:Results:• Measure the diameter of each
inhibition zone• The diameter of the inhibition zones are directly
proportional to the susceptibility of the microorganism to the antibiotics(sensitive, intermediate, resistant).
ResultsResults
CefAz
VanAug
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