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

77

Smearing out of the Smearing out of the samplesample

88

Smear FixationSmear Fixation

99

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.

1010

Gram’s StainGram’s Stain

1111

G-ve bacilliG-ve bacilli

Gm+ve cocci

1212

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

1313

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)

1414

Gram Staining Gram Staining TechniqueTechnique

1515

Gram Stain Gram Stain [single][single]

• Procedure:Procedure:

ss

CVCViodineiodine

30-60 30-60 secsec

2 min2 min10 sec10 sec

safraninsafranin

30 30 secsec

1616

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

1717

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))

1818

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))

1919

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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