MCR PRS Post Lab Prepn of Smear and Staining

8/18/2019 MCR PRS Post Lab Prepn of Smear and Staining

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Post LabDiscussion

Examination of Microorganismsand Staining Techniques


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Preparation of Specimens for Compound

Light Microscope Examination:

1. Wet Mount

drop of medium with microorganisms is

placed on a slide and covered withcoverslip

shape, movement of microorganisms

Advantage: live organisms are observed

Disadvantage: dries out quickly


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2.Hanging drop

drop of liquid containing microorganism is

placed on a cover slip and inverted and

suspended on a slide with a

concavity/depression Shape, movement of microorganisms

(organisms with flagella)

more complex technique

Advantage: allows for longer-term observation

and more reliable observation of motility


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3. Preparation of Bacterial Smear

thin film material containing microorganismsspread on a slide

heat fixed to kill microbes

study the morphology


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Preparation and Staining of

Specimens

specimens must be fixed and stained to

enhance their visibility and to distinguishmorphological properties


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Purpose of Fixation:

Preservation of internal and external

features of cells

Cellular enzymes are inactivated Cell structures are hardened

Organism dies AND adheres strongly to

the glass slide


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2 Types of Fixation:

1.Heat fixation

flame heating bacterial smear

preservation of morphology but NOT internalstructures

2.Chemical fixation

chemical fixatives penetrate cells and preservesintracellular components

• Acetone

• Ethanol

• Acetic acid

• Mercuric chloride

• Formaldehyde

• Glutaraldehyde


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Dyes and Simple Staining

Stains and bind to cells by ionic, covalent andhydrophobic forces

Basic dyes (positively charged) bind to

negatively charged groups – Work best at high pH

Acidic dyes (negatively charged) bind to

positively charged groups – Work best at low pH


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Common Dyes

Basic DyesSafranin

Carbol fuchsinCrystal violet

Methylene blue

Malachite green- commonly used

Acidic Dyes

Eosin

Acid fuchsinCongo red


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Staining Techniques

1. Simple Stain basic dye

Purpose:

cell shape and basic structures become

visible

Highlight microorganisms shapes andarrangements


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Morphology

Shape

cocci

bacilli

Curved and spiral

Arrangement cocci in pairs, in clusters,

in chains, tetrads andsarcinae

bacilli in singles, in pairsand in chains

spirillum in singles


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Morphologic arrangement of Cocci:

Cocci in cluster

Staphylococci


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Cocci that remain in

pairs after dividing

are called diplococci

Neisseria gonorrhoea


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• Cocci in chainlike

patterns are calledstreptococci


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Cocci in groups of

four are called tetrads

Cocci attached in

groups of 8 are called

sarcinae


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Bacilli in singles, in

pairs or in chains

Diplobacilli

streptobacilli


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2. Differential Stain

differentiates between types of organisms uses 2 or more dyes to stain organism

a. Grams Stain

- Hans Christian Gram

- Gram positive and Gram negative

b. Acid fast stain

- Acid fast organisms

- Non-acid fast organisms


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Gram Positive Gram Negative

Color after G. staining

procedure

purple red

Peptidoglycan in c. wall Thick layer Thin layer

Lipopolysaccharide in cell

walls

Absent Present


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Gram positive

CV-I complex is difficult toremove during

decolorization Purple in color

Gram negative

decolorizer dissolves lipidsin cell wall of Gramnegative bacteria

CV-I Complex is removed

Colorless retain the color of

counterstain

red


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Gram Positive Gram negative


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Erroneous Result

1. Film is too thick

2. Decolorization not completed.

3. Film is over decolorized.4. Use of old culture which gives variable

results.


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Common Gram positive bacteria

Streptococcus pyogenes

Streptococcus pneumoniae

Staphylococcus aureus Enterococcus fecalis

Bacillus cereus

Bacillus subtilis

Clostridium sp.


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Common Gram negative bacteria:

Salmonella sp.

Shigella sp.

Neisseria meningitidis

Haemophilus influenzae

Escherichia coli

Klebsiella pneumoniae

Proteus sp.

Pseudomonas aeruginosa


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Acid-Fast Staining Some organisms do not stain well with conventional dyes

(e.g. Mycobacterium and Nocardia)

Mycobacterium tuberculosis

Mycobacterium leprae

Harsher treatment required (slide is heated for severalmins.)

Steps:

1. Slide is heated while using carbol fuchsin (Ziehl-Neelsen technique)

2. Decolorize: Acid alcohol

3. Counterstain with methylene blue

Acid-fast cells remain red due to high mycolic acidcontent (lipid)

Non-acid-fast cells = blue (counterstain)


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Ziehl-Neelsen’s Method

Acid fast organism

retain red color because

carbolfuchsin is soluble

in cell wall lipids (red)

Non-acid fast organism cell wall lacks lipid

components and carbol

fuchsin is removed

retain the color of the

counterstain (blue)


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3. Special Stains

Used to color and isolate specific parts of

microorganisms

Endospores, flagella, capsule


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a. Negative Staining for Capsules

CAPSULE

– gelatinous covering of microorganisms

Mix bacteria with colloidal suspension ofcolored particles (India ink or nigrosin) to

provide dark background

Bacteria

– stained with simple stain

Capsules appear as halos surrounding stained

bacterial cell


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B. Endospore (spore) staining

Spore staining

• detects endospores produced by

Bacillus and Clostridium• various sizes, shapes and locations

• dormant structures within the cell

• endospores protect the organism fromadverse environmental conditions

• endospores cannot be stained by

ordinary methods


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Steps (Schaffer-Fulton Method)

Preparation of a Bacterial Smear

Air dry

Heat fix

Application of Primary Dye (Malachite Green)

5 minutes w/ concurrent application of heat

Rinse with water

Application of Counterstain (Safranin) 1minute


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Endospore

Most commonly used endospore stain

(Schaeffer-Fulton endospore stain)

Malachite green (primary stain)

safranin (counterstain)

Spores ( green) , bacterial cells (red)


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Spore Staining: Bacillus subtilis stained using

Schaeffer Fulton Method. Note the central elliptical

green spores within the red cells (X 1,000)


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MOTILITY TEST

Positive result - test tube exhibits growth radiating fromthe stab; E. coli

Negative result - test tube exhibits growth along the stab- Staphylococcus aureus


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CATALASE TEST

Test to detect whether the bacteria

produce enzyme catalase

Differentiation between Staphylococcusand Streptococcus genera

Catalase is an enzyme used by bacteria to

induce the reaction of reduction ofhydrogen peroxide into water and oxygen


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CATALASE TEST

Positive result - evolution of gas bubbles

- Staphylococcus aures


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COAGULASE TEST

• Pathogenicity test

• Tests to detect if bacteria produce

coagulase enzyme

• Coagulase is an enzyme produced bymicroorganism that enables the

conversion of fibrinogen to fibrin

Two methods:

1. slide method

2. tube method


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+ Result- coagulation or forming a clot

Staphylococcus aureus

- Result – the plasma remains liquid

- Staphylococcus epidermidis

MCR PRS Post Lab Prepn of Smear and Staining

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