Staining

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STAINING Gunjan Mehta,

Dept. of Biotechnology,

VSC, Rajkot.

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STAINING Staining is a technique used in

microscopy to enhance contrast in the microscopic image

Stains are dyes which are used to highlight microorganisms or biological tissues for viewing with the help of a microscope

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AIMS OF STAINING IN MICROBIOLOGY To increase visibility & contrast To study the morphology To detect extracellular and intracellular

components of microbes To help in the primary level of diagnosis

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METHODS OF MAKING FILM/SMEAR Film / smear preparations are made

usually on 3x1’’ glass slides Slides should be superior quality and

grease free If the grease free slide is not provided

with, should clean the slides

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CLEANING OF SLIDESMethod 1 Wipe the slide with dry cotton cloth Pass the slide through blue flame of

Bunsen flame 16-12 times

Method 2 Moisten the finger with water, rub it on

the surface of fine sand soap Smear it over the slide Remove soapy film with clean cotton

cloth

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COVER SLIPS Should be ¾ or 7/8 inches 0.1 mm thickness Should be used only for wet

preparations

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MAKING FILMS Take a clean slide A target circle of approximately 1.5 cm

diameter should be marked on the under surface of slide

Place a loopful of liquid bacterial suspension on the circled area

Spread the suspension with a Nichrome wire loop

Allow the slide to air dry completely

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MAKING SMEARS FROM VARIOUS SPECIMENS Purulent specimen

Use sterile wire loopMake a thin smear If needed put a drop of saline

Non Purulent specimenMake a smear from a drop of well mixed

specimen Culture

Emulsify a colony in sterile distilled waterUse sterile wire loopMake a thin smear

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MAKING SMEARS FROM VARIOUS SPECIMENS .. .. .. Sputum

Use a broom stickTransfer a purulent part to the slideSpread evenly

SwabsRoll the swab on a slide If needed put a drop of saline

FaecesUse a broom stickTransfer a mucopurulent part to the slideSpread evenly

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FIXING OF SLIDES

Heat fixationBy passing the slide over flame

Chemical fixationUsing ethanolMethanolPicric acidPotassium permanganateFormaldehyde vapour

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FIXATION - PROCEDURE

Three quick passes over the flame is sufficient for fixation

Chemical fixation is just applying few drops of chemicals and allow to evaporate

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FIXATION - USES

It kills bacteria rendering safe handling It prevents autolysis by inactivating the

autolytic enzymes It increases the permeability of cells to

stain It makes cells rigid It unfolds the globular proteins and

exposing reactive groups & increasing affinity for stain

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METHODS OF STAINING Simple staining Differential staining Special staining

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SIMPLE STAINING

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TYPES Monochrome staining / positive staining Negative / indirect staining

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MONOCHROME STAINING / POSITIVE STAINING - PRINCIPLE

Basic dyes are used for this Basic dyes are Positively charged These dyes get attached to negatively

charged cytoplasm of microbial organism

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SIMPLE STAINING

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NEGATIVE / INDIRECT STAININGPRINCIPLE Acidic dyes are used for this Acidic dyes are negatively charged These dyes get repelled by the

negatively charged cytoplasm of microbial organism

So the dye get gathered around the organism

They give a contrast back ground Organism stands unstained

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MONOCHROME STAINING

The use of single stain to colour the bacteria is known as monochrome staining

Usual stains used areCrystal violetLoeffler’s Methylene blueDilute carbol fuchsin

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CRYSTAL VIOLET Crystal violet 10 gm Absolute alcohol 100ml Distilled water 1 litre Dissolve the dye in alcohol Filter through filter paper Add to water

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LOEFFLER’S METHYLENE BLUE

Saturated solution of methyleneblue in alcohol 300 ml

KOH, 0.01% in water 1 litre Dissolve & mix thoroughly

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CARBOL FUCHSIN - STRONG Phenol 85 gm Basic fuchsin 15 gm Ethanol 250 ml Distilled water 1250 ml Mix Phenol and Basic fuchsin , heat

gently to dissolve. Add ethanol and distilled water. Filter in to a bottle.

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CARBOL FUCHSIN - WEAK Dilute 1 volume of strong Carbol fuchsin

with 10-20 volumes of distilled water.

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PROCEDURE MONOCHROME STAINING Cover the fixed smear with stain

If Crystal violet keep stain for 2-6 sec If Methylene blue 3-5 min If Dilute Carbol fuchsin 15-30sec

Wash under running water Blot dry Examine under oil immersion

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NEGATIVE / INDIRECT STAINING

Usual stains used areNigrosin Indian ink

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NIGROSIN

Nigrosin 100gm Formalin 5ml Distilled water 100ml Dissolve nigrosin in formalin Add to Distilled water

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INDIA INK A dense homogenous India ink free from

large particles is to mix with quarter of its volume of grade 12 ballotini glass beads 0.22 mm and shake well for one hour in a Mickle tiss disintegrator.

This ink may be improved by evaporation to concentrate.

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NEGATIVE STAINING - PROCEDURE Wipe the slide clean Place a large drop of stain on the slide Emulsify small portion of bacterial

culture Place a cover slip Look under high power objective

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NEGATIVE STAINING

India ink Nigrosin

Cryptococcus spore B. subtilisCocci

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NEGATIVE STAINING

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DIFFERENTIAL STAINING

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DIFFERENTIAL STAINING

Gram’s method Acid fast staining

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SALUTES .. .. ..Hans Christian Gram 13-09-1853 to 14-11-1938. Danish bacteriologist. The work with Gram staining gained him international reputation

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GRAM’S STAINING Gram staining is a microbiological

procedure that categorizes bacteria based on the physical and chemical structure of their outer surface

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GRAM’S METHOD - PRINCIPLE

The cell wall of gram positive bacteria contains high amount of Peptidoglycan and negligible amount of lipids

The cell wall of gram negative bacteria have very negligible Peptidoglycan and high amount of lipids

The primary basic Stain, stains both kind of cells

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GRAM’S METHOD – PRINCIPLE .. In second step when iodine add, it act

as a mordant and form crystal violet iodine Peptidoglycan complex in gram positive bacteria

This complex is not formed in gram negative bacteria as Peptidoglycan is much less in their cell wall

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GRAM’S METHOD – PRINCIPLE .. In the next step, application of

decolorizer extract the lipids from the gram negative bacteria

Removal of lipid makes the cell porous Porosity allows the release of primary

stain from cell Gram negative cells become colourless

at this step This colourless bacteria took the counter

stain

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GRAM’S METHOD – PROCEDURE Cover the smear with *Basic

pararosaniline violet dye for1min Wash with water Cover with Gram’s iodine for 1 min Wash with water Decolourise with acetone for 2 sec

*Crystal violet *Methyl violet *Gentian violet

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GRAM STAINING CONTD Wash with water Cover with *Counter stain Wash with water Blot dry Observe under oil immersion

*Basic fuchsin*Neutral red*Safranine

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GRAM STAIN

Figure 4.17.1

GRAM STAIN

Figure 4.17.2

GRAM STAIN

Figure 4.17.3

GRAM STAIN

Figure 4.17.4

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CRYSTAL VIOLET Crystal violet 10 gm Absolute alcohol 100ml Distilled water 1 litre Dissolve the dye in alcohol Filter through filter paper Add to water

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METHYL VIOLET Methyl violet 10 gm Absolute alcohol 100ml Distilled water 1 litre Dissolve the dye in alcohol Filter through filter paper Add to water

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GENTIAN VIOLET Crystal violet 5 gm Methyl violet 5gm Absolute alcohol 100ml Distilled water 1 litre Dissolve the dye in alcohol Filter through filter paper Add to water

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LUGOL’S IODINE Iodine 5 gm Potassium iodide 10 gm Distilled water 100 ml

Dissolve Iodine & Potassium iodide in some water.

Add remaining water. For use dilute 1/5 with Distilled water

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BASIC FUCHSIN Basic fuchsin 0.5 gm Distilled water 1 litre Dissolve the dye in water Recommended for general use

Apply for 10 -30 sec

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NEUTRAL RED Neutral red 1 gm 1% Acetic acid in water 2ml Distilled water 1 litre Dissolve dye in water Add Acetic acid Recommended for Gonococci & other

intracellular gram negative bacteria

Apply for 2-4 min

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SAFRANINE Safranine 0.5 gm Distilled water 1 litre Dissolve the dye in water Recommended for general use

Apply for 10 -30 sec

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GRAM STAINING - RESULTSReport should include Number of bacteria Gram reaction of bacteria Morphology Presence and number of pus cells Presence of yeast cells Presence of epithelial cells

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GRAM STAINING - RESULTS When reporting Gram’s staining,

consider the morphology and colour of organism.

Spherical purple organism - report as GPC

Rod shaped purple organism - report as GPB

Spherical pink/red organism - report as GNC

Rod shaped pink/red organism -report GNB

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GPC GPB

GNCGNBGNB

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VARIATIONS IN GRAM REACTIONSPositivity of organism can loss Cell wall damage Over de-colorization Use of too old iodine Smear from old cultureNegative organism can appear positive When smear is too thick

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MODIFICATIONS

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MODIFICATIONS OF GRAM’S STAINING Kopeloff & Beerman’s Gram method for

films Kopeloff & Beerman’s Gram method for

sections Jensen’s Gram method for smears Preston & Morrel’s Gram method Quick gram method for single slide

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KOPELOFF & BEERMAN’S GRAM METHOD FOR FILMS Apply methyl violet for 5 min Wash with iodine solution Cover slide with fresh iodine solution - 2

min Decolorize with acetone - 2-3 sec Apply basic fuchsin - 30 sec Wash under tap - 5 sec Blot dry with out rubbing Complete drying by waving in warm air

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KOPELOFF & BEERMAN’S GRAM METHOD FOR SECTIONS Remove paraffin wax with xylene Remove xylene by flooding with

absolute alcohol Flood with 50% alcohol Apply methyl violet for 5 min Cover slide with fresh iodine solution - 2

min Decolorize with acetone for 2-3 sec Apply basic fuchsin for 30 sec

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KOPELOFF & BEERMAN’S GRAM METHOD FOR SECTIONS … Wash under tap 5 sec Blot dry without rubbing Dehydrate quickly by flooding with 95%

alcohol Drain and add 50% alcohol Immerse slide in xylene Wipe away excess xylene Mount No.1 cover slip in Canada balsam

or DPX

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JENSEN’S GRAM METHOD FOR SMEARS

Recommended for smears of gonococci and meningococci

Cover the slide with 0.5% methyl violet 30 sec

Pour Lugol’s iodine 1% to wash away stain

Cover with fresh iodine - 30 sec

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JENSEN’S GRAM METHOD FOR SMEARS ….

Wash off iodine with ethanol

Pour fresh alcohol 30 sec

Wash with water

Pour neutral red 0.1% 2 min

Wash & dry

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PRESTON & MORREL’S GRAM METHOD Cover slide with ammonium oxalate

crystal violet 30 sec Wash with Lugol’s iodine 1%

Cover with fresh iodine Wash with iodine acetone 30 sec

Wash with tap water Add Carbol fuchsin 30 sec

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QUICK GRAM METHOD FOR SINGLE SLIDE Held the fixed slide with forceps through

out the staining Flood the slide with basic stain Allow to act for 5 sec Tip off stain Flood the tilted slide with iodine solution

for 5 sec Flood the tilted slide with acetone for 2

sec Flood the tilted slide with counter stain

for 5 sec

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AMMONIUM OXALATE CRYSTAL VIOLET STAIN Solution A

crystal violet 10 gmEthanol 95% 100 mlMix and dissolve

Solution BAmmonium oxalate 1% aqueous

solution

Mix 20 ml of solution A and 80 ml of Solution B

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ACETONE-IODINE SOLUTION Strong iodine solution

Iodine 10 gmPotassium iodide 6 gmDistilled water 10 mlEthanol 90% to 100 ml

Strong iodine solution - 3.5 ml Acetone 96.5 ml

Mix well before use

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ACID-FAST STAINING

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SALUTES.. .. ..Paul Ehrlich 14-03-1854 to 20-08-1915. German scientist in the fields of hematology, Immunology, chemotherapy. Got Nobel prize for Medicine in 1908

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ACID-FAST STAINING Is a differential staining developed in

1882 by Paul Ehrlich.

Improved by Ziehl and Neelsen 1885

It is an important diagnostic procedure for identification of Mycobacterium and Nocardia.

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ACID-FAST STAINING - PRINCIPLE The cell wall of Mycobacterium & Nocardia

is composed of unique type of lipids.

One of which, a fatty acid called Mycolic acid

This impact a waxy nature to the cell wall

It is responsible for the characteristic feature of acid-fast staining

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ZIEHL NEELSEN METHOD FOR TUBERCLE BACILLUS

Flood the slide with strong Carbol fuchsin. Heat until steam arise. Care not to boil.

Keep for 5-7 minutes

Wash under running water Decolorize with 20% H2SO4 for 1 minute

Counter stain with methylene blue / malachite green for 1 minute

Wash and stand on to drain. Do not blot.

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ACID-FAST STAINING The results:

- Acid-fast cells retain the primary dye and appear pink- Non Acid-fast cells appear blue/green

AFB

Cells that retain a basic stain (carbolfucsin) in the presence of acid-alcohol are called acid-fast.

Non–acid-fast cells lose the basic stain when rinsed with acid-alcohol, and are usually counterstained (with methylene blue) to see them.

DIFFERENTIAL STAINS: ACID-FAST STAIN

Figure 3.11

• used to identify bacteria in the genus Mycobacterium (two members are pathogens which cause tuberculosis and leprosy).

Endospores are stained with the primary stain malachite green.

The vegetative cells (actively growing cells) are counterstained with safranin (pink cells).

SPECIFIC STAINING

Figure 3.11

• used to identify bacteria in the genera :

1. Clostridium: members are pathogens which cause gangrene, tetanus, and botulism.

2. Bacillus: one member is a pathogen that causes anthrax.

FLAGELLAR STAIN

Figure 4.21

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ZIEHL NEELSEN SMEAR EVALUATIONAND AFB REPORT

No. of AFB Oil immersion field

Report

0 300 AFB not seen

1-2 300 Doubtful , repeat

1-9 100 Scanty exact bacilli

1-99 100 1+

1-9 Each field 2+

10/ more Each field 3+

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ZIEHL NEELSEN CARBOL FUCHSIN Basic fuchsin 5gm Phenol 25 gm Alcohol 95% 50 ml Distilled water 500ml Dissolve fuchsin in phenol Add alcohol, mix thoroughly Add distilled water

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SULPHURIC ACID 20% Concentrated sulphuric acid

250 ml Distilled water 1 litre Pour water into a large flask Place the flask in 5-8 cm of cold water in

the sink Add acid very slowly through the side of

the flask Mix gently

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LOEFFLER’S METHYLENE BLUE

Saturated solution of methyleneblue in alcohol 300 ml

KOH, 0.01% in water 1 litre Dissolve & mix thoroughly

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MALACHITE GREEN Malachite green 5 gm Distilled water 500ml Dissolve the dye into water to get stock

solution

Working solutionStock solution 40mlDistilled water 360ml

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MODIFICATIONS

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ZIEHL NEELSEN METHOD FOR TISSUE SECTIONS

Treat slide with xylene Wash well with alcohol & then with

water

Flood the slide with strong Carbol fuchsin. Heat until steam arise. Care not to boil.

Keep for 5-7 minutes

Wash under running water Decolorize with 25% H2SO4 for 1 minute

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ZIEHL NEELSEN METHOD FOR TISSUE SECTIONS CONTD…. …. Counter stain with methylene blue for 1

minute Gently blot slide

Flush with absolute alcohol Wipe with fresh paper

Clear in xylene Mount in Canada balsam or DPX

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ZIEHL NEELSEN METHOD FOR WEAKLY ACID FAST ORGANISMS For Leprosy bacilli use 5% H2SO4

For Actinomycetes & Nocardia use 1%

For Brucella abortus Stain with dilute carbol fuchsin without

heatingDecolourise with 0.5% acetic acid 15 secCounter stain with Loffler’s methylene blue

1 min

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SPECIAL STAINING

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SPECIAL STAINING

Spore staining Flagella staining Staining for metachromatic granules Leishman’s stain Giemsa’s stain Staining for spirochaetes

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SPORE STAINING

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SPORE STAINING

Modified Ziehl Neelsen method

Malachite green stain

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MODIFIED ZIEHL NEELSEN METHOD

Flood the slide with strong Carbol fuchsin. Heat until steam arise. Care not to boil.

Keep for 5-7 minutes Wash under running water Decolourise with 0.25% H2SO4 for 1

minute Counter stain with methylene blue for 1

minute Wash and stand on to drain. Do not blot. Pink spores on blue stained bacteria

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MALACHITE GREEN STAIN

Place the slide over a beaker of boiling water

When there is condensation under the slide, flood with 5% aq.solution of Malachite green stain 1min

Wash in tap water Add 0.5% safranine / 0.05% basic

fuchsin 30 sec Wash & dry Spores appears green & bacterium red /

pink

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MALACHITE GREEN STAIN

spore

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FLAGELLA STAINING

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FLAGELLA STAINING

Grow bacteria for 16-24 hr on a non inhibitory medium

Touch a loopful of water on to the edge of a colony and let motile bacteria swim into it

Transfer the loopful in to a loopful of water on a slide

Cover with a cover slip [ to detach flagella ]

After 10 min apply 2 drops of Ryu's stain to the edge of cover slip, stand for 5-15 min

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RYU’S FLAGELLA STAIN Solution A

Tannic acid powdered 10 gmPhenol 5 % aq. Solution 50 mlAluminum potassium sulphate 50 ml

Solution BCrystal violet 12 gmEthanol saturated solution 100 gm

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RYU’S FLAGELLA STAIN .. .. ..

Mix 10 parts of Solution A one part of Solution B

Store at ambient temperature Mixture is stable indefinitely Does not require filtration Allow to stabilize by standing Fresh stain is potent

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FLAGELLA STAINING

flagella

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STAINING FOR SPIROCHAETES

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STAINING FOR SPIROCHAETES

Larger spirochaetes stain by ordinary methods

Smaller spirochaetes are best observed under dark ground microscope

If permanent preparation of small spirochaetes are required use Fontana or Levaditi methods

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FONTANA STAINING - FILMS Treat the film with fixative three

times,30 sec each Wash with absolute alcohol - 3 min

Drain off excess alcohol

Pour on the mordant Heat for 30 sec until steam arises Wash in distilled water and dry the slide

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FONTANA STAINING.. .. .. Treat with ammoniacal silver nitrate Heat till steam arise Keep for 30 sec [till the film becomes

brown]

Wash well with distilled water, dry and mount in Canada balsam

The spirochaetes are stained brownish – black on a brownish – yellow background

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FONTANA’S STAIN Fixative

Acetic acid 1mlFormalin [40%] 2mlDistilled water 100ml

MordantPhenol 1gmTannic acid 5gmDistilled water 100ml

Ammoniacal silver nitrate

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FONTANA’S STAIN … … … Add 10% ammonia to 0.5% solution of

silver nitrate in distilled water until the precipitate formed just dissolves.

Add more silver nitrate solution drop by drop until the precipitate returns and does not re-dissolve.

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SPIROCHAETES

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LEVADITI’S STAINING - TISSUES Fix 1mm thick tissue in 10% formalin

for 24 hrs Wash the tissue for 1 hr in water Place in 96 -98 % alcohol for 24 hrs

Place the tissue in 1% solution of silver nitrate for 2hrs

There after at about 500C for 4-6 hrs Rapidly wash the tissue in 10% pyridine

solution

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LEVADITI’S STAINING.. .. .. Transfer to the reducing fluid, keep for 2

days at room temperature in dark

Wash and dehydrate the tissue with alcohol and embed in paraffin

Cut thin sections, remove paraffin using xylene, mount in Canada balsam

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STAINING FOR META CHROMATIC GRANULES

Fix the dried smear using alcohol Cover the smear with the toluidine blue

malachite green for 3-5 min Wash with distilled water Cover smear with Albert’s iodine for 1 min Wash with distilled water Air dry

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ALBERT’S STAIN Malachite green 0.2 gm Toluidine blue 0.15 gm Ethanol 95% 2 ml Glacial acetic acid 1ml Distilled water 100 ml

Dissolve the dyes in ethanol. Mix the acid & water and allow to stand

for 24 hours and filter

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LEISHMAN’S STAIN - FILM Pour the undiluted stain on the unfixed

film and allow it to act for 1 min Add double volume of distilled water to

the slide Allow to stand for 12 min Flood gently with distilled water Keep for 30 sec Remove excess water by blotting Air dry

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LEISHMAN’S STAIN - SECTION Treat the section with xylene Treat with ethyl alcohol Treat with distilled water Drain & stain for 5-10 min with 1 part of

Leishman’s stain and 2 parts distilled water

Wash with distilled water Blot, dehydrate with a few drops of

absolute alcohol, clear in xylene and mount in Canada balsam

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LEISHMAN’S STAIN Leishman’s powder 0.15 gm Methanol 100ml The powder is ground in a mortar with a

little methanol The residue of undissolved stain is

allowed to settle Fluid decanted in to a bottle Residue in the mortar is treated with

more methanol Repeat till stain goes into solution

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GIEMSA’S STAIN

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GIEMSA’S STAIN – RAPID METHOD Fix film in methanol for 3 min Stain in a mixture of 1 part stain and 10

parts buffer solution for 1 hr Wash with buffer solution 30 sec This is excellent for malaria parasites

and trypanosomes

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GIEMSA’S STAIN – RAPID METHOD WITH HEAT Fix preparation with absolute alcohol 15

min Prepare fresh solution of 10 drops of

Giemsa’s solution with 10 ml of buffer solution

Cover the fixed film with the above solution

Heat till steam arise Allow to cool for 15 sec Pour off and replace with fresh stain,

heat again Repeat 5 times, wash, dry & mount

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GIEMSA’S STAIN –SLOW METHOD Fix the film in methanol for 3 min Mix 1 ml stain with 20 ml buffer solution

in a petri dish Place a piece of thin glass rod in the

stain in the dish Place the fixed slide downwards in the

stain with the help of glass rod Leave for 24 hrs Wash with buffer solution Dry and mount

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GIEMSA STAIN – STOCK SOLUTION Giemsa stain powder 1 gm Glycerol 60 ml Absolute methanol 60 ml Heat glycerol to 55-600C in a water bath. Add the stain powder and mix

thoroughly. Incubate the mixture at the same

temperature for 2 hours. Cool and add methanol. Keep to mature for about 2 weeks.

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GIEMSA STAIN .. .. ..

Stock solution 1 part 0.01 M-Phosphate buffer [ ph 7 ] 10

parts Mix and allow to stand

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GIEMSA STAIN

Plasmodium vivax Plasmodium falciparum

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GIEMSA STAIN

Trypanosoma cruzi Trypanosoma brucei

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FLUOROCHROME TECHNIQUE Auramine – phenol technique

Acridine – orange technique

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AURAMINE – PHENOL TECHNIQUE

Cover smear with auramine phenol 10 min

Wash with tap water Decolourise with 1% acid alcohol Wash with tap water Add 0.1% potassium permanganate

stain 15 sec Wash with tap water Dry- do not blot Exam by fluorescence microscopy

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AURAMINE PHENOL STAIN Auramine O powder 3 gm Crystalline Phenol 30 gm Distilled water 1 liter Dissolve phenol in water with gentle

heat Add auramine slowly Shake vigorously until dissolved Filter & store in stoppered bottle

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POTASSIUM PERMANGANATE STAIN Potassium permanganate 2 gm Distilled water 2 liters Add Potassium permanganate to

distilled water Shake to dissolve Keep in stoppered bottle

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1% ACID ALCOHOL DECOLORIZER Conc.HCl 20 ml Methylated spirit 1980 ml Pour Methylated spirit into large flask Place flask in cold water Add Conc.HCl and cover the top Leave for 10 min Keep in stoppered bottle

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FLUOROCHROME STAINING

Cryptococcus spore Tubercle bacilli

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ACRIDINE – ORANGE TECHNIQUE

Cover the unfixed dried smear with acridine orange acid stain for 5 – 10sec

Wash off stain Decolorize with alcohol saline solution

for 5-10 sec Rinse with saline Put a cover glass Examine by fluorescence microscopy

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ACRIDINE – ORANGE STAIN

Acridine – orange 0.13gm Glacial acetic acid 10 ml Distilled water 490 ml Mix well filter

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ALCOHOL SALINE SOLUTION Absolute ethanol 5ml Saline 245 ml Fill the flask with saline Add Ethanol

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SALINE Sodium chloride 8.5 gm Distilled water 1000ml Mix well Filter

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RESULT T. vaginalis - orange red with yellow

green nucleus Yeast cells – orange Bacteria – orange Pus cells – yellow green Epithelial cells – yellow green

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ACRIDINE – ORANGE STAIN

Trypanosoma brucei Clostridium tetani

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Thanks….