3- gram staining

8/12/2019 3- Gram Staining

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GRAM STAINING

DR NIVEEN ASHER


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Importance

Helps identify & differentiate between gram

positive and negative organisms.

Original method by Christian Gram in 1884


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BASIC TECHNIQUE

Number of ways to perform gram staining

Basic4 steps

1. Addition of basic aniline dye

2. Lugols iodine.

3. Decoloriser.

4.

Counter stain


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Why few organisms Gram positive

Certain organisms take up the basic dye .

When followed by decolorisation .Some

retain color /Some lose the color

Those that retain----Gram positive

Those that lose-----Counter stained.

They become pink-----Gram negative.


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Cell wall


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MODIFICATIONS

Kopeloff &Beermans (1922) Modification

Jensens Modification.

Weigerts modification

Preston and Morrells modification(1962)


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Differences in modifications

Other Modifications

Decoloriser either

alcohol or acetone

Wait till color stopscoming out of the slide.

Over decolorisation

Preston and Morell

modification

Acetone Iodine

Specific time for everystep

Less chance of false

results


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Preparation of Gram Stain

Preparation of stain

Appropriate amounts of ingredients 5.Dilute carbol Fuchsin

1;Ammonium oxalate crystal violet Zn Carbol Fuchsin 50ml

Crystal violet 20 g Distilled water 950 ml

Methylated spirit 200ml

Ammonium oxalate800ml

2..Lugols Iodine solution

Iodine 10 g

Potassium iodide 20g

Distilled water 10ml

3.Liqou iodi fortis

Iodine 10 gPotassium iodide 6g

Methylated spirit 90ml

Distilled water 10ml

4.Iodine acetone

Liqour iodi fortis 35ml

acetone 965 ml


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Labelling of slides

.

Lab Number

Name of stain

Correlate properly withpatients slip

Use special engraving

pencil or permanent

marker


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Smear making

Spread evenly

Area


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Type of smears

Direct smearPurulent specimens

Non purulent

specimens

Sputum

swabs

Deposit after

centrifugation CSF

and all body fluids

(peritoneal, pleural,

synovial )
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Drying of smears

Air dry

Avoid contamination due to flies and insects.

If delay in staining expected ..put in a

covered container..

Transport also in covered containers


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HEAT Fixation

To preserve the smear

To avoid washing

away of smear duringstaining


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Heat fixation

Air dry the smear

Rapidly pass slide withsmear up

at least threetimes,through the flame

Allow to cool

Do not over heat.Thismay destroy organisms,give false results.

Unfit for intra cellularorganisms


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Method of Gram staining

Air Dry and heat fix the

smear

Cover with crystal violet

for 30 sec


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Wash with clean water - 30 sec


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Cover with lugols iodine for 30 sec


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Wash off with tap water


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Cover with dilute carbol fuchsin(freshly

prepared)

30 sec


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Wash off with clean water


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Wipe the back of the slide and put in

draining rack


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Examine the slide

40 x to check the

staining.

100x oil immersion
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Results

Gram positive bacteria-----dark purple

Yeast cells -----dark purple

Gram negative -----pale to dark red

Nuclei of pus cells ------red

Epithelial cells -------pale red


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Gram positive cocci


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Quality control

Filtration of Stain

To reduce artefacts

Increase appreciability of organism


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QC Check the stain with a

known Gram positive +Known Gram negative

Organism on the same

slide
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Storage

Screw capped leak proof amber

coloured bottle

Older the better (mature)


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Why Gram positive appear gram negative

Cell wall of gram positive damaged

Over decolorisation

Old grams iodine

Old bacterial culture

3- gram staining

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