3- gram staining
TRANSCRIPT
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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 )
http://images.google.com.pk/imgres?imgurl=http://biotek.com.au/products/catalog/images/50%2520ml%2520Centrifuge%2520Tube.jpg&imgrefurl=http://www.biotek.com.au/products/catalog/index.php%3FcPath%3D85&usg=__JdRRI4Ea29xQD8xFMGBLXFYgS6c=&h=440&w=336&sz=11&hl=en&start=12&um=1&tbnid=lEzuua_lmLtMqM:&tbnh=127&tbnw=97&prev=/images%3Fq%3DCENTRIFUGE%2BIN%2BMICROBIOLOGY%26hl%3Den%26sa%3DX%26um%3D1http://images.google.com.pk/imgres?imgurl=http://www.marketlabinc.com/files/products/images/medium/ml5400Centrifuge.jpg&imgrefurl=http://www.marketlabinc.com/products/details/752&usg=__WTXwze9uFy3EvyK3cEBU22ad_Pg=&h=290&w=290&sz=24&hl=en&start=16&um=1&tbnid=foADykWnQWB4jM:&tbnh=115&tbnw=115&prev=/images%3Fq%3DCENTRIFUGE%2BIN%2BMICROBIOLOGY%26hl%3Den%26sa%3DX%26um%3D1 -
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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
http://images.google.com.pk/imgres?imgurl=http://www.medical-supplies-equipment-company.com/files/images/product/M5-P.jpg&imgrefurl=http://www.medical-supplies-equipment-company.com/lab-equipment-supplies/product/ppf/id/21643/new_prod_full.asp&usg=__NzXflBlk6PL6pmxxGiDdZAnJ28Q=&h=301&w=250&sz=15&hl=en&start=10&um=1&tbnid=L2W8lobJiuie0M:&tbnh=116&tbnw=96&prev=/images%3Fq%3DCENTRIFUGE%2BIN%2BMICROBIOLOGY%26hl%3Den%26sa%3DX%26um%3D1 -
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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
http://images.google.com.pk/imgres?imgurl=http://scienceprofonline.googlepages.com/SafrininstepGramstain.JPG/SafrininstepGramstain-full.JPG&imgrefurl=http://scienceprofonline.googlepages.com/virtualmicrobiologyclasslaboratory23&usg=__u5PZEOQ2dco2JDGpndRbhsuICms=&h=375&w=500&sz=50&hl=en&start=25&um=1&tbnid=Tu6Z04u5-ZikHM:&tbnh=98&tbnw=130&prev=/images%3Fq%3Dgram%2Bstain%2Bin%2Bmicrobiology%26ndsp%3D20%26hl%3Den%26sa%3DN%26start%3D20%26um%3D1 -
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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