gram stain demonstration
DESCRIPTION
Gram staining, Gram's staining, Gram staining in dermatology, Gram staining methodTRANSCRIPT
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Gram staining (technique and applications)
Dr. Sanjay Singh
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Hans Christian Joachim Gram Danish Bacteriologist
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Gram staining
• A staining technique used to classify bacteria• Those that retain the gentian violet are Gram-positive and those that
do not retain it are Gram-negative
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Cell Wall in GRAM +VE & GRAM –VE Bacteria Cell Wall Structures Gram Positive
organismsGram Negative
organisms
Inner cytoplasmic membrane
Present Present
Peptidoglycan layer Thick Thin
Teichoic Acid Present Absent
Outer membrane layer Absent Present
Lipid A, LPS , Lipo-protien components
Absent Present
Peri-plasmic space Absent Present
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REAGENTS USED IN GRAM STAIN
1. Gram Crystal Violet 0.5%
2. Gram Iodinea) Potassium Iodide 2%b) Resublimed Iodine 1%
3. Gram Decolorizera) Methanol 80%b) Acetone 20%
4. Gram Safranine 1%
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1. CRYSTAL VIOLET• Primary stain• Violet colored, stains all micro-organism
2. GRAM IODINE• Mordant• Forms Crystal violet iodine complexes
3. DECOLORIZER• Acetone + Methanol• Removes Crystal violet iodine complex from thin peptidoglycan layers• Dissolves outer layer of Gram negative org
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4. GRAM SAFRANINE • Counter stain• Red colored• Stains thin walled Gram neg organism• Pus cells cytoplasm & lobes of nuclei also stain red
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• Step 1 - Prepare a Smear
• Put some of the material to be stained on a microscope slide, make thin smear. Allow to air dry.
•Heat fix by gently warming
The Gram Stain Procedure
Watch what happens to the “Bacteria” at each step
“Bacteria”
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Step 2 - Apply the Primary Stain
Flood the Smear with Crystal Violet
Allow to stand for 1 min
Rinse with water to remove excess stain
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Step 3 - Apply the Mordant
Flood the Smear with Iodine solution
Allow to stand 1 min
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Step 4 - Rinse
Rinse with water to remove excess Iodine
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Step 5 - Decolorize
Drip Decolorizer (Acetone) across the slide about 5 sec
The effluent should appear pale or clear
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Step 6 - Rinse
Rinse with water to remove excess acetone
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Step 7 - Counterstain
Flood the slide with Safranin solution
Let stand for 1 minutes
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Step 8 - Rinse, Dry and Observe
Gram-Positive Gram-Negative
Rinse with water to remove excess stain
Blot dry
Observe under Oil Immersion
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Choosing a Right Smear
Before choosing a field for microscopic examination, it is important to look at the smear macroscopically
Note that the smear is easily visible in ordinary light
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Correct preparation
• Proper smear preparation produce a monolayer of organisms sufficiently dense for easy visualization but thin enough to reveal characteristic morphological characteristics.
• Use clean, new glass slides.
• The length of time that crystal violet and Gram’s iodine are left on the smear is not critical.
• In general, the decolorizing solution is rinsed across the smear until the decolorizing fluid is no longer blue.
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Colors makes the Difference in Gram staining
• Bacteria that manage to keep the original purple are called Gram positive. • Bacteria that lose the original
purple dye and can therefore take up the second red dye are called Gram negative
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Report as follows
If no microorganisms are seen in a smear of a clinical
specimen, report “No microorganisms seen.”
If microorganisms are seen, report relative numbers and describe morphology.
Observe predominant shapes of microorganisms
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S. Pneumonie
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• S aureus
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S. salivarius
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Vaginal Gram Stain of candida InfectionEpithelial cells with yeast cells (purple) and yeast pseudohyphae (long purple thread-like structure).
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• Candida albicans seen as a pseudohyphae at lower left and individual yeast cells at upper right
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• C. diptheriae
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Nocardia : form filaments (reminiscent of fungal hyphae) and exhibit branching & beaded appearance in the gram stain
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• Actinomycetes
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Cryptococcus neoformans
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GRAM VARIABLE
Gram variability Old cultures
Decolorize improperly
Dead and alive bacteria together
Autolytic organisms e.g. Streptococcus pneumoniae
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• Thank You