Gram staining (technique and applications)

Dr. Sanjay Singh

Hans Christian Joachim Gram Danish Bacteriologist

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

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

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%

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

4. GRAM SAFRANINE • Counter stain• Red colored• Stains thin walled Gram neg organism• Pus cells cytoplasm & lobes of nuclei also stain red

• 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”

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

Step 3 - Apply the Mordant

Flood the Smear with Iodine solution

Allow to stand 1 min

Step 4 - Rinse

Rinse with water to remove excess Iodine

Step 5 - Decolorize

Drip Decolorizer (Acetone) across the slide about 5 sec

The effluent should appear pale or clear

Step 6 - Rinse

Rinse with water to remove excess acetone

Step 7 - Counterstain

Flood the slide with Safranin solution

Let stand for 1 minutes

Step 8 - Rinse, Dry and Observe

Gram-Positive Gram-Negative

Rinse with water to remove excess stain

Blot dry

Observe under Oil Immersion

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

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.

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

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

S. Pneumonie

• S aureus

S. salivarius

 Vaginal Gram Stain of candida InfectionEpithelial cells with yeast cells (purple) and yeast pseudohyphae (long purple thread-like structure).

• Candida albicans seen as a pseudohyphae at lower left and individual yeast cells at upper right

• C. diptheriae

Nocardia : form filaments (reminiscent of fungal hyphae) and exhibit branching & beaded appearance in the gram stain 

• Actinomycetes

Cryptococcus neoformans

GRAM VARIABLE

Gram variability Old cultures

Decolorize improperly

Dead and alive bacteria together

Autolytic organisms e.g. Streptococcus pneumoniae

• Thank You