IBN PUTRA DWIJA, S.SI.,M.Biotech

Gram staining named after Danish Microbiologist Hans Cristian Gram (1853-1938)

1st used to differentiate of Pneumococci and Klebsiella pneumonia in lung specimen

Devised in 1882, but published in 1884 in Fortschitte der Medicin; 1884, Vol. 2, pages 185-189

Not the same with the method that we used today

Gram staining is differential staining which is differentiate microorganism (bacteria) into two large group (Gram positive and Gram negative) based on their chemical and physical properties of the cell walls

Gram staining is not used to staining Archaea because of variable response

Gram result can be used to decided empirical treatment, before culture of bacteria are finished as an definitive treatment

Gram staining can be used to decided of culture medium because Gram +ve/ -ve need different medium.

CELL WALL OF GRAM POS & NEG

CELL WALL IN GRAM +VE AND GRAM –VE BACTERIA Cell Wall Structures Gram

Positive organisms

Gram 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

Equipment for Gram stainingSlide glass (kaca slide/object glass)Wire loop (ose)Bunsen Gram setPencils glassMicroscope Immersion oilStaining rack

Gram setConsist of :

1. Primary stain (Crystal violet, methyl violet or Gentian violet)

2. Mordant (Gram's Iodine)/lugol 3. Decolourizer (ethyl alcohol, acetone or

1:1ethanol-acetone mixture) 4. Counterstain (Dilute carbol fuchsin, safranin

or neutral red)

The Gram Stain ProcedureStep 1 - Prepare a Smear

Watch what happens to the “Bacteria” at each step

“Bacteria”

Before take the specimen, make sure the loop is sterile

(burn wire loop on the Bunsen burner until red, cooling down a while) Remember : sterile your loop in every single step!!

Take one loop of bacterial culture/ specimen to be stained

Spread on the glass slide into proportional size

Allow to air dry. Heat fix by gently warming

The Gram Stain ProcedureStep 2 - Apply the Primary Stain

Flood the Smear with Crystal Violet

Allow to stand for 1-3 min

Rinse with water to remove excess stain

The Gram Stain ProcedureStep 3 - Apply the Mordant

Flood the Smear with Iodine / Lugol solution

Allow to stand 0,5-1 min

The Gram Stain ProcedureStep 4 - Rinse

Rinse with water to remove excess Iodine

The Gram Stain ProcedureStep 5 - Decolorize

Drip Decolorizer (80% Methanol +20% Acetone)/ alcohol 96% across the slide about 15-30 sec

The effluent should appear pale or clear

The Gram Stain ProcedureStep 6 - Rinse

Rinse with water to remove excess alcohol

The Gram Stain ProcedureStep 7 - Counterstain

Flood the slide with Safranin/Carbol Fuchsin solution

Let stand for 1-3 minutes

The Gram Stain

Step 8 - Rinse and Dry

Gram-Positive Gram-Negative

Rinse with water to remove excess stain

Dry with tissue, don’t swap the smear, let it air dry.

Observe your Gram slide under microscope with 100x lens

Add immersion oil during observation

Immersion oil make cell more clearly under microscope

Gram positive bacteria : blue-blackGram negative bacteria : red

If you want to make Gram stain from the specimen with a small amout of bacterial cell (e.q. urine, CSF etc) centrifuges its first to suspend the cell and make the smear as usually.

REMEMBER : Gram staining is only used for empiricallytreatment. Bacterial culture is the gold

standart

Thank you