Gram stainGram stain

عمل الطالبة : ديانا عمل الطالبة : ديانا شكري القدرةشكري القدرة

بإشراف الدكتور: عبد بإشراف الدكتور: عبد الرؤوفالرؤوف

History of the GramHistory of the Gram

In 1884, Hans Christian Gram, a In 1884, Hans Christian Gram, a Danish doctor working in Berlin, Danish doctor working in Berlin, accidentally stumbled on a method accidentally stumbled on a method which still forms the basis for the which still forms the basis for the identification of bacteria. While identification of bacteria. While examining lung tissue from patients examining lung tissue from patients who had died of pneumoniawho had died of pneumonia

The Gram staining method, named The Gram staining method, named after the Danish bacteriologist who after the Danish bacteriologist who originally devised itoriginally devised it

It is one of the most important staining It is one of the most important staining techniques in microbiologytechniques in microbiology....

It is almost always the first test performed It is almost always the first test performed for the identification of bacteriafor the identification of bacteria....

Gram developed a staining procedure Gram developed a staining procedure which divided almost all bacteria into which divided almost all bacteria into

two large groups ..(two large groups ..(Gram-positiveGram-positive and and Gram-negativeGram-negative))

Gram positive and negativeGram positive and negative

We first must to know what theWe first must to know what the

Different betweenDifferent between.. ..

Gram positive andGram positive and

gram negative cellsgram negative cells……

Gram positive cellGram positive cell

The gram-positive cell envelope The gram-positive cell envelope consists of a thick layer of consists of a thick layer of peptidoglycan embedded with peptidoglycan embedded with techoic acids and a plasma techoic acids and a plasma membrane comprised of membrane comprised of phospholipids with integral phospholipids with integral membrane proteins traversing the membrane proteins traversing the bilayerbilayer....

Gram negative cellGram negative cell

The gram-negative cell envelope consists The gram-negative cell envelope consists of a thin layer of peptidoglycan of a thin layer of peptidoglycan surrounded by two phospholipid surrounded by two phospholipid membranes, one interior and one membranes, one interior and one exteriorexterior . .

Polysaccharide chains are bound to the Polysaccharide chains are bound to the phosphate heads of the outer phosphate heads of the outer membrane to form lipopolysaccharidesmembrane to form lipopolysaccharides....

Both the Both the membranes membranes contain integral contain integral membrane membrane proteinsproteins . .

Place cursor overPlace cursor over

each membraneeach membrane

for IDfor ID……

Gram staining is based on the ability of Gram staining is based on the ability of bacteria cell wall to retaining the bacteria cell wall to retaining the crystal violet dye during solvent crystal violet dye during solvent treatmenttreatment....

Gram stain procedureGram stain procedure

****Place a slide with a bacterial smear Place a slide with a bacterial smear on a staining rackon a staining rack

****stain the slide withstain the slide with

crystal violetcrystal violet

for 30-60 secfor 30-60 sec..

****Pour off the stainPour off the stain

****Flood slide withFlood slide with

Gram'sGram's iodine iodine

for 1-2 minfor 1-2 min

****Pour off the iodinePour off the iodine....

****Decolourize by washing the slideDecolourize by washing the slide

brieflybriefly with acetone (2-3 seconds) with acetone (2-3 seconds)....

****Wash slideWash slide

thoroughly withthoroughly with

water to removewater to remove

the acetonethe acetone – –

do not delaydo not delay

with this stepwith this step....

****Flood slide withFlood slide with safraninsafranincounterstaincounterstain for 30 secfor 30 sec....

****Wash with waterWash with water....****Blot excess waterBlot excess water

and dry in handand dry in hand over bunsen flameover bunsen flame....

What’s happen to gram negative What’s happen to gram negative cell when we stain itcell when we stain it.. ..

****The cells are floodedThe cells are flooded

with crystal violet dyewith crystal violet dye . .

******The individual crystalThe individual crystal

violet ions penetrate the thin violet ions penetrate the thin peptidoglycan layer of the cell as peptidoglycan layer of the cell as well as the plasma membrane, well as the plasma membrane, making their way through the matrix making their way through the matrix created by the crosslinking of created by the crosslinking of polysaccharides and proteins within polysaccharides and proteins within the peptidoglycan layerthe peptidoglycan layer....

****Gram's iodine is added and penetrate Gram's iodine is added and penetrate the thin peptidoglycan layer of the the thin peptidoglycan layer of the cell. the iodide ions mix with the cell. the iodide ions mix with the crystal violet dyecrystal violet dye....

****The crystal violet and iodide ions react, The crystal violet and iodide ions react, forming a crystal violet-iodine forming a crystal violet-iodine complex. This complex is insoluble in complex. This complex is insoluble in water and produces particles much water and produces particles much larger than either the iodide ions or larger than either the iodide ions or the crystal violet ions individuallythe crystal violet ions individually..

****A decolorizing solution, normally A decolorizing solution, normally consisting of a mixture of ethyl alcohol consisting of a mixture of ethyl alcohol and acetone, is addedand acetone, is added . .

****The mixture displaces water in the The mixture displaces water in the peptidoglycan layer, resulting in peptidoglycan layer, resulting in dehydration. This loss of water causes dehydration. This loss of water causes the thin peptidoglycan layer to shrink the thin peptidoglycan layer to shrink slightly, tightening the matrix created slightly, tightening the matrix created by the crosslinking of polysaccharides by the crosslinking of polysaccharides and proteins. The mixture also and proteins. The mixture also disrupts and dissolves the outer disrupts and dissolves the outer membrane, exposing the membrane, exposing the peptidoglycan layer to the peptidoglycan layer to the environmentenvironment..

Although the thin peptidoglycan layer Although the thin peptidoglycan layer of the gram-negative envelope is of the gram-negative envelope is dehydrated, the crystal violet-iodide dehydrated, the crystal violet-iodide complex can escape through the complex can escape through the large pores that remain. The complex large pores that remain. The complex is eventually washed away, leaving is eventually washed away, leaving colorless, unstained cells, unlike colorless, unstained cells, unlike gram-positive cells which appear gram-positive cells which appear purple at this steppurple at this step..

** **The counterstain, normally safranin, The counterstain, normally safranin, is addedis added..

****The cells are flooded with crystal violet The cells are flooded with crystal violet dye. Crystal violet is a water-soluble, dye. Crystal violet is a water-soluble, basic dyebasic dye

****The individual crystal violet ions The individual crystal violet ions penetrate the thick peptidoglycan layer penetrate the thick peptidoglycan layer of the cell as well as the plasma of the cell as well as the plasma membranemembrane..

****Gram's iodine solution is addedGram's iodine solution is added****the iodide ions are also able to penetrate the iodide ions are also able to penetrate

the thick peptidoglycan layer of the cellthe thick peptidoglycan layer of the cellThe crystal violet and iodide ions forming The crystal violet and iodide ions forming

large complexlarge complex.. ..

What’s happen to gram positive What’s happen to gram positive cell when we stain itcell when we stain it.. ..

decolorizing solution, is addeddecolorizing solution, is added . .It displaces water in the peptidoglycan It displaces water in the peptidoglycan layer, resulting in dehydration. This layer, resulting in dehydration. This loss of water causes the thick loss of water causes the thick peptidoglycan layer to shrink, peptidoglycan layer to shrink, tightening the matrix created by the tightening the matrix created by the crosslinking of polysaccharides and crosslinking of polysaccharides and proteinsproteins..Because of its larger size, the crystal Because of its larger size, the crystal violet-iodine complex is blocked from violet-iodine complex is blocked from moving easily through the thick layers moving easily through the thick layers of dehydrated peptidoglycan and of dehydrated peptidoglycan and exiting the bacterial cellexiting the bacterial cell . .

****The counterstain, normally safranin, The counterstain, normally safranin, is addedis added because of its small sizebecause of its small size

of safranin is ableof safranin is able to penetrate theto penetrate the dehydrateddehydrated peptidoglycan layerpeptidoglycan layer

** ** When viewed under a microscope, When viewed under a microscope, gram-positive cells appear purple gram-positive cells appear purple due to the crystal violet-iodine due to the crystal violet-iodine complex retained insidecomplex retained inside

The endThe end