Gram staining

Gram staining ismethodstaining samplesbacteria that differentiates betweentwo main typesbacterial cell wall.

Itnamed afterinventor,Danish scientist Hans Christian Gram (1853-1928), who developedtechnique1884discriminate between pneumococciKlebsiella pneumoniae bacteria.

Gram StainingStep by Step Procedure

1. Heat fixspecimen.
2. Addbasic dyestainsample. Crystal violet or gentian violetsuitable. Allowstain1 minute. The slide should look violetcolour.
3. Rinse offwater.
4. Add iodine solution (1% iodine, 2% potassium iodidewater]])1 minute. This acts asmordantfixesdye.
5. Rinsewater.
6. Apply 95% ethanol ormixtureacetonealcohol several times until no more colour appearscome fromsample.This leaves Gram-positive organisms stained purpleGram-negative organisms unstained.
7. Rinsewater.
8. Applysuitable counterstain. Opinions vary as tobest choice but suitable stains include safranin or fuchsin.This stainsgram negative organisms.
9. Blot gentlyallowdry. Do not smear.

Results:

Inspectslide undermicroscope

Gram positive organisms will appear blue-black or purple.

Gram negative organisms will appear red.

Gram-positive bacteria havethick meshlike cell wall madepeptidoglycan whichcapableretainingviolet dye. Gram negative bacteria havethin cell wall made oflayerpeptidoglycan surrounded by an outer membrane containing lipids.

Asrulethumb (which has exceptions), Gram-negative bacteriamore dangerous as disease organisms, because their outer membrane acts as "camouflage";human body does not contain peptidoglycanin fact produces an enzyme called lysozyme which attacksopen peptidoglycan layerGram-positive bacteria. Gram-positive bacteriaalso much more susceptiblepenicillin.

• Gram-positive bacteria
• Gram-negative bacteria