Question

Steps of Gram Staining Bacteria containing thick cell wall Violet color Bacteria containing thin cell wall (LPS) Violet color Crystal violet treatment lodine Purple color Purple color Decolorization Purple color colorless Safranin Purple color pink 3. A fellow student showed you a gram stained slide where cells containing thick cell walls were stained pink. What would you tell her about the staining procedure? Why? 4. A fellow student showed you a gram stained slide where cells containing LPS were stained purple. What would you tell her about the staining procedure? Why?

Answer 1

Gram Staining Gram strainig is the process in which gram Positive bacteria having thick peptidoglycan cell wall are differentiated from gram negative bacteria having thin peptidoglycan layer and LPS layer. In gram stainig gram positive bacteria strain purple in gram stainig and gram negative stain pink.

Steps of gram Staining

I) Primary stain (Crystal violet) is applied to to heat fixed smear of bacterial culture.

II) Add the iodine. Iodine bind to crystal violet,form complex and that complex retain in the cell. Till this step all bacteria appear purple coloured.

III) decolurization is performed by 70 percent alcohal. After decouration reaction gram positive bacteria still appears purple coloured but gram negative bacteria become colourless. This is because gram positive bacteria has thick cell wall so ethanol or acetone decourazing agent won't be able to remove crystal violet + iodine complex from gram postive bacterial cell wall. But remove compelx from gram negative cell wall.

IV) Counterstaing with saferinin. Gram negative bacteria get saferinin and become pink coloured but gram postive doesn't get saferinin as they have crystal violet so apper purple.

Q.3 In this slide of gram postive bacteria( having thick cell wall) appear pink. This is possibly due to change in steps of gram stainig .

After adding primary stain (crystal violet) instead of adding iodine he has performed decolurization reaction first and then added iodine and after that adding saferinin counterstain. The cell appear pink because crystal violet- iodine complex will not be formed. only this complex is retained in cell of gram postive as complex size is larger. As he applied decourazing agent crystal violet decourized. then applying iodine doesn't necessory.cells appear colourless. After applying saferinin these thick walled cells appear pink.

One reason may be he has skipped one of crystal violet or iodine step.

Q.4 In LPS and thin cell wall containing gram negative cells, purple colouration occur if decolurization step is not performed. As I mentioned upto addition of iodine all cells appear same purple coloured. If decolurization not done it is then only possible that gram negative cells retain purple colour and not colourless. So these cell will not get saferinin stain as they have purple colour because of crystal violet iodine complex. So appear purple.