3.1 gram staining

Question 1

step 1 of gram staining

Answer 1

sample is spread on slide and left to airdry
– slide is flooded with crystal violet dye, which adheres to the cell surface of all present bacteria – incubated for 1 minute then rinsed with water.

Question 2

step 2 gram staining

Answer 2

treatment with iodine complexes the dye within the cell wall, intercalating into the peptidoglycan layer and all of the cells remain purple
– after 30 seconds, sample is rinsed with water.

Question 3

step 3 gram staining (gram neg)

Answer 3

– acid alcohol treatment disrupts the cell membrane on the surface of GRAM NEG cells, thus causing the outer membrane, the thin pept layer, and the small smount of bound crystal violet to wash away, gram NEG are colourless.
– slide next washed away with water.

Question 4

step 3 gram staining (gram pos)

Answer 4

the peptidoglycan layer of gram positive cells is very thick, so while some of it may wash away at this step, a large amount still remains and the cells remain purple/ – slide is next washed away with water.

Question 5

step 4 gram staining

Answer 5

sample is then washed with saranin – this pink dye attaches to all of the cell walls.
– excess dye is washed away with water and the sample can be blotted dry.

Question 6

when viewed under microscope after gram staining –

Answer 6

gram - cells appear pink.
because it is faint dye, does not appear on dark purple stained gram + cells.