Three factors that may affect gram stain reaction?

Answer 1

(i) The age of bacterial culture should not be more than 24 h.

At older age cell loses Gram positivity and will appear as Gram negative.

(ii) Application of heat during the fixation of smears is another important step. Too much heating during this step will lead to loss in, Gram positiveness.

(iii) Overcrowding of cells in smear also affects the result, due to improper decolourization.

(iv) Staining reagents should be freshly prepared.

(v) In Gram staining decolourizing step is very important. To obtain satisfactory differentiation, the nature and the exposure time of decolourizing agent should be standardized with the material to be stained. Acetone alone is more powerful decolourizing agent than ethanol.

(vi) It is also important not to allow a .bacterial smear to dry.

There are many variations of original Gram staining procedure

Answer 2

the important factor which effect gram stain reactions is reaction time i.e. if the timing of dyes to be implied like meythlene blue and also timing of iodine expousre effects straining procedure then it comes to the concentraion of dyes which effect gram stain reaction

Answer 3

Using actively growing cells while old cells lose their ability to hold the stain. Prepare thin smears and adjust decolorization time when cells are crowded they resist decolorization. Bacteria in thin smears decolorize faster than thick smears. Avoid overheating cells. Don't rinse too long water is a decolorize. Gram positivity is a characteristic that can be lost.

Answer 4

The age of the culture, the thickness of the smear, and the presence of excess decolorizer can all affect the outcome of a Gram stain reaction. Older cultures may lose their ability to retain the crystal violet stain, thick smears may result in difficulty penetrating the cells with the staining reagents, and excess decolorizer can wash away the crystal violet stain from Gram-positive bacteria.