Question

What is a gram stain and when is it useful?

Answer 1

Gram stain is an inexpensive and most effective method to differentiate b/w gram positive and gram negative bacteria.

Explanation:

Hans Christain Gram was the first person who used gram staining technique to differentiate b/w gram positive and gram negative bacteria. This method is also known as differential stain.

Following is concept to steps of gram stain procedure:

• Heat Fixation:
  In `1^(st)` step, slide with biological sample containing two classes of bacteria(gram -ve & +ve) is passed through a flame. So that bacteria properly stick to the surface of slide.

• Crystal-violet dye(Primary dye):
  In `2^(nd)` step, heat fixed sample of bacteria is stained with a crystal-violet`(CV^+)` dye . The crystal violet dye(a purple colour dye) interact with peptidoglycan layer of both gram positive and gram negative bacteria and render them with `color(purple)("purple")` colour.

• Gram's iodine(Mordant):
  In `3^(rd)` step, gram's iodine(a solution potassium iodide and Iodine) is added. Iodine is negatively charged i.e `I^-`. Thus, it gets attached to positively charged `CV^+` and form `"CV-I"` complex. Hence, it enhances affinity of bacterial cells for primary dye. In simple words, it help crystal violet dye to link properly with peptidoglycan layer in bacterial cell walls or fixes primary dye.

• Decolorisation solution:
  In `4^(th)` step, a decolorizing solution i.e ethyl alcohol or acetone is added to sample. This will wash out the crystal violet dye from gram negative bacteria while gram positive bacteria will retain `color(purple)("primary dye")`. The reason is: gram positive bacteria have `50%` peptidoglycan while cell wall of gram negative bacteria constitutes `10%` peptidoglycan(considering dry weight). This, thinner peptidoglycan layer of gram negative bacteria is easily degraded and bacteria lose the primary dye.

• Safranin(counter stain):
  In `5^(th)` `&` final step, `color(hotpink)("safranin")` is added for further distinction b/w two types of bacteria. The gram negative bacteria bearing no colour are stained `color(hotpink)("pink")` after it's addition while gram negative bacteria having their thick peptidoglycan layer retain the purple dye at the end of procedure.

Hope it helps!