Question

After performing a gram stain of a mixed culture of gram positive and gram negative cells, you shout: "oops, i forgot the iodine step!" what would you expect to see if you observed the slide under the microscope?

Answer 1

Gram's staining is a differential staining technique that employs a primary stain like crystal violet and a counter stain like safranin along with the decolourizing agent alcohol and a mordant called the Gram's iodine.

Iodine is a mordant added after the primary stain. It fixes the stain by combining with it to enchance the staining ability. This forms an insoluble crystal violet iodine complex appearing purple under the microscope. These microorganisms are classified as Gram positive.

If addition of iodine is skipped, crystal violet is not fixed on the slide and the insoluble complex is not formed. The cells are decolourized by alcohol and are stained by the counter stain safranin making the Gram positive cells wrongly indentified as Gram negative due to its pink colouration. Thus, the slide will show all the cells as pink coloured Gram negative cells.