If iodine step missed in gram stain what will happen?

Answer 1

Iodine (I - or I3 - ) interacts with CV+ and forms large complexes of crystal violet and iodine (CV-I) within the inner and outer layers of the cell. Iodine is often referred to as a mordant, but is a trapping agent that prevents the removal of the CV-I complex and, therefore, color the cell.[10]

Answer 2

Staining mechanism

Gram positive bacteria have a thick mesh-like cell wall made of peptidoglycan (50-90% of cell wall), which stains purple while Gram-negative bacteria have a thinner layer (10% of cell wall), which stains pink. Gram-negative bacteria also have an additional outer membrane which contains lipids, and is separated from the cell wall by the periplasmic space. There are four basic steps of the Gram stain, which include applying a primary stain (crystal violet) to a heat-fixed smear of a bacterial culture, followed by the addition of a trapping agent (Gram's iodine), rapid decolorization with alcohol or acetone, and counterstainingwith safranin or basic fuchsin.

Crystal violet (CV) dissociates in aqueous solutions into CV+ and chloride (Cl - ) ions. These ions penetrate through the cell wall and cell membrane of both Gram-positive and Gram-negative cells. The CV+ ion interacts with negatively charged components of bacterial cells and stains the cells purple.

Iodine (I - or I3 - ) interacts with CV+ and forms large complexes of crystal violet and iodine (CV-I) within the inner and outer layers of the cell. Iodine is often referred to as a mordant, but is a trapping agent that prevents the removal of the CV-I complex and therefore color from the cell

When a decolorizer such as alcohol or acetone is added, it interacts with the lipids of the cell membrane. A Gram-negative cell will lose its outer membrane and the lipopolysaccharide layer is left exposed. The CV-I complexes are washed from the Gram-negative cell along with the outer membrane. In contrast, a Gram-positive cell becomes dehydrated from an ethanol treatment. The large CV-I complexes become trapped within the Gram-positive cell due to the multilayered nature of its peptidoglycan. The decolorization step is critical and must be timed correctly; the crystal violet stain will be removed from both Gram-positive and negative cells if the decolorizing agent is left on too long (a matter of seconds).

After decolorization, the Gram-positive cell remains purple and the Gram-negative cell loses its purple color. Counterstain, which is usually positively charged safranin or basic fuchsin, is applied last to give decolorized Gram-negative bacteria a pink or red color.

Some bacteria, after staining with the Gram stain, yield a Gram-variable pattern: a mix of pink and purple cells are seen. The genera Actinomyces, Arthobacter, Corynebacterium, Mycobacterium, and Propionibacterium have cell walls particularly sensitive to breakage during cell division, resulting in Gram-negative staining of these Gram-positive cells. In cultures of Bacillus, Butyrivibrio, and Clostridium a decrease in peptidoglycan thickness during growth coincides with an increase in the number of cells that stain Gram-negative[10] In addition, in all bacteria stained using the Gram stain, the age of the culture may influence the results of the stain.

Answer 3

Gram's iodine is used to enhance retention to the bacterial cells. Upon using crystal violet and washing, it is vital that these cells can actually up take the CV or Safranin O that is typically used as a counterstain. One of the easiest ways to distinguish gram positive cells from gram negative cells is to look at the color. GN are pink. GP are purple. This is of course assuming that the other steps were carried out correctly and you havent over decolorized with the EtOH. Hope this helps.

Answer 4

The gram cells would get discolored by the alcohol treatment and they would be seen as gram - cells.

Answer 5

If you do not put the iodine on a gram + stain the gram cells would get discolored by the alcohol treatment and they would be seen as gram - cells.