Why do gram negative cells stain pink?

Answer 1

Gram negative bacteria actually stain red because they have a less complex cell wall than Gram positive bacteria and they are easily decolorised by the decoloriser acetone alcohol. Hence they take up the colour of the counter stain safranin which is red during the Gram staining procedure.

Answer 2

As a culture ages, bacteria die, therefore the cell wall structure which retains the primary stain (crystal violet) deteriorates. When the loss of cell wall structure occurs so goes the ability of the cell to retain the primary stain. Primary stain washes away, cell retains secondary stain....looks pink.

Answer 3

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 (death by heat) smear of a bacterial culture, followed by the addition of a trapping agent (Gram's iodine), rapid decolorization with alcohol or acetone, and counterstaining with safranin. Basic fuchsin is sometimes substituted for safranin since it will more intensely stain anaerobic bacteria but it is much less commonly employed as a counterstain.

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 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 lipopolysaccharide membrane and the inner peptidoglycan 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. In addition, in all bacteria stained using the Gram stain, the age of the culture may influence the results of the stain.

Answer 4

Why will gram positive cells more than 24 hours old stain gram negative?

Answer 5

Contamination

Answer 6

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