What can result in a gram-positive organism staining gram-negative?

Answer 1

The following characteristics are generally present in a Gram-positive bacterium.

1. cytoplasmic lipid membrane
   • teichoic acids and lipoids are present, forming lipoteichoic acids which serve to act as chelating agents, and also for certain types of adherence.
2. thick peptidoglycan layer
3. capsule polysaccharides (only in some species)
   • if present, it contains two rings for support as opposed to four in Gram-negative bacteria because Gram-positive bacteria have only one membrane layer.
4. flagellum (only in some species)

Although there is only difference in gram positive and negative is of cell wall as :

The Gram positive cell wall

The Gram positive cell wall is characterised by the presence of a very thick peptidoglycan layer, which is responsible for the retention of the crystal violet dyes during the Gram staining procedure. It is found exclusively in organisms belonging to the Actinobacteria (or high %G+C Gram positive organisms) and the Firmicutes (or low %G+C Gram positive organisms). Bacteria within the Deinococcus-Thermus group may also exhibit Gram positive staining behaviour but contain some cell wall structures typical of Gram negative organisms. Imbedded in the Gram positive cell wall are polyalcohols called teichoic acids, some of which are lipid-linked to form lipoteichoic acids. Because lipoteichoic acids are covalently linked to lipids within the cytoplasmic membrane they are responsible for linking the peptidoglycan to the cytoplasmic membrane. Teichoic acids give the Gram positive cell wall an overall negative charge due to the presence of phosphodiester bonds between teichoic acid monomers.

The Gram negative cell wall

Unlike the Gram positive cell wall, the Gram negative cell wall contains a thin peptidoglycan layer adjacent to the cytoplasmic membrane, which is responsible for the cell wall's inability to retain the crystal violet stain upon decolourisation with ethanol during Gram staining. In addition to the peptidoglycan layer the Gram negative cell wall also contains an additional outer membrane composed by phospholipids and lipopolysaccharides which face into the external environment. The highly charged nature of lipopolysaccharides confer an overall negative charge to the Gram negative cell wall. The chemical structure of the outer membrane lipopolysaccharides is often unique to specific bacterial strains (i.e. sub-species) and is responsible for many of the antigenic properties of these strains.

As a phospholipid bilayer, the lipid portion of the outer membrane is largely impermeable to all charged molecules. However, channels called porins are present in the outer membrane that allow for passive transport of many ions, sugars and amino acids across the outer membrane. These molecules are therefore present in the periplasm, the region between the plasma membrane and outer membrane. The periplasm contains the peptidoglycan layer and many proteins responsible for substrate binding or hydrolysis and reception of extracellular signals. The periplasm is thought to exist as a gel-like state rather than a liquid due to the high concentration of proteins and peptidoglycan found within it. Because of its location between the cytoplasmic and outer membranes, signals received and substrates bound are available to be transported across the cytoplasmic membrane using transport and signalling proteins imbedded there.

Answer 2

The age of the isolate can affect the stain. Also, over de-colorizing can cause the stain to look red vs. purple.

A gram (+) bacteria staining as a gram (-) bacteria is known as gram variability.

Gram variability can be due to:

1. Old structure of gram positive bacteria

2. 2. Changes in the environment of the organism

3. 3. Modification of staining technique

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Answer 3

Gram-positive organisms staining gram-negative can result from over-decolorization during the Gram staining process, leading to the loss of the crystal violet-iodine complex from the thick peptidoglycan layer of the gram-positive cell wall. This can happen if the decolorizer is left on for too long or if the cell wall of the gram-positive organism is damaged, making it more porous.

Answer 4

Yes, a gram may also test negative. However, this may happen if it is correctly stained. This is mostly due to some corruption on the part of the organism.

Answer 5

If the culture you are staining is more than 24 hours old, Gram positive bacteria can decolorize and stain Gram negative.