GRAM STAINING : Mechanism , Limitations and Modified techniques

GRAM STAINING: Mechanism , Limitations and Modified techniques

Gram stain or Gram staining, also called Gram’s method, is a method of staining used to classify bacterial species into two large groups: gram-positive bacteria and gram-negative bacteria. The name comes from the Danish bacteriologist Hans Christian Gram, who developed the technique.

Gram staining differentiates bacteria by the chemical and physical properties of their cell walls. Gram-positive cells have a thick layer of peptidoglycan in the cell wall that retains the primary stain, crystal violet. Gram-negative cells have a thinner peptidoglycan layer that allows the crystal violet to wash out with the addition of ethanol. They are stained pink or red by the counterstain, commonly safranin or fuchsine. Lugol’s iodine solution is always added after the addition of crystal violet to strengthen the bonds of the stain with the cell membrane. Gram staining is almost always the first step in the preliminary identification of a bacterial organism. While Gram staining is a valuable diagnostic tool in both clinical and research settings, not all bacteria can be definitively classified by this technique. This gives rise to gram-variable and gram-indeterminate groups.

Staining mechanism

Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan (50–90% of cell envelope), and as a result are stained purple by crystal violet, whereas gram-negative bacteria have a thinner layer (10% of cell envelope), so do not retain the purple stain and are counter-stained pink by safranin. There are four basic steps of the Gram stain:

 

1. Applying a primary stain (crystal violet) to a heat-fixed smear of a bacterial culture. Heat fixation kills some bacteria but is mostly used to affix the bacteria to the slide so that they don’t rinse out during the staining procedure.

2. The addition of iodine, which binds to crystal violet and traps it in the cell

3. Rapid decolorization with ethanol or acetone

4. Counterstaining with safranin. Carbol fuchsin is sometimes substituted for safranin since it more intensely stains anaerobic bacteria, but it is less commonly used as a counterstain.

Crystal violet (CV) dissociates in aqueous solutions into CV+ and chloride (Cl−)  ions. These ions penetrate the cell walls of both gram-positive and gram-negative cells. The CV+ Ion interacts with negatively charged components of bacterial cells and stains the cells purple.

Iodide (I− Or I−3) 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, colors 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 loses 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 is 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 fuchsine, is applied last to give decolorized gram-negative bacteria a pink or red color. Both gram-positive bacteria and gram-negative bacteria pick up the counterstain. The counterstain, however, is unseen on gram-positive bacteria because of the darker crystal violet stain.

Gram-positive bacteria

Gram-positive bacteria generally have a single membrane (monoderm) surrounded by a thick peptidoglycan. This rule is followed by two phyla: Firmicutes (except for the classes Mollicutes and Negativicutes) and Actinobacteria.In contrast, members of the Chloroflexi (green non-sulfur bacteria) are monoderms but possess a thin or absent (class Dehalococcoidetes) peptidoglycan and can stain negative, positive or indeterminate; members of the Deinococcus–Thermus group stain positive but are diderms with a thick peptidoglycan.

Historically, the gram-positive forms made up the phylum Firmicutes, a name now used for the largest group. It includes many well-known genera such as Lactobacillus, Bacillus, Listeria, Staphylococcus, Streptococcus, Enterococcus, and Clostridium. It has also been expanded to include the Mollicutes, bacteria such as Mycoplasma and Thermoplasma that lack cell walls and so cannot be Gram-stained, but are derived from such forms.

Some bacteria have cell walls that are particularly adept at retaining stains. These will appear positive by Gram stain even though they are not closely related to other Gram-positive bacteria. These are called acid-fast bacteria, and can only be differentiated from other gram-positive bacteria by special staining procedures.

Gram-negative bacteria

Gram-negative bacteria generally possess a thin layer of peptidoglycan between two membranes. Lipopolysaccharide (LPS) is the most abundant antigen on the cell surface of most Gram-negative bacteria, contributing up to 80% of the outer membrane of E. coli and Salmonella. Most bacterial phyla are gram-negative, including the cyanobacteria, green sulfur bacteria, and most Proteobacteria (exceptions being some members of the Rickettsiales and the insect-endosymbionts of the Enterobacteriales).

Gram-variable and Gram-indeterminate bacteria

Some bacteria, after staining with the Gram stain, yield a gram-variable pattern: a mix of pink and purple cells are seen. 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.

Gram-indeterminate bacteria do not respond predictably to Gram staining and, therefore, cannot be determined as either gram-positive or gram-negative. Examples include many species of Mycobacterium, including Mycobacterium bovis, Mycobacterium leprae, and Mycobacterium tuberculosis, the latter two of which are the causative agents of leprosy and tuberculosis, respectively. Bacteria of the genus Mycoplasma lack a cell wall around their cell membranes, which means they do not stain by Gram’s method and are resistant to the antibiotics that target cell wall synthesis.

Limitations of Gram staining:

Some Gram-positive bacteria may lose the stain easily and therefore appear as a mixture of Gram-positive and Gram-negative bacteria (Gram-variable). When over-decolorized, even Gram-positive bacteria may appear pink and when under-decolorized gram-negative bacteria may appear Gram-positive.

The Gram reaction also depends on the age of the cell. Old cultures of Gram-positive bacteria (where cell walls may be weakened) may readily get decolorized. Gram-positive cells affected by cell wall active agents such as lysozyme or antibiotics may become Gram-negative. Gram-positive bacteria such as Actinomyces, Arthobacter, Corynebacterium, Mycobacterium, and Propionibacterium have cell walls particularly sensitive to breakage during cell division, resulting in Gram-negative staining of these cells. In cultures of Bacillus, and Clostridium a decrease in peptidoglycan thickness during cell growth may cause some of them to appear Gram-negative.

Certain groups of bacteria can display variable responses to the stain, which can be due to growth stress (e.g., unsuitable nutrients, temperatures, pHs, or electrolytes) that results in a number of nonviable, gram-negative cells in a gram-positive culture, but certain bacterial species are known for their gram variability even under optimal growth conditions. Some bacteria tend to appear Gram-negative when grown in an acidic medium.

Loss of cell walls in Gram-positive bacteria may render them Gram-negative (L-forms). Bacteria totally devoid of cell walls (Mycoplasma) are always Gram-negative. Bacteria such as Mycobacterium that have extra waxy content in their cell wall are difficult to stain. Small and slender bacteria such as Treponema, Chlamydia, and Rickettsia are often difficult to stain by Gram’s method. Gram-positive bacteria that have been phagocytosed by polymorphs may also appear Gram-negative.

MODIFIED GRAM STAINING TECHNIQUES :

Following are the modified gram staining techniques :

1.      Kopeloff and Beerman’s modification

2.      Jensen’s modification

3.      Preston’s and Morrell’s modification

4.      Weigert modification