Mechanism of Gram Staining & L forms

 

Mechanism of Gram Staining

• Christian Gram - Gram stain in 1884- based on the differences in cellwall

• The difference between gram-positive and gram-negative bacteria is due to the physical nature of their cell walls. If the cell wall is removed from gram- positive bacteria, they become gram negative. 

                            

• During the procedure the bacteria are first stained with crystal violet and next treated with Gram’s iodine (mordant) to promote dye retention. 

• When gram-positive bacteria then are decolorized with ethanol, the alcohol shrinks the pores of the thick peptidoglycan. Thus the dye-iodine (CV-I) complex is retained during the short decolorization step and the bacteria remain purple. 

• In contrast, gram-negative peptidoglycan is very thin, not as highly cross-linked, and has larger pores. Alcohol treatment also dissolves lipid from the gram- negative wall to increase its porosity further. For these reasons, alcohol more readily removes the purple crystal violet-iodine complex from gram-negative bacteria. They then absorb the colour of counter stain – Safranin. 
  

• Thus, Gram-positive bacteria stain purple, whereas gram-negative bacteria colour pink or red 

  

Effect of Lysozyme, Penicillin on Cellwall

The enzyme lysozyme attacks peptidoglycan by hydrolysing the bond between N-acetylmuramic acid with N-acetylglucosamine

• Penicillin inhibits peptidoglycan synthesis; inhibits transpeptidases which perform crosslinking of peptidoglycan . 

In an osmotically protective medium, Gram-positive bacteria are converted to protoplasts which completely lack a wall.  Gram-negative cells retain their outer membrane after penicillin treatment and are classified as spheroplasts because some of their cell wall remains.

• Protoplasts and spheroplasts are osmotically sensitive - in a dilute solution, they will lyse due to uncontrolled water influx

• Mycoplasmas lack a cell wall (pleomorphic) and are osmotically sensitive, can grow in dilute media or terrestrial environments because of stronger plasma membranes; the presence of sterols in the membranes provide added strength.

 Such protoplasts and spheroplasts are non-viable and cannot multiply, If they grow and divide, they form L forms

L-phase bacteria, L-phase variants, and cell wall-deficient (CWD) bacteria

• bacteria that lack cell walls

• When bacteria are treated with enzymes that hydrolyze the cell wall (e.g. lysozyme) or antibiotics that interfere with biosynthesis of peptidoglycan (penicillin), wall-less bacteria are often produced. 

• Such a treatment of bacteria in osmotically protective medium liberates protoplasts from gram positive bacteria and spheroplasts from gram negative bacteria. Spheroplasts retain the outer membrane.  

• Usually these treatments generate wall-less non-viable organisms that do not multiply. However, if such cells can grow and divide, they are called L forms.

• L forms were first reported by Klieneberger Nobel in cultures of Streptobacillus monoliformis. They are named L forms after Lister Institute, where they were discovered. 

• They are produced more readily with penicillin than with lysozyme.

• Some L forms are stable and some are unstable.  

• Unstable forms (spheroplasts) are those which revert back to cell wall containing state when inducing stimulus (penicillin) is removed. Such forms usually have small amounts of residual peptidoglycan that serves as primer for building cell wall. 

• Stable forms ( protoplasts) do not revert back to normal form since they completely lack peptidoglycan. 

Significance of L forms: L forms may produce chronic infections in the host. They may persist in protective regions of the body. Since L forms are relatively resistant to antibiotics, they are difficult to treat. Their reversion to normal form can result in relapse of infection.

• Since they lack cell wall, they don’t have a definite shape. 

• L forms are difficult to cultivate and require medium that has right osmotic strength and low concentration of agar, inactivated serum and sucrose. 
• L forms resemble mycoplasma in morphology, type of growth on agar “fried-egg colony”.

• Mycoplasma, also lack a cell wall but not considered L-forms since they are not derived from bacteria that normally have cell walls. While mycoplasma lack cell wall and have sterols in their membrane, the L forms may have reminiscent of cell wall but do not have sterols in their membrane.