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.

 

Hepatitis B - Laboratory diagnosis & Prophylaxis

 Laboratory diagnosis

 
HBsAg first appears in the blood during the incubation period, while the virus is actively 'replicating' in liver cells. The antigen is produced in vast excess - it is associated with new infectious virus particles as well as occurs in the serum as small non-infectious spherical and filamentous forms.

In acute infection, the HBsAg usually disappears within 3 months of onset. 

The HBeAg and HBV DNA can also be detected in blood while the virus is actively replicating in the liver . Symptoms usually appear as the concentrations of bilirubin, alanine aminotransferase and each of the major viral components peak in the serum. These events coincide with the first appearance of antibodies to HBV proteins

Antibody to HBcAg (anti-HBc) rises first. As a generalisation, the detection of IgM antibody specific to the hepatitis B core is the primary indicator of acute infection. It usually appears at or just before the onset of symptoms and remains detectable for at least 6 months. The IgG component of anti-HBc usually persists for life. 

Anti-HBe is the second antibody to appear and is associated with the rapid clearance of HBeAg. Later, anti-HBe declines and persists for only a few months or years if there is no active viral replication.

The antibody to HBsAg, anti-HBs, may not become detectable for 3-6 months after acute infection. It is associated with resolution of the illness. This antibody is recognised as the marker of immunity to HBV.

Detection of HBsAg + IgM HBcAg (anti-HBc) -recent/acute infection

Detection of HBsAg + IgG HBcAg (anti-HBc) -remote infection

Detection of HbeAg - High infectivity

Nondetection of HbeAg-low infectivity

Primary & secondary tests to diagnose/monitor hepatitis B virus (HBV) infection

	Marker	Incubation period	Acute infection	Past/resolved infection	Chronic infection	Vaccination
For diagnosis and monitoring
	HBsAg	±	+	–	+	–*
	Anti-HBs	–	–	+	–	+
	Anti-HBc- Total	–	±	+	+	–
	Anti-HBc- IgM	–	+	–	±†	–
	HBeAg	±	+	–	±	–
	Anti-HBe	–	–	±	± ‡	–
	HBV-DNA	±	+	±	+	–

^*Recent HBV vaccination within one to two weeks can lead to a false-positive test. The vaccine antigen can be detected at low levels;

^†May be positive in chronically infected individuals;

^‡Patients with chronic HBV infection usually have detectable Hepatitis B e antigen (HBeAg) or antibody to hepatitis B e protein (anti-HBe). Rarely, both HBeAg and anti-HBe can be detected simultaneously;

Interpretation of HBV Immunologic Markers

Markers				Interpretation
HBsAg*	HBcAb†	HBsAb‡
–	–	–	HBV infection
–	–	+	Immune because of vaccination
–	+	+	Immune because of natural HBV infection
+	+	–	Acute or chronic HBV infection
–	+	–	Interpretation unclear; four possibilities: l  Resolved HBV infection (most common) l  False-positive HBcAb l  “Low-level” chronic HBV infection l  Resolving acute HBV infection

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HBcAb = hepatitis B core antibody; HBsAb = hepatitis B surface antibody; HBsAg = hepatitis B surface antigen; HBV = hepatitis B virus; + = positive test result; – = negative test result.

*— The presence of HBsAg indicates that the person is infectious.

†—HBcAb appears at the onset of acute HBV infection. Presence may also indicate chronic HBV infection or a false-positive test.

‡—The presence of HBsAb indicates recovery and immunity from HBV infection or successful immunization against HBV.

A recently infected chronic carrier will show evidence of ongoing viral replication in liver cells. HBeAg and HBV DNA can be detected in blood for months or years after acute infection.

Treatment

No specific antiviral treatment  for acute HBV infection. The main treatment is symptom relief.  Special consideration should be given to any medications that a patient takes to see the effect it may have on an impaired liver.

Interferon alpha alone or in combination with other antiviral agents such as lamivudine or famciclovir is useful in treating chronic hepatitis.  There is no treatment for carrier state though some show spontaneous resolution. 

Patients should avoid alcohol and cigarettes which can aggravate the liver.  

The only safe and effective measure for prevention is universal active immunisation. In 1992, WHO has recommended the integration of Hepatitis B vaccine into the immunisation programmes of all nations. India is lagging behind due to the high cost of imported vaccine. Development of low cost vaccine in India should help include this in national immunisation schedule.