Description of a Tuberculosis Gram Stain

Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis. Tuberculosis is a respiratory disease characterized by formations of tubercules, necrosis, abscesses, fibrosis and calcification in the lungs. It can also infect the kidneys, lymph nodes, bone, joints and skin.

Primary laboratory diagnosis of Mycobacterium tuberculosis involves staining to visualize the bacteria under the microscope. The specimen of choice is sputum or bronchoalveolar lavage to identify a respiratory infection or an actual tissue sample.

Physiology

• Mycobacterium tuberculosis is a rod-shaped (bacilli), non-motile, non-sporing, obligate aerobe. They measure 0.4x3 micrometers in length. Mycobacterium tuberculosis undergoes binary fission (reproductive cell division) every 15 to 24 hours, which is slow compared to other bacteria. Because of this, the mycobacterium can take up to nine weeks to appear on culture media.

  Mycobacterium tuberculosis is sensitive to UV light and heat (e.g. pasteurization) but resistant to chemical disinfectants.

Gram Stain

• Mycobacterium tuberculosis cannot truly be classified as gram positive or gram negative. The gram classification is based upon the amount of peptidoglycan in a bacterium's cell wall. A gram positive bacterium has a thick peptidoglycan layer (about 90 percent), while a gram negative bacterium has a thin peptidoglycan layer (5-20 percent). The gram dyes penetrate the cell wall and bind with the chemical components in the cell wall. The stained bacteria will appear purple or pink under the microscope.

  With Mycobacterium tuberculosis, the cell wall is composed of peptidoglycan but also complex lipids, which inhibit gram dye penetration. The cell wall also resists decolorization by acid or alcohol, so the mycobacterium are termed "acid-fast." The cell wall lipids are mycolic acid, cord factor and wax-D. If the bacteria were to be stained, the bacterial cells would appear weakly gram-positive or pale with very little color.

  To visualize the bacteria, special stains such as the Aumarine stain or the Zhiel-Neelson stain must be performed.

Auramine-rhodamine stain

• The Auramine-rhodamine process uses a yellow fluorescent dye to visualize Mycobacterium tuberculosis under a fluorescence microscope. Potassium permanganate or acridine orange can be used as a counterstain. Under the lens, the bacterial cells will appear green.

  The Auramine-rhodamine stain is more sensitive than the Zhiel-Neelson and more cost effective.

Ziehl-Neelson stain

• The Ziehl-Neelson stain originated from scientists Franz Ziehl and Friedrich Neelsen. It uses heated carbol fuschin, high concentration acid-alcohol and a counterstain such as malachite green or methylene blue. Under a fluorescence microscope, the bacterial cells will appear bright pink.

  The Ziehl-Neelson is the gold standard for primary identification of Mycobacterium tuberculosis and Mycobacterium leprae.

Agar

• Secondary to microscopy identification, Mycobacterium tuberculosis must be grown on special culture agar. Lowenstein-Jensen agar has complex organic substances (e.g. egg, potato), salts, glycerol and malachite green (inhibits other bacteria). Antibiotics may be added to further inhibit contaminating bacteria. The specimen is placed onto this agar, and the bacteria grow in three to nine weeks.

  A culture solution of nutrients such as albumin and biotin can be used to support the growth of even the smallest quantity of the target bacteria. This is known as Middlebrook 7H9 (or 7H12) medium.

  The colonies will appear as small, buffed, light brown colonies on both.

  With advanced technology, Mycobacterium tuberculosis can now be grown using an automated reader. A liquid solution of Middlebrook 7H9 medium and a fluorescent indicator compound is used to monitor the growth and report positive or negative infection.