Monday, 27 September 2010

Mycobacterium tuberculosis in Gram Stain

Mycobacterium tuberculosis
(In Gram Stain Of Peritoneal Dialysis Fluid)

So, you find yourself the lone technologist manning the micro lab during the evening shift. A dialysis fluid arrives for routine analysis and a gram stain is performed on a cytospin(1) of the sample. Under the microscope no bacteria are initially evident during repeated scans - yet something catches the technologist’s eye. It may have been the odd string of purple-blue dots or perhaps thin strands of clearing where the surrounding material uniformly retains the safranin gram counterstain.

An astute colleague recently encountered that very scenario and immediately knew something was amiss. She immediately suspected the presence of either Nocardia, or due the site, more likely Mycobacterium species. A fluorescent acid-fast(2) Auromine-O stain was performed to confirm her suspicious of presence Mycobacterium species.

The patient was a 72 year old Oriental gentleman with chronic renal failure receiving peritoneal dialysis. The specimen was sent to the provincial health laboratories the following morning where further analysis by AMTD(3) confirmed the identity as Mycobacterium tuberculosis (TB).
The diagnosis came as a total surprise to the doctor in charge.

Below are some photographs I took of this rather interesting specimen. Additional cytospins were made with more material deposited on the slide resulting in more Mycobacteria per field. The initial gram stain was even more challenging than what is seen in the gram stains that follow.

Gram Stain of Peritoneal Dialysis Fluid ;
(All photos 1000X Magnification)

Can you spot the purple-blue dots in the lower right quadrant of the photograph above?
(click on photo to enlarge)

If one examines the cell wall of a Mycobacterium under an electron microscope, it resembles the gram positive cell wall structure. Mycobacteria, however, have a high content of mycolic acid associated with the cell wall which resists staining by the traditional gram stain method. For this reason the gram stain is not routinely used to visualize Mycobacteria. Alternative stains are employed such as the Ziehl-Neelsen, or Rhodomine-Auromine stains. The Mycobacteria are stained using these stains and the high mycolic acid content resists decolourization using a mild acid-alcohol solution.

'Ghost Cells' A clue to the presence of Mycobacteria in a gram-stained specimen
(Click on photo to enlarge for better viewing)

One clue found in a gram stain that may suggest the presence of Mycobacteria are the ghost like cells. These appear as a clear (unstained) line in the shape and size of a bacillus and are due to the mycolic acids resisting retention of the gram stain. As the bacterial cells are not stained they appear as a clear line or 'ghost cell' surrounded by material retaining the counterstain. Look carefully at the photo above (click on it to enlarge for better viewing) and look for the clear ghost cells in the left. Focusing the microscope up and down may bring areas retaining the gram stain into better focus thereby revealing the parts of the cell wall staining purple-blue as seen on the right. (arrows point identical areas to where the Mycobacteria are visible under varying focus) [contrast this appearance with the 'dots' that Streptococcus anginosus may exhibit in direct specimen gram stains]

Gram stain retained as purple-blue dots spaced between clear areas of high mycolic acid content that resists the stain. Easy to overlook by the untrained eye.

A cytospin of dialysis fluid containing a larger amount of material resulting in more bacterial cells per microscopic field. Short rows of dots are visible, however they are unlike the chain of cocci as a Streptococcus would appear.

Mycolic acids also assist Mycobacterium's ability to survive. They are similarly responsible in resisting the uptake of antibiotics used to eradicate the organism. The also resist being engulfed and killed by macrophages - a cellular defense mechanism of the body.

(1) Cytospin - a fluid specimen is added to a miniature funnel that is clamped to a microscope slide above a blotting spacer. The assembly is placed in a special centrifuge which then, under force deposits the liquid, under force onto the microscope slide. Solid material such as cells and bacteria are concentrated and somewhat flattened onto the glass slide and excess fluid is wicked away by the blotting material. This microscope slide is then stained by the desired method and examined under the microscope. This maximizes detection of any bacteria if present in the specimen.

(2) Acid-Fast/Auromine-O -Auromine-O, Auromine-Rhodomine, and Ziehl-Neelsen stains are known as 'Acid-Fast' stains. The two former stains are fluorescent stains which glow a bright yellow to apple-green under a particular fluorescent wavelength as seen under a fluorescent microscope. The later (Z-N) is examined under a traditional light microscope and Mycobacteria will appear bright red against a green (malachite green) or blue (methylene blue) counterstained background. Mycobacteria resist being decolourized by a mild acid-alcohol solution and as they retain these stains, the cells are said to be 'acid-fast'.

(3) AMTD - An acronym for Amplified Mycobacterium tuberculosis Direct test. This is a DNA probe test that looks for a particular and unique sequence of nucleic acids within the microbes genome that only occurs in Mycobacterium tuberculosis. Finding its presence confirms the organism is Mycobacterium tuberculosis and not a 'MOT' (Mycobacterium Other than Tuberculosis). Other species of Mycobacteria exist and not all have the same devastating consequences assiciated with TB. AMTD is a very rapid test for identifying TB in a sample compared to the weeks to months required to isolate and identify the bacilli by conventional culture techniques.

Our fluorescent microscope does not permit the attachment of the camera at this time so I'm currently unable to take photographs of the bacilli fluorescing in the acid-fast stain employed by our laboratory.

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