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Mycobacterium tuberculosis

Mycobacterium tuberculosis microscopy

Transmitted light microscopy on sputum samples is the most widely diffused method to diagnose pulmonary tuberculosis in low-income countries. However, this method is complex and has low sensitivity compared with culture, while the more-sensitive fluorescence microscopy method is a standard diagnostic tool in high-income countries. The present case study describes a new device for fluorescence microscopy, which can be employed on small microscopes at an affordable price. Furthermore it can be adapted on existing microscopes. Battery operation is also possible.

Introduction

The Mycobacterium genus includes pathogens responsible for severe human diseases such as Mycobacterium tuberculosis – the agent of tuberculosis (TB), and Mycobacterium leprae – the agent of leprosy. The rod-shaped Mycobacterium tuberculosis is a non-motile bacteria about 3 to 4 um in length and less than 0.5 um in width. The species are classified as acid-fast bacteria and a widely diffused staining method is the Ziehl-Neelsen, where bacilli are observed in transmitted light. Illumination is typically provided by the built-in light source (mainly halogen lamp) or even sunlight reflected by a mirror. The relative complexity of this method and the need of high magnification lenses are major drawbacks, while fluorescence microscopy is credited with improved sensitivity. The identification of mycobacteria with the fluorescent dye Auramine O is due to the affinity of the fluorochrome to mycolic acid in the cell walls. Recent investigations also indicate a potential affinity to the bacteria DNA. Auramine O is excited by blue light and emits in the region from ~500nm to ~650nm (green-yellow-red).
Fluorescence microscopy displays some important advantages:
1.High contrast fluorescence images allow easier detection of bacteria.
2.The use of low to medium power lenses (typically 10x, 20x, and 40x) permits a larger field of view than conventional microscopy, where typically a 100x lens is used.
3.The fluorochrome staining method is simpler than the Ziehl-Neelsen method.
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A potential shortcoming of fluorescence microscopy is the risk of false-positive results: all acid-fast organisms can be stained by Auramine O, including some parasites. Furthermore inorganic objects may incorporate the fluorochrome too.

FLUOLED® Easy and FLUOLED® 21: transmitted light fluorescence for TB microscopy

A unique, proprietary illumination system using high-power solid-state (LED) light sources to replace the traditional mercury arc-lamps.

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FLUOLED® Easy (fig. on left, on Olympus CX31 microscope): the LED fluorescence module is designed to attach to a standard bright field microscope and is used in transmission mode.

FLUOLED® 21 (fig. on right): in this modified version of Olympus’ CX21 microscope the LED illumination system is fully integrated into the microscope base. A truly plug-and-play unit for TB.

The mycobacteria will appear as bright luminous rods on a dark background. A potassium permanganate counterstain helps prevent non-specific fluorescence.
The mycobacteria will appear as bright luminous rods on a dark background. A potassium permanganate counterstain helps prevent non-specific fluorescence.

Results

Transmitted-light fluorescence microscopy proved to be a valuable technology for observation of Auramine O stained Mycobacterium tuberculosis.

  • Light source lifetime: typically 30.000 hours, thus allowing many years of operation and cost savings.
  • No need of any special alignment procedure.
  • Adjustable light illumination intensity (on FLUOLED® 21).
  • No warm-up time required for the light source.
  • Allows transmitted light bright field observation.
  • Battery pack option for field operation.


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