Bacilloscopy: Definition and Advantages of Fluorescence Microscopy

Medical procedure in the laboratory to determine if there is tuberculosis.

Throughout the world, the most common diagnostic test used to detect tuberculosis is the microscopic examination of stained sputum or other clinical material stained on a glass slide.

When present in sufficiently high concentrations, the bacteria can be easily identified by a trained technician using this technique called Bacilloscopy, which has changed little since it was invented more than 100 years ago.

Baciloscopy is cheap to perform, specific enough to indicate treatment in countries where TB prevails, and can be completed in a matter of hours if necessary.

Bacilloscopy requires a large number of bacilli for the result to be positive (5000-10,000 per ml of sputum) and identifies the most infectious subset of patients.

Diagnosis of traditional sputum smear and TB manual:

Microscopy of light source ZN tinting Sputum

  • It is the fastest and cheapest method available
  • Good specificity
  • Weak sensitivity
  • You need trained personnel
  • It takes a lot of time and is tedious

Fluorescent microscope

  • Greater sensitivity
  • More expensive
  • Faster, less tedious
  • Maintenance requirements (mercury vapor lamps)

The inherent low sensitivity of the test is exacerbated by the conditions under which it is commonly performed: poor equipment, heavy workload and inexperienced or unmotivated personnel.

The proportion of cases detected by microscopy is usually as low as 40-50% of all cases. Duplicate or tripled sputum tests are requested to help overcome this problem.

This need for multiple testing, each of which requires sputum collection, drying, staining and meticulous examination, causes delays in reporting and a relatively large number of patients does not complete the test or is lost in the health system despite have a positive test.

Several methods are being used that may somewhat increase the speed or sensitivity of smear microscopy, including the use of fluorescence microscopes. Due to the higher cost of the necessary equipment, traditional fluorescence microscopes (gas lamps) are mainly used in industrialized countries.

Advantages of fluorescence microscopy

Fluorescence microscopy has several advantages over light microscopy using ZN staining and is widely used in most developed nations.

The fluorochrome staining procedure used is simpler than that of ZN staining, and fluorescence microscopy has approximately 10% higher sensitivity to detect AFB bacilli.

One of the most important advantages is that fluorescence microscopy can be examined at a lower magnification, and then reading ZN slides (30-60 versus 100x) more quickly and efficiently with FM, a time gain can be achieved considerable.

This advantage is very important for the overloaded laboratory configuration in many low resource environments.

Unfortunately, it is these low resource environments where MF was not widely implemented. A simple explanation for this is the high cost of conventional mercury vapor (MV) fluorescence microscopes, even if there is considerable savings with the use of these due to greater sensitivity and greater time efficiency.

Fluorescent mercury vapor microscopes require significant maintenance, must be maintained in a clean environment, light bulbs, despite their high costs, have a limited lifetime (200 h) that is further shortened by the fluctuating power supply and They also present a toxic hazard if they break.

The user’s acceptance worsened further by concerns about the production of UV light, the production of heat and the requirement to work in a dark room.

Alternative to VM lamps used in MF

It has recently been shown that low-cost ultra-bright light-emitting diodes (LEDs) could be a viable alternative to VM lamps used in MF. LEDs can produce very narrow spectrum light and can excite auramine and other fluorescent stains commonly used without the production of UV light.

The LEDs have an expected lifetime of up to 50,000 h (compared to 200 h of a conventional mercury bulb), produce minimal heat and do not contain any hazardous material.

The energy consumption is much lower, to the point where the operation of the portable battery or solar energy is feasible.

Finally, it is shown that the quality of the image is still good even without the use of a dark room, which is a significant advantage when the limitations of space and the lack of air conditioning are important barriers for user acceptance.