under the microscope
under the microscope
By Priya Dhagat, MS, MLS( ASCP) CM, CIC
Tuberculosis: Principles of Laboratory Detection for Healthcare Professionals
Tuberculosis remains a leading infectious disease killer despite being an ancient disease dating back thousands of years. According to the WHO, over 10 million people fell ill from TB 1.25 million people died from TB in 2023. The U. S. CDC reported 9,633 cases of TB disease, representing a 15.6 % increase in case count compared with 2022. With stark increases in TB disease and deaths, rapid and accurate detection of MTB is critical for prompt clinical diagnosis and treatment as well as for public health and prevention of transmission within highly affected areas. While healthcare professionals are educated routinely about airborne isolation precautions, PPE, use of negative pressure rooms, and other infection prevention measures for MTB, it is equally as important to understand the basics of laboratory detection. In this article, we will take a step into the laboratory and focus on three areas: AFB smear microscopy, MTB culture, and molecular diagnostics.
AFB Smear Microscopy
Mycobacteria are characteristically acid-fast, meaning they resist decolorization by acids routinely used during staining procedures. This is due to their waxy, lipid rich mycolic acid cell wall which retains primary stains. Historically, three specific staining techniques have been used to detect acid-fast bacilli: Ziehl-Neelsen, Kinyoun, and Auramine-rhodamine. Both Zeilh-Neelsen utilize carbol-fushin as a primary stain and a methylene blue counter stain which allows for the visualization of red stained mycobacteria against a blue background when using brightfield microscopy. The difference between these two techniques is the usage of heat: Ziehl-Neelson uses heat to force the stain into the waxy cell wall whereas the Kinyoun stains does not use heat, but rather a higher concentration of carbol-fushin. A newer and more common staining technique uses auramine-rhodamine- a fluorescent stain that contains a mixture of both auramine and rhodamine which increases the contrast between tubercle bacilli and other microorganisms. It uses a potassium permanganate as a counter stain, resulting in yellow or orange mycobacteria against a dark background when using fluorescent microscopy. If AFB are seen in a smear examination after staining, they are counted and classified as 4 +, 3 +, 2 + or 1 +, according to the number of AFB seen under the microscope.
Culture
Mycobacterial culture is the gold standard for detection of MTB. Since mycobacteria are intracellular bacteria that depend heavily on the many host cell nutrients, media must be complex to mimic an environment suitable for growth and detection. There are two categories of media: solid egg and agar-based media and liquid / broth media. These specific media types allow for differentiation of species based on pigment production, colony morphology, and inhibition of contaminating bacteria and fungi. Lowenstein-Jensin media is an egg-based selective solid media that contains proteins and fatty acids required for mycobacterial growth and malachite green to inhibit growth of contaminating bacteria and fungi. Middlebrook media is agar based solid composed of amino acids, vitamins, and other growth factors, along with antibacterial and antifungal properties to enhance the growth of mycobacteria tuberculosis. Like solid media, liquid / broth media contains supplements to enrich growth of mycobacteria but in a shorter time. Average time for growth can be between 12-16 days compared to up to 8 weeks for solid media. Colonies growing on solid media have an off-white or cream color and appear crumbly or dry with many wrinkles. Click here to see images of MTB colonies.
Molecular Diagnostics
Polymerase Chain Reaction( PCR) is a rapid and sensitive method to detect MTB DNA in clinical samples. In general, PCR relies on several key steps to separate, amplify, and detect DNA by either conventional methods( e. g., gel electrophoresis) or in real-time using fluorescent signals. PCR offers high specificity and sensitivity and faster results than smear microscopy and culture but should be used in conjunction with these methods for accurate diagnosis. Antibiotic susceptibly and shortened time to treatment are two major benefits of nucleic acid amplification tests.
Priya Dhagat, MS, MLS( ASCP) CM, CIC, is an infection preventionist and the associate director of the system-wide Special Pathogens Program within the Department of Emergency Management at New York City Health + Hospitals, overseeing special pathogen preparedness and response efforts across New York City Health + Hospitals frontline healthcare facilities. Additionally, she supports and offers subject matter expertise for infection prevention topics for the National Emerging Special Pathogens Training and Education Center( NETEC).
With stark increases in TB disease and deaths, rapid and accurate detection of MTB is critical for prompt clinical diagnosis and treatment as well as for public health.”
10 • www. healthcarehygienemagazine. com • nov-dec 2025