![]() In the Ziehl-Neelsen method, smeared slides are first stained with carbolfuchsin (CF). The carbolfuchsin staining comprises of the Ziehl-Neelsen method and the Kinyoun method. Īcid-fast structures can be visualized under a microscope using two principal methods, the carbolfuchsin staining, and the fluorochrome procedure. Training staff to make good smears is pivotal for accurate and valid testing. The smeared slides should also be properly heat-fixed before proceeding on to the staining process. Ideally, a smear of uniform thickness should be made at the center of the slide to facilitate visualization using a microscope. A typical smear is 3 cm by 2 cm in size, however, depending on the individual laboratory guidelines, it can be as small as 2 cm by 1 cm as well. Smearing must be done by pressing and applying the sputum uniformly on the slide. ![]() Microscopic evaluation of sputum for acid-fast bacilli begins with making a smear. This is to facilitate the early or “same day” diagnosis of tuberculosis patients from the community. ![]() In those countries with an established and well-implemented external quality assessment (EQA) program but limited human resources, the World Health Organization recommends using two specimens for diagnosis. According to the American Centers for Disease Control and Prevention, at least 3 consecutive sputum samples, each collected at 8 to 24-hour intervals, with at least one sample being an early morning expectorate, are required for diagnosis. The collected sample should be stored at 2 to 8☌ until transported to the laboratory. Appropriate labeling of the sample with the patient’s name and date of the collection must be ensured. The transparency of the container allows for visual inspection of the specimen to assess its consistency and quality. Sputum specimens must be collected in appropriate containers. A 50-ml plastic, screw-capped, transparent container is usually used to foster a secure containment. Inadequate volume affects the sensitivity of the test, thus reducing its medical utility. Patients must be requested to repeat the collection until an acceptable specimen is achieved. Besides the quality, the amount of the specimen is also important and should be a minimum of 5 milliliters. Medical laboratory personnel should also regularly evaluate specimen quality before performing TB microscopy. However, blood-tinged or bloodstained sputum is considered acceptable. The presence of food debris and contaminants is not advised. It is important to differentiate salivary sputum specimens from saliva and mucus, as the latter are not representative of lung status and may give false-negative results. ![]() Suboptimal samples include mucoid, mucosalivary, and salivary specimens. Purulent and mucopurulent sputum are considered good specimens for microscopy. Sputum collection must be performed in dedicated areas that are well ventilated to prevent the nosocomial inhalation of aerosols by uninfected people. To yield a better sample, gastric aspiration should be done early morning, when the patient has not had anything to eat. This procedure is particularly useful in children. Gastric aspiration can be used to obtain sputum previously expectorated and swallowed by the patient.
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