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You are watching: What are the large blue stained areas on the sputum slide

StatPearls . Treacertain Island (FL): StatPearls Publishing; 2021 Jan-.


1 Adventist College of the Philippines-College of Medicine; Cavite State University-Med Tech Dept.


Acid-fast bacteria, additionally well-known as acid-fast bacilli or sindicate AFB, are a team of bacteria sharing the characteristic of acid fastness. Acid fastness is a physical home that provides a bacterium the ability to resist decolorization by acids in the time of staining actions. This means that once the bacterium is stained, it cannot be decolorized utilizing acids routinely provided in the procedure. This necessary and distinct feature of particular bacteria offers the capability to classify and also detect them making use of reasonably basic laboratory procedures such as microscopy.<1> Bacteria displaying acid fastness include:

Genus Mycobacterium – M. leprae, M. tuberculosis, M. smegmatis, M. Avium complex, M. kansasii.
Genus Nocardia – N. brasiliensis, N. cyriacigeorgica, N. farcinica, and also N. nova.

Acid fastness have the right to likewise be attributed to various other frameworks not classified as bacteria. These include:

Bacterial endospores
Head of sperm
Cryptosporidium parvum
Isospora belli
Cyclospora cayetanensis
Taenia saginata eggs
Hydatid cysts
Nuclear inclusion bodies in lead poisoning

Even though acid fastness deserve to be attributed to many different bacteria, correlation via background renders it a reasonably distinctive characteristic of M. tuberculosis in clinical practice.<2> This renders acid-fast staining sensitive and particular, gave clinical correlation is part of the equation. This writing will certainly emphasis on the acid-rapid bacteria M. tuberculosis. The diagnosis of M. tuberculosis using this characteristic is described as TB microscopy, acid-fast smear microscopy, and direct sputum near microscopy.<3> Even though the usage of highly advanced molecular diagnostic tests has actually come into play, the value of this staining strategy cannot be overproclaimed, particularly for low and middle-income nations.<4>

Specimen Collection

Mycobacterium tuberculosis is generally a lung pathogen; therefore, for all forms of tuberculosis, the main specimen compelled in acid-fast smear microscopy is sputum. Tright here are 4 chief ways of collecting sputum.

Coughing is the most common and extensively provided approach for sputum repertoire. A healthtreatment worker wearing proper individual protective devices need to supervise and also guide this process. The patient must be described that the sputum should be brought out from the lungs and that mucus from the nose and also saliva from the mouth is not correct samples.
Bronchoscopy is an invasive procedure supplied to visualize the respiratory passages of the lungs. This procedure may be used to extract sputum via bronchial washing, brushing, and/or biopsying. This procedure is avoided in patients who are infectious or have actually not undertaken other methods of sputum collection. In other words, this procedure need to not be provided as a substitute for sputum arsenal but rather as an adjunct examination for diagnosis.

Sputum arsenal need to be performed in dedicated locations that are well ventilated to proccasion the nosocomial inhalation of aerosols by uninfected people. Purulent and also mucopurulent sputum are thought about excellent specimens for microscopy. Suboptimal samples include mucoid, mucosalidiffer, and also salidiffer specimens. It is vital to differentiate salidiffer sputum specimens from saliva and also mucus, as the latter are not representative of lung status and may give false-negative outcomes. The existence of food debris and also contaminants is not advised. However before, blood-tinged or bloodstained sputum is taken into consideration acceptable. Medical laboratory personnel have to additionally on a regular basis evaluate specimales quality before percreating TB microscopy.<7> Patients need to be requested to repeat the arsenal till an acceptable specimales is achieved. Besides the top quality, the amount of the specimen is also necessary and also have to be a minimum of 5 milliliters.<8> Inadequate volume affects the sensitivity of the test, thus reducing its medical utility.

Sputum specimens need to be accumulated in proper containers. A 50-ml plastic, screw-capped, transparent container is normally supplied to foster a secure containment. The transparency of the container permits for visual inspection of the specimales to assess its consistency and quality. Suitable labeling of the sample through the patient’s name and also date of the arsenal must be ensured. The collected sample have to be stored at 2 to 8°C till transported to the laboratory. According to the American Centers for Disease Control and also Prevention, at leastern 3 consecutive sputum samples, each gathered at 8 to 24-hour intervals, with at leastern one sample being an early morning expectorate, are forced for diagnosis. In those countries via an establiburned and well-applied outside high quality assessment (EQA) regimen but restricted human resources, the World Health Organization recommends making use of two specimens for diagnosis. This is to facilitate the early or “exact same day” diagnosis of tuberculosis patients from the area.<9>


Microscopic evaluation of sputum for acid-fast bacilli begins via making a smear. A typical smear is 3 cm by 2 cm in size, yet, relying on the individual laboratory guidelines, it have the right to be as tiny as 2 cm by 1 cm as well. Smearing should be done by pushing and also applying the sputum uniformly on the slide. Ideally, a smear of uniform thickness have to be made at the center of the slide to facilitate visualization using a microscope. The smeared slides must also be correctly heat-addressed prior to proceeding on to the staining procedure. Training staff to make good smears is pivotal for specific and also valid testing. <10><11>

Acid-rapid structures have the right to be visualized under a microscopic lense using two major methods, the carbolfuchsin staining, and the fluorochrome procedure.

The carbolfuchsin staining comprises of the Ziehl-Neelsen technique and the Kinyoun approach. In the Ziehl-Neelsen method, smeared slides are first stained with carbolfuchsin (CF). <12> This is done by submerging the smear in a drop of carbolfuchsin and also ultimately heating it utilizing an alcohol lamp till steam have the right to be viewed increasing. This facilitates the penetration of the stain inside each bacterium. Care have to be taken that boiling does not take place as that may transform the outcomes of the test. The stain and also smear need to remajor in contact for roughly 10 minutes and be enabled to cool down after that, thus, trapping the stain within the bacterial cell wall. These steps make the whole smear, consisting of acid-fast bacilli, red. After stain fixation, the second action concentrates on washing the excess stain off from the smear. This is done by gently washing the smear in a stream of water and also then spanning it through acid alcohol for 2 to 3 minutes. Acid alcohol has actually the capability to entirely decolorize all non-acid-rapid organisms, for this reason only leaving behind red-colored acid-rapid organisms, like M. tuberculosis. The slides are then stained a second time via methylene blue that serves as a counterstain. The recommfinished time for stain to smear contact is 1 minute however is largely dependant on the high quality of methylene blue. Counterstaining creates an reliable visual contrast of red acid-quick bacilli during microscopy. The Ziehl-Neelsen approach of staining is likewise dubbed the warm method as it entails heating the carbolfuchsin stain. In comparison, the historical method of staining referred to as the Kinyoun technique does not involve heating and is therefore known as the cold approach. Right now, the cold method is currently obsolete.

The fluorochrome procedure mostly utilizes among 2 dyes, the auramine-O dye, or the auramine-rhodamine dye. Auramine-O is a hydrochloride dye that reasons stained AFB to emit fluorescence (green or yellow) when perceived under a fluorescence microscopic lense. Unlike the Ziehl-Neelsen approach, heating is not compelled for the penetration of the stain right into the bacteria. The stain to smear contact time, yet, should be a minimum of 20 minutes for the acid-fast organisms to pick up the stain correctly. After the auramine dye has completely stained the smear, a drop of acid alcohol is applied for one to two minutes to decolorize the smear. Methylene blue or potassium permanganate is used as a counterstain to administer background color. Potassium permanganate is preferred as it offers a darker background offering it a better contrast and also sensitivity as compared to methylene blue. <13> The last step is to gently wash the slide through slow running water and also letting it dry. Blotting the slide is avoided as it might damages the stained smear.

Multiple studies have compared the Ziehl-Neelsen strategy with the fluorochrome procedure. The results acquired by both approaches are thought about extremely reproducible. This means that both approaches are equally qualified of detecting acid-rapid bacilli in a sputum sample. <14> Although, it is exciting to note that a few studies imply that fluorescence microscopy is more likely to positively detect a sputum sample through a fewer number of acid-fast bacilli. It additionally has actually the benefit of taking half as long as light-microscopy to declare a sample negative of acid-quick bacilli. <14><15>


AFB-microscopy is suggested for suspected situations of tuberculosis. Confident microscopy confirms the visibility of acid-fast bacilli. Care have to be taken not to translate positive outcomes as M. tuberculosis bereason the Ziehl-Neelsen and auramine stains just show the existence of acid-fast bacteria/structures. Other acid-rapid organisms for which Ziehl-Neelsen stain may test positive are mentioned in the introduction.

Potential Diagnosis

The outcome of acid-fast microscopy must always be clinically correlated with the patient’s background, examicountry, and other appropriate investigations.<16> Acid-quick microscopy is an efficient and also dependable tool for monitoring the patient"s response to therapy. For a patient through tuberculosis, the confirmation of cure is establimelted by negative acid-rapid microscopy at the end of the treatment regimales. Similarly, the infectivity of a tuberculosis-positive patient deserve to also be assessed by means of sputum microscopy. On the various other hand, multidrug-resistant tuberculosis and very drug-resistant tuberculosis cannot be differentiated from prone M. tuberculosis via this method. Identification of these strains requires particular and also sensitive investigations such as culture and drug susceptibility tests (DST).

In some settings, the Xpert MTB/Rif assay,<17> a nucleic acid amplification test (NAAT), has reput sputum microscopy as a major tool of diagnosis.<18> Having said that, monitoring the progression of the illness using this molecular innovation has not been found reliable. While additional diagnostic tests such as the lateral flow urine lipoarabinomannan assay (TB LF LAM), loop-mediated isothermal amplification (TB LAMP), Truenat tests, and the choose, have actually started to be presented in many settings, the prestige of acid-quick microscopy, in addition to AFB culture, still holds via within the diagnostics pipeline for TB.<19><20>

Typical and Critical Findings

Reading smears is a crucial step in sputum microscopy. The Global Laboratory Initiative (GLI) handbook for acid-fast microscopy states guidelines for reading, recording, and reporting outcomes for both, Ziehl-Neelsen and the auramine techniques.<21> The recommended number of visual areas to be examined is 150 for a smear size of 3 cm x 2 cm and also 100 for a smear dimension of 2 cm x 1 cm (Note: 150 and also 100 visual areas have the right to be designated as "1 length" meaning the visual fields are examined from one finish to another). It need to, yet, be noted that some nations have actually their own guidelines for interpreting and reporting results. The results of acid-fast microscopy might be reported according to the complying with criteria based on the World Health Organization and also International Union Against Tuberculosis and also Lung Diseases (WHO-IUTLD):For the Ziehl-Neelsen Method (through light/brightfield microscopy)

No Acid-quick bacilli (AFB) seen - Report as "0". It means no AFB was oboffered in 2 lengths (i.e., 300 visual fields), hence, conferring a "negative" outcome.
1-9 AFB in 1 length - Record the actual variety of AFB checked out (e.g. +1, +2, +9). Keep in mind that the plus sign must precede the number. This is likewise described as a scanty positive outcome.
10-99 AFB in 1 length - Report as "1+". Note that the plus sign have to come after the number. This is a positive outcome.
1-10 AFB per area in at leastern 50 visual fields - Report as "2+". Keep in mind that the plus authorize must come after the number. This is a positive outcome.
More than 10 AFB per area in at least 20 visual fields - Report as "3+". Keep in mind that the plus sign must come after the number. This is a positive outcome and is highly infectious. 
No Acid-fast bacilli (AFB) seen - Report as "0". It means no AFB was observed in 1 size, thus, a "negative" outcome. Only 1 length is forced because it has actually a more comprehensive visual area compared to the brightfield microscopy.
1-2 AFB in 1 length - Confirmation should be done prior to reporting through the slide reading of another clinical technologist (i.e., clinical laboratory scientist, microscopist) or by preparing a brand-new slide for examicountry.
3-24 AFB in 1 length - Record the actual number of AFB watched (e.g. +3, +4, +24). Keep in mind that the plus authorize have to precede the number. This is likewise described as a scanty positive result.
1-6 AFB in 1 length - Report as "1+". Keep in mind that the plus authorize must come after the number. This is a positive result.
7-60 AFB in 1 length - Report as "2+". Keep in mind that the plus authorize have to come after the number. This is a positive outcome.
More than 60 AFB in 1 length - Report as "3+". Note that the plus sign must come after the number. This is a positive result and is very contagious. 

Keep in mind that in examining slide smears, a sepaprice reporting for 200x magnification using a fluorescence microscopic lense is additionally easily accessible and also might be supplied by more professional and knowledgeable personnel.

Interfering Factors

Even though the sensitivity and specificity of the Ziehl-Neelsen stain for pulmonary tuberculosis have the right to be as much as 70% and 97.1%, respectively, numerous components have the right to interfere with precise and valid reporting of results.<22>

Pre-analytical factors such as mistakenly labeling the sample, inappropriately storing and also transporting the specimen, and also poor strategy for sample repertoire can all result in a discrepancy of the microscopy results. Sample for sputum microscopy should be expectorated from the lungs; faiattract to execute so might significantly affect the sensitivity of this test also. Expocertain to straight sunlight and the extreme warm have the right to additionally ruin a far-reaching number of acid-rapid bacilli in the sputum sample, thereby rendering the outcomes jeopardized.

Analytical determinants such as bad smearing, staining, and microscopy can additionally hamper the results of acid-quick microscopy. Suitable smearing requires correct smear dimension, thickness, and also fixation. Imappropriate staining of the slides deserve to be caused by slide contamination, incorrect staining time, over or under-heating the stain, and blotting the smear through paper for drying. <23> The report of microscopy is likewise dependent on the microscopic lense itself and the person analyzing the sample. The reported variety of bacilli can differ from perchild to person, and also therefore, appropriate training is also essential for exact and valid results.

Post-analytical factors can involve mix-up and release of mismatched patient outcomes and various other clerical mistakes (e.g., inas necessary reporting a scanty result of "+3" as "3+"). Long turnapproximately time and also incorrect outcome interpretation can also perhaps hinder the use of acid-quick microscopy as a diagnostic tool.

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False-positive and also false-negative outcomes of acid-rapid microscopy may have actually grave repercussions on the patients individually and additionally the society all at once. False-positive results (i.e., a positive result for an actually negative patient) will certainly result in uncrucial therapy through anti-tuberculosis drugs. These drugs can reason substantial side results ranging from mild elevation in liver enzymes to full-blvery own hepatic faientice. Optic neuritis, shade blindness, and peripheral neuropathy are simply some of the other potential side results of anti-tuberculosis medications.<25><26>