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

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


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


Acid-fast bacteria, also known together acid-fast bacilli or merely AFB, are a group of bacteria share the properties of acid fastness. Acid fastness is a physical building that provides a bacter the capacity to withstand decolorization by acids throughout staining procedures. This method that as soon as the bacter is stained, it cannot be decolorized using acids routinely provided in the process. This important and unique function of details bacteria gives the capability to classify and also detect castle using fairly easy activities 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 N. Nova.

Acid fastness can also be meeting to various other structures no 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 mountain fastness have the right to be deadline to numerous different bacteria, correlation with history makes it a reasonably unique characteristics of M. Tuberculosis in clinical practice.<2> This provides acid-fast staining sensitive and specific, detailed clinical correlation is part of the equation. This composing will focus on the acid-fast bacteria M. Tuberculosis. The diagnosis of M. Tuberculosis making use of this characteristic is described as TB microscopy, acid-fast smear microscopy, and also direct sputum near microscopy.<3> Even though the use of highly progressed molecular diagnostic tests has come into play, the value of this staining technique cannot it is in overstated, specifically for low and middle-income countries.<4>

Specimen Collection

Mycobacterium tuberculosis is mainly a lung pathogen; therefore, for all types of tuberculosis, the primary specimen forced in acid-fast smear microscopy is sputum. There are 4 chief ways of collecting sputum.

Coughing is the many common and also widely used technique for sputum collection. A health care worker put on appropriate personal protective devices must supervise and guide this process. The patient must be described that the sputum must be carried out indigenous the lungs and that mucus from the nose and also saliva from the mouth is not appropriate samples.
Bronchoscopy is an invasive procedure offered to visualize the respiratory tract passages the the lungs. This procedure might be provided to extract sputum via bronchial washing, brushing, and/or biopsying. This procedure is avoided in patients who are transmittable or have not undertaken other techniques of sputum collection. In other words, this procedure have to not be provided as a substitute for sputum collection but rather together an adjunct examination for diagnosis.

Sputum collection should be carry out in specialized areas that are well ventilated to protect against the nosocomial inhalation of aerosols by uninfected people. Purulent and also mucopurulent sputum are considered great specimens because that microscopy. Suboptimal samples encompass mucoid, mucosalivary, and also salivary specimens. It is vital to distinguish salivary sputum specimens from saliva and mucus, as the last are not representative of lung status and also may offer false-negative results. The existence of food debris and contaminants is not advised. However, blood-tinged or bloodstained sputum is considered acceptable. Medical laboratory personnel should also regularly evaluate specimen quality prior to performing TB microscopy.<7> Patients should be requested to repeat the repertoire until an agree specimen is achieved. Besides the quality, the amount the the specimen is also important and also should be a minimum that 5 milliliters.<8> insufficient volume influence the sensitivity that the test, for this reason reducing its medical utility.

Sputum specimens have to be built up in ideal containers. A 50-ml plastic, screw-capped, transparent container is usually supplied to foster a for sure containment. The transparency of the container permits for intuitive inspection the the specimen to evaluate its consistency and also quality. Proper labeling that the sample with the patient’s name and also date the the collection must be ensured. The built up sample have to be stored at 2 come 8°C until transported come the laboratory. According to the American Centers for disease Control and Prevention, at the very least 3 continually sputum samples, each gathered at 8 come 24-hour intervals, v at least one sample being very early morning expectorate, are forced for diagnosis. In those nations with one established and also well-implemented external quality evaluate (EQA) regimen but limited human resources, the people Health organization recommends using two specimens because that diagnosis. This is to facilitate the early on or “same day” diagnosis of tuberculosis patient from the community.<9>


Microscopic evaluation of sputum for acid-fast bacilli starts with do a smear. A usual smear is 3 cm by 2 centimeter in size, however, depending upon the individual activities guidelines, it have the right to be as small as 2 cm by 1 cm together well. Smearing must be excellent by pressing and applying the sputum uniformly top top the slide. Ideally, a smear that uniform thickness need to be made in ~ the center of the on slide to facilitate visualization using a microscope. The smeared slides should likewise be effectively heat-fixed before proceeding on come the staining process. Training staff to make good smears is pivotal for accurate and also valid testing. <10><11>

Acid-fast structures can be visualized under a microscope using two primary methods, the carbolfuchsin staining, and also the fluorochrome procedure.

The carbolfuchsin staining comprises that the Ziehl-Neelsen an approach and the Kinyoun method. In the Ziehl-Neelsen method, smeared slides are an initial stained through carbolfuchsin (CF). <12> This is done by submerging the smear in a autumn of carbolfuchsin and also subsequently heater it making use of an alcohol lamp until steam can be checked out rising. This facilitates the penetration of the stain inside each bacterium. Care must be taken that boiling does not happen as the may change the outcomes of the test. The stain and smear must remain in contact for around 10 minutes and also be enabled to cool down thereafter, thus, trapping the stain within the bacterial cell wall. These procedures make the entire smear, consisting of acid-fast bacilli, red. After ~ stain fixation, the 2nd step focuses on washing the overfill stain off from the smear. This is excellent by gently washing the smear in a stream of water and then covering it v acid alcohol because that 2 to 3 minutes. Mountain alcohol has the ability to fully decolorize all non-acid-fast organisms, therefore only leave behind red-colored acid-fast organisms, like M. Tuberculosis. The slides are then stained a 2nd time with methylene blue that serves together a counterstain. The encourage time for stain come smear contact is 1 minute however is mostly dependant ~ above the top quality of methylene blue. Counterstaining creates an efficient visual contrast of red acid-fast bacilli throughout microscopy. The Ziehl-Neelsen an approach of staining is likewise called the hot method as it involves heating the carbolfuchsin stain. In contrast, the historic method of staining called the Kinyoun method does no involve heating and also is for this reason known together the cold method. Currently, the cold an approach is already obsolete.

The fluorochrome procedure mainly utilizes one of 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 viewed under a fluorescence microscope. Unlike the Ziehl-Neelsen method, heater is not forced for the penetration of the stain right into the bacteria. The stain come smear call time, however, must be a minimum the 20 minutes because that the acid-fast biology to choose up the stain properly. After ~ the auramine dye has completely stained the smear, a drop of acid alcohol is used for one to 2 minutes to decolorize the smear. Methylene blue or potassium permanganate is offered as a counterstain to carry out background color. Potassium permanganate is preferred as it offers a darker background offering it a far better contrast and also sensitivity as contrasted to methylene blue. <13> The last step is to gently to wash the slide with slow running water and also letting it dry. Blotting the slide is avoided as it may damages the stained smear.

Multiple studies have compared the Ziehl-Neelsen method with the fluorochrome procedure. The results obtained by both methods are taken into consideration highly reproducible. This means that both techniques are equally capable of detecting acid-fast bacilli in a sputum sample. <14> Although, the is exciting to keep in mind that a few studies imply that fluorescence microscopy is more likely to positively detect a sputum sample v a fewer number of acid-fast bacilli. It also has the benefit of taking fifty percent as lengthy as light-microscopy to declare a sample an adverse of acid-fast bacilli. <14><15>


AFB-microscopy is shown for suspected instances of tuberculosis. Confident microscopy confirms the presence of acid-fast bacilli. Care must be taken no to translate positive outcomes as M. Tuberculosis because the Ziehl-Neelsen and auramine stains only suggest the existence of acid-fast bacteria/structures. Other acid-fast organisms because that which Ziehl-Neelsen stain may test positive are pointed out in the introduction.

Potential Diagnosis

The an outcome of acid-fast microscopy must always be clinically associated with the patient’s history, examination, and also other appropriate investigations.<16> Acid-fast microscopy is one effective and reliable tool for security the patient"s response to therapy. Because that a patient v tuberculosis, the check of cure is established by an unfavorable acid-fast microscopy in ~ the finish of the therapy regimen. Similarly, the infectivity of a tuberculosis-positive patience can also be assessed via sputum microscopy. Top top the various other hand, multidrug-resistant tuberculosis and also extremely drug-resistant tuberculosis can not be differentiated from at risk M. Tuberculosis via this technique. To know of this strains requires specific and also sensitive investigations together as culture and medicine susceptibility exam (DST).

In some settings, the Xpert MTB/Rif assay,<17> a nucleic mountain amplification check (NAAT), has actually replaced sputum microscopy together a major tool of diagnosis.<18> having said that, security the development of the an illness using this molecular modern technology has not been uncovered effective. While additional diagnostic tests such together the lateral circulation urine lipoarabinomannan assay (TB LF LAM), loop-mediated isothermal amplification (TB LAMP), Truenat tests, and the like, have started to be presented in many settings, the importance that acid-fast microscopy, along with AFB culture, still holds with within the diagnostics pipeline for TB.<19><20>

Normal and an important Findings

Reading smears is a vital step in sputum microscopy. The an international Laboratory initiative (GLI) handbook for acid-fast microscopy specifies guidelines because that reading, recording, and reporting results for both, Ziehl-Neelsen and also the auramine methods.<21> The recommended number of visual fields to be check is 150 because that a smear size of 3 centimeter x 2 cm and also 100 for a smear dimension of 2 cm x 1 centimeter (Note: 150 and also 100 visual fields can be designated as "1 length" meaning the visual fields are examined from one end to another). That should, however, be listed that some nations have their very own guidelines for interpreting and reporting results. The outcomes of acid-fast microscopy may be reported according to the complying with standards based upon the civilization Health Organization and also International Union versus Tuberculosis and also Lung conditions (WHO-IUTLD):For the Ziehl-Neelsen method (via light/brightfield microscopy)

No Acid-fast bacilli (AFB) seen - Report together "0". It way no AFB to be observed in 2 lengths (i.e., 300 visual fields), thus, conferring a "negative" result.
1-9 AFB in 1 length - document the actual variety of AFB watched (e.g. +1, +2, +9). Keep in mind that the to add sign must precede the number. This is also referred to together a scanty hopeful result.
10-99 AFB in 1 length - Report as "1+". Note that the to add sign have to come after ~ the number. This is a optimistic result.
1-10 AFB per field in at least 50 visual fields - Report together "2+". Note that the plus sign should come ~ the number. This is a positive result.
More 보다 10 AFB per ar in at least 20 intuitive fields - Report as "3+". Note that the plus sign need to come after the number. This is a positive an outcome and is extremely infectious. 
No Acid-fast bacilli (AFB) seen - Report as "0". It means no AFB to be observed in 1 length, thus, a "negative" result. Just 1 length is required due to the fact that it has actually a broader visual field compared to the brightfield microscopy.
1-2 AFB in 1 length - Confirmation need to be done before reporting v the slide analysis of another medical technologist (i.e., clinical laboratory scientist, microscopist) or by prepare a brand-new slide for examination.
3-24 AFB in 1 length - Record the actual number of AFB seen (e.g. +3, +4, +24). Note that the to add sign need to precede the number. This is likewise referred to as a scanty confident result.
1-6 AFB in 1 length - Report as "1+". Note that the to add sign should come ~ the number. This is a positive result.
7-60 AFB in 1 length - Report together "2+". Note the the to add sign have to come after the number. This is a confident result.
More than 60 AFB in 1 length - Report together "3+". Note that the plus sign must come after ~ the number. This is a positive result and is extremely infectious. 

Note the in assessing slide smears, a different reporting for 200x magnification making use of a fluorescence microscopic lense is also available and may be provided by more skilled and experienced personnel.

Interfering Factors

Even though the sensitivity and also specificity that the Ziehl-Neelsen stain because that pulmonary tuberculosis have the right to be approximately 70% and 97.1%, respectively, several components can interfere with accurate and valid reporting of results.<22>

Pre-analytical components such as wrongly labeling the sample, inappropriately storing and transporting the specimen, and poor an approach for sample collection can all an outcome in a discrepancy that the microscopy results. Sample because that sputum microscopy must be expectorated native the lungs; fail to execute so may significantly influence the sensitivity the this test as well. Exposure to direct sunlight and also the too much heat can also destroy a far-ranging number the acid-fast bacilli in the sputum sample, in order to rendering the outcomes compromised.

Analytical components such as bad smearing, staining, and also microscopy can additionally hamper the results of acid-fast microscopy. Appropriate smearing requires correct smear size, thickness, and fixation. Improper staining of the slides deserve to be brought about by on slide contamination, incorrect staining time, over or under-heating the stain, and blotting the smear with record for drying. <23> The report the microscopy is also dependent ~ above the microscopic lense itself and also the person analyzing the sample. The reported number of bacilli can differ from person to person, and also therefore, appropriate training is additionally essential for accurate and also valid results.

Post-analytical factors can involve mix-up and release of mismatched patience results and other clerical mistake (e.g., inappropriately reporting a scanty result of "+3" together "3+"). Long come back time and incorrect result interpretation can additionally potentially hinder the use of acid-fast microscopy together a diagnostic tool.

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False-positive and also false-negative outcomes of acid-fast microscopy may have grave consequences on the patient individually and likewise the society as a whole. False-positive results (i.e., a positive an outcome for an actually an adverse patient) will an outcome in unnecessary treatment with anti-tuberculosis drugs. These drugs can cause far-ranging side results ranging from mild elevation in liver enzymes to full-blown hepatic failure. Optic neuritis, color blindness, and also peripheral neuropathy are just some the the various other potential side impacts of anti-tuberculosis medicines.<25><26>