Afb staining procedure for sputum pdf

Laboratory diagnosis of tuberculosis by sputum microscopy through tb. Notes on sputum examination helpful to undergraduate and postgraduate medical students. Author summary lung fluke paragonimus infection causes similar symptoms to pulmonary tb and is an important differential diagnosis in endemic areas. Amyloid stain kit used in the detection of amyloid ffpe as well as frozen tissue sections cut at 10 microns.

Use fluorescent acidfast staining and microscopic examination goal reporting afb smear result within 24 hrs from receipt of specimen in lab use rapid broth system for primary culture detection of afb goal to reduce culture detection of afb from 34 wks to 12 wks use rapid id. Fix the smear onto the slide by passing over the flame of spirit lamp or bunsen burner. Use millipore filtered water in the waterbath and staining procedure. Control tissue is alzheimers or other known amyloidosis. Afb smear refers to the microscopic examination of a fluorochrome stain of a clinical specimen. Cdc recommends fluorochrome staining for detection of afb in primary patient specimens. Detection of acidfast bacilli afb in stained and acidwashed smears examined microscopically may provide the initial bacteriologic evidence of the presence of mycobacteria in a clinical specimen. Place slides on dryer with smeared surface upwards, and air dry for about 30 minutes.

According to rntcp guidelines, 2 samples are collected, one stat and one early next morning sample. Afb stain or ziehlneelsen acidfast staining procedure. Key words afb pulmonary tuberculosis smears sputum containers zn staining. It is not saliva or mucous from the back of the throat. Efficiency of cold staining method of afb in sputum a comparison with ziehl neelsen method under field conditions article pdf available january 1990 with 444 reads how we measure reads. Complete kit to aid in the staining of tissue for amyloidosis. Number of specimens for the detection of acid fast bacilli. Afb stain urine test test results, normal range, cost. Number of acidfast bacilli observed in smears, concentrations of culturable bacilli in sputum specimens and probability of positive result number of bacilli. Sputum microscopy culture, staining, requirements and observations.

Call the physician when these organisms are identified. Mycobacteriology laboratory manual world health organization. Staining methods histology and cytology 152 notes sources of errors irregular distribution of the blood smear on a glass slide may result in an erroneous cell counts. Its also known as an acidfast bacillus afb stain or a tuberculosis tb smear. At concentration below afb per ml of the sputum, the chance of observing the bacilli in a smear is less than 10%. We compared the sensitivity and specificity of a modified ziehlneelsen modifiedzn staining method for acidfast bacilli afb with that of the standard ziehlneelsen standardzn staining method, using culture results with mycobacterium tuberculosis as the gold standard.

Note that seeing 19 afb per 100 hpf is reported as scanty and the exact number of bacilli should be recorded. Essentially, sputum is composed of a mixture of saliva and mucus. Your doctor can use the results to help diagnose the cause of respiratory symptoms. Standard manual for laboratory technicians on sputum smear microscopy. If you find that you have been suffering from the following symptoms, then you need to consult your physician right away and he would, after a physical examination, order a afb stain urine test. In a sputum smear stained by ziehlneelsen method, afb appears as a red or. With the aid of a nebulizer, have patients inhale about 25 ml of 3 to 10% sterile saline. Staining techniques for detection of acid fast bacilli herbert. Zn staining and 38 37 sputum, 1 cervical abscess yielded positive results. If a mycobacterium species afb, tb, fungus or a virus is suspected, refer to afb culture, fungal culture or viral culture.

Binay thapa, microbiologist, public health laboratory 3. Acid fast bacteria afb, kinyoun staining procedure. The technique need a skilled reader artefacts are frequent. The evaluator must understand appropriate methods and ensure they are being followed. Pdf assessment of sputum staining techniques for the diagnosis.

Standard manual for laboratory technician on sputum smear. A comparative study on the different staining methods. Have patient rinse mouth with water after brushing gums and tongue. Neelsen in 1883 used ziehls carbolfuchsin and heat then decolorized with. Three consecutive early morning sputum samples were processed by pick and smear method and stained with classical pap staining technique. Acid fast bacilli afb smear and culture are two separate tests always performed together at the msphl, tuberculosis tb unit. Microscopic detection of mycobacterium tuberculosis in.

Sputum examination for tuberculosis by direct microscopy. Acid fast staining in formalinfixed tissue specimen of. Clinical and radiological findings and histopathological results were collected and cytology results were correlated with it. Mycobacteria are called acidfast bacilli because they are a group of rodshaped bacteria bacilli that can be seen under the microscope following a staining procedure where. Over 90% of tuberculosis tb cases occur in low and middleincome countries lmics. To enhance sensitivity by smear, current guidelines recommend the collection of early morning sputum specimens on 3 consecutive days with a minimum of 8 hours between collections. Evaluation of a cold staining method for detecting acid. Demonstration of ziehl neelsen staining, a method of acid fast staining. Transport specimen to the microbiology laboratory immediately at room. Do not let the specimens dry at any stage of the staining procedure. A technical guide for sputum smear microscopy, based on one initiated in 1969 by dr j holm, the then director of the international union against tuberculosis, was first published in 1978 by the iuat as the technical guide for sputum examination for tuberculosis. Methods assessed include specimen collection, smear preparation, acidfast stain procedure, reading, and reporting. To overcome the cumbersome process involved in collection and processing of the sputum specimen, the time consumed for reporting of sputum by ziehl neelsen zn method and to introduce a routine screening test in suspected.

It is named for two german doctors who modified the stain. Pdf efficiency of cold staining method of afb in sputum. Whereas in cold zn staining, instead of heating, we increase the concentration of the basic dye and phenol and incorporate a wetting agent chemical. Grading of acidfast bacilli afb by three staining was done before and after concentration according to rntcp guidelines. Alcohols used for wiping the skin may cause hemolysis and artifacts. The afb culture is the inoculation of a clinical specimen onto culture media bectondickinson mycobacteria growth. A novel method of staining acidfast bacilli in sputum. Analysis of ds is easy to perform, quick, and inexpensive, but has limited sensitivity, with a detection threshold of 5000 to 10,000 afbml of sputum2. Afb sputum test afb detection test qbc fast afb kits. Ziehlneelsen staining technique can diagnose paragonimiasis. Ziehlneelsen staining is a type of acidfast stain, first introduced by paul ehrlich. Negative staining technique principle, procedure, results.

Ziehlneelsen staining is a bacteriological stain used to identify acidfast organisms, mainly mycobacteria. The afb stain test is often ordered to determine if you are currently infected with tuberculosis or nocardia. So this method is also called ziehlneelsen staining techniques. The stained smear is the easiest and quickest procedure that can be performed for diagnosis of tb and is economical especially in developing countries. The stains are either auramine o, or auraminerhodamine. The procedure of negative staining technique take a clean, dry, scratch and grease free microscopic glass slide and place a drop of india ink or nigrosin on it at one end near the edge. Sputum smear examination plays a key role in routine diagnosis and treatment of. The evaluator needs to have a good understanding of acidfast bacilli afb smear microscopy and good laboratory practice glp. Perform qc use positive controls every day to check staining procedure. For the last fifty years, ziehlneelsen stain zns has been believed to destroy paragonimus eggs. Ziehlneelsen zn method of acid fast staining technique is used to stain mycobacterium species including m. Mycobacterium tuberculosis part 4 afb stain acid fast. Do not allow sections to dry out at any point during staining procedure.

Patients that are sputum smearpositive are 510 times more infectious. This will interfere with proper decolorisation, and counterstain may mask the. Total 587consecutive sputum samples both spot and early s were collected and were stained with both zn stain and cold afb stain and results were compared. Hot zn stain is the usual method in which we heat the smear to enhance the dye penetration. Also referred to as phlegm, sputum is produced in the bronchi and bronchioles of the respiratory system and coughed up from the lower airways and should not be confused or mistaken for saliva, which is secreted in the mouth for digestion purposes. It is the differential staining techniques which was first developed by ziehl and later on modified by neelsen. And then, there is bright field staining, where carbolfuchsin is used as the primary stain. Most samples that are submitted for acidfast bacilli afb testing are collected because the healthcare practitioner suspects that a person has tuberculosis tb, a lung infection caused by mycobacterium tuberculosis. Sputum specimens are best if coughed up first thing in the morning, after you have been sleeping at night. Laboratory diagnosis of tuberculosis by sputum microscopy through tb care i under the terms of agreement no. A sputum stain for mycobacteria is a laboratory test performed on a sample of your sputum, or phlegm. However, our investigation of stored zns slides and our prospective.

Carbolfuchsin solution, microwave 80 power, 45 seconds, allow slides to stand in hot. Then smear from sputum is fixed by heat or alcohol. Laboratory diagnosis of tuberculosis by sputum microscopy. Afb microscopy examination of smears is a rapid, convenient and inexpensive test all types of specimens can be evaluated sputum, tissue, body fluids, etc. Same day sputum smear microscopy for the diagnosis of. Mycobacteria become more concentrated in the sputum as patients sleep, so.

Drain staining rackslides after each step to prevent solution carry over. Procedure of acidfast stain prepare bacterial smear on clean and grease free slide, using sterile technique. Afb kits are unique systems of reagents, supplies, and instruments that couple timeproven fluorescence technology with unique innovations that provide significant advantages over conventional methods. Early detection of tuberculosis is important for reducing its morbidity and mortality especially in the patients with nonproductive cough. Sonam wangchuk, microbiologist, head of public health laboratory 2. Due to simplicity, rapidity, and low cost, zn is the commonly used technique for the diagnosis of pulmonary tb in lmics. The afb culture is the inoculation of a clinical specimen onto culture media bectondickinson mycobacteria growth indicator tube bd mgit broth and lowensteinjensen lj media slant, incubation at 37c for up to six 6 weeks and detection of growth or no growth during this incubation period. Standard diagnosis is wet film wf microscopic examination of sputum samples. Ziehlneelsen acid fast staining procedure spread the sputum evenly over the central area of the slide using a continuous rotational movement.

Tb laboratory techniques for diagnosing tuberculosis. There is two basic techniques but both have the same principle. Tuberculosis bacilli stain red on a blue background, are straight or slightly curved, and often cluster in groups of 3 to. Demonstration of mycobacterium tuberculosis in sputum and. This publication was made possible by the generous support of the american people through the united states agency for international development usaid. Take a small portion of the bacterial colony with the help of sterilized inoculating loop. Acidfast stain principle, procedure, interpretation and. These stains can be read at lower magnification, with less fields examined. Prepare a thin smear of the specimen on a clean grease free glass slide. Sputum collection instructions sputum is the mucous or phlegm coughed up from your lungs. Acidfast bacilli afb smear microscopy globally recognized as a transmissionrisk indicator, the afb smear exists as a rapid, less expensive answer to tuberculosis tb control strategy when culture and antimicrobial susceptibility testing ast are not available. Routine stool examination stains are not recommended. A sputum grams stain is a quick, lowrisk laboratory test.