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Reporting Of AFB (Acid Fast Bacilli) In Sputum Smears

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Hey, Good to see you here 😀 …… In this Article, We’re gonna discuss the Interpretation of Results of AFB Staining in Sputum Specimen and thereafter Reporting of Results….. If you have any queries, don’t forget to mention in Comments….. Thanks

Acid-fast staining is the most commonly used staining technique for the identification of Mycobacterium species especially the Mycobacterium tuberculosis in the sputum samples.



After making an Acid fast stained smear of the specimen a Laboratory technologist has to carefully observe under the microscope for the Red-colored Thin, Beaded curved rods which is a characteristic morphology of Mycobacterium tuberculosis and thereafter counting the no. of bacteria observed per High Power field (HPF) of the microscope.

In this Article, I’m gonna explain in detail about the Interpretation and Reporting of AFB in Acid Fast Staining Results….

The Interpretation Of Acid Fast Staining Results

 The appearance of Acid Fast Bacilli / Bacteria Red, straight or slightly curved rods, occurring singly or in small groups, may appear beaded curved rods.

 The appearance of Non-Acid fast bacteria and other Cells in the Specimen – Green Colored (if you are using the malachite green as counterstain) or Blue colored (if you are using the Methylene blue as counterstain)

 The appearance of BackgroundGreen colored (if you are using the malachite green as counterstain) or Blue colored (if you are using the Methylene blue as counterstain)

ACID FAST STAINING - mycobacterium tuberculosis - laboratory hub
Acid Fast Stained Sputum Smear Microscopic View Showing Acid Fast Bacilli

The Reporting Of Afb In Acid Fast Staining Results

 For precise and accurate reports of the test, the Acid fast stained smears are observed at least for 300 High Power Fields (HPF) before reporting. After observing the 300 HPF the Reporting is done as per the no. of AFB observed which is as follows:

When no AFB is seen after examining 300 fields

 Report the Examined smear as ‘AFB NOT SEEN’. Don’t report it as ‘Negative’ because organisms may be present but not seen in those fields which were examined.




 Up to three specimens (collected as an early morning specimen) may need to be precisely and accurately examined to detect Mycobacterium tuberculosis in sputum specimen.

When any definite AFB are seen

 Report the smear as ‘AFB POSITIVE’, and give a grade to the observations as per the no. of bacteria seen which is described below:

NUMBER OF AFB SEEN ( AT HPF - 1000X MAGNIFICATION)REPORTED AS FOLLOWS -
0 AFB per 300 FieldAFB Not Seen
1-2 AFB per 300 FieldsDoubtful; repeat with another specimen
1-9 AFB per 100 Fields1+
1-9 AFB per 10 Fields2+
1-9 AFB per Field3+
>9 AFB per Field4+

When very few AFB are seen

 Sometimes when only one or two AFB is seen in the Acid fast stained smears, request for a further specimen to examine for the confirmation of the test.

 This is because In Tap water and in deionized water (deionized using ‘old’ resin) sometimes contains AFB that resembles tubercle bacilli, and occasionally stained scratches on a slide can be mistaken for AFB.

 Moreover, AFB can be transferred from one smear to another when the same piece of blotting paper is used to dry several smears, which is usually avoided as it increases the chances of erroneous results. 



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