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 the Interpretation and Reporting of AFB in Acid Fast Staining Results….
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 Background – Green colored (if you are using the malachite green as counterstain) or Blue colored (if you are using the Methylene blue as counterstain)
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 specimens.
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 Field AFB Not Seen
1-2 AFB per 300 Fields Doubtful; repeat with another specimen
1-9 AFB per 100 Fields 1+
1-9 AFB per 10 Fields 2+
1-9 AFB per Field 3+
>9 AFB per Field 4+
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.