WIDAL TEST

(QUALITATIVE SLIDE AGGLUTINATION METHOD)

Aim
To diagnose enteric fever caused by Salmonella species using qualitative slide
agglutination test.

Principle

The Widal test is applied for the diagnosis of enteric fever that includes typhoid
and paratyphoid caused by Salmonella typhi and Salmonella paratyphi respectively. For
the slide agglutination test, stained Salmonella antigens are used to detect the presence of
specific agglutinin in the patient’s serum. The slide agglutination test is used as a primary
screening procedure. There are some agglutinins that are produced in the patient’s serum
during the fever period, which react with somatic antigen O of Salmonella typhi, A or B
of Salmonella paratyphi and then with flagellar antigen H which is common in most of
the Salmonella species. In this test four specific antigen suspensions are used viz H,
Salmonella typhi (O antigen), Salmonella paratyphi - A and Salmonella paratyphi - B. If
agglutination occurs in ‘O’ antigen added circle, then it is considered positive for
Salmonella typhi. If agglutination occurs in A or B antigen added circle, then it is
confirmed as positive for paratyphoid. Agglutination will occur in H antigen circle for all
the cases of antigens like O, A, and B.

Materials required
1. Widal test kit
2. Widal test slide
3. Suspected serum sample
4. Pipette
5. Applicator stick

Procedure
1. A clean glass marked with six circles was taken.
 2. The circles were labeled as “H”, “O”, “A”, “B”, positive control and negative
control from the first circle to the last.
3. One drop of undiluted serum was placed in the first four circles with the help of a
sterile pasture pipette.
4. One drop of postive control serum and one drop of negative control serum was
placed in the fifth and the last circle respectively.
5. One drop of H antigen was added to the first circle and one drop of Salmonella
typhi O antigen was added in the second circle. Salmonella paratyphi A antigen
and Salmonella paratyphi B antigen were added in the third and fourth circles
respectively.
6. One drop of ‘H’ antigen was placed at the fifth circle and sixth circle.
7. With separate applicator sticks serum and the antigen was mixed together and was
spread well to fill the whole of the individual circle. Then slide was observed for
agglutination.
Result:
The given test patient’s serum is positive for typhoid.
The given test patient’s serum is positive for paratyphoid A or B
The given test patient’s serum is negative for typhoid and paratyphoid.

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(Draw and write in the left side of your observation note book)
Observation:

DRAW THE SLIDE PICTURE HERE

The agglutination has (occurred / not occurred) in H antigen circle.

The agglutination has (occurred / not occurred) in O antigen circle.
The agglutination has (occurred / not occurred) in A antigen circle.
The agglutination has (occurred / not occurred) in B antigen circle.