Exp. – 3.
WIDAL Test
Exp. No:
Date:
Introduction
The WIDAL test is a serological technique which tests for the presence of Salmonella antibodies
in the patients serum when facilities for culturing of antigen testing are not available, the
WIDAL test is performed for reliability and interpreted with care. It can be of value in
diagnosing typhoid and paratyphoid in endemic areas. It is of no value in the investigation of
Salmonella in food poisoning when the investigation is carried out for typhoid, the patients
serum is tested for ‘O’ (somatic) and ‘H’ (flagella) antibodies (agglutinins) against the following
antigen for O and H suspensions (stained suspensions)
Salmonella typhi ‘O’ antigen
Salmonella tyhpi ‘H’ antigen
Salmonella paratyphi ‘AH’ antigen
Salmonella paratyphi ‘BH’ antigen
Testing for Paratyphoid
The following antigen suspensions are required
Salmonella paratyphi ‘AO’ antigen suspension
Salmonella paratyhpi ‘AH’ antigen suspension
Salmonella paratyphi ‘BO’ antigen suspension
Salmonella paratyphi ‘BH’ antigen suspension
Salmonella paratyphi ‘CO’ antigen
Salmonella paratyphi ‘CH’ antigen
In enteric fever, specific agglutinins are usually detectable in the patient’s blood after 6 days of
fever. Serum from individuals vaccinated with TAB may also show moderately elevated titre of
all three H agglutinins.
Stained Salmonella Antigens are used to detect, identify and quantitate specific antibodies in
serum sample from patients suffering from Pyrexia of Udermined Origin (PUO). These antigens
are standardized, smooth suspensions of killed bacilli which have been stained to facilitate
reading of agglutination. They are suitable to be used in the standard WIDAL tube test as well
as in the rapid slide test. They may also be used as control antigens while carrying out
serological identification of bacterial isolates from clinical specimens.
Principle
Antibodies in serum, produced in response to exposure to Salmonella organisms will agglutinate
bacterial suspension which carries homologous Antigens.
Sample
Fresh serum, store at 2-8oC in case of any delay in testing. Serum should be clear and should not
be heated or inactivated.
Precautions
1. Bring all the reagents to room temperature before use.
2. Use clean and dry glassware.
3. For greater proficiency in the interpretation of test result, always include positive and
negative control sera.
4. Serum samples to be tested should be clear and free from any bacterial contamination.
5. Do not inactivate sera prior to testing.
6. Shake antigen vials well before sue, to make the suspension homogenous.
7. Very rarely reagent may show fibric structure (appearance like fungus), which will not
interfere in the specificity and sensitivity of the test performance.
Procedure
A. Rapid slide (Screening) Test. (Write this in observation book)
1. Clean the glass slide and wipe it free of water.
2. Place one drop of undiluted test serum in each of the 1st four circles (1-4) and one drop of
positive control serum in each in each of the last two circles (5&6).
3. Place one drop of antigens O, H, A (H) and B (H) in circle 1, 2, 3 and 4 respectively and
O antigen in circle 5 and any one of the H antigens H, A (H) or B(H) in circle 6.
4. Mix the contents of each circle with separate wooden applicator stick and spread to fill
the whole area of the individual circle.
5. Rock the slide for one minute and observe for agglutination. If agglutination is visible
within one minute, proceed for quantitative slide or tube test or for the quantitative
estimation of the titre of the appropriate antibody.
B. Quantitative Slide Test. (Don’t write this in observation book)
1. Clean the glass slide and wipe it free of water.
2. Place 0.005 ml, 0.01ml, 0.02ml, 0.04ml & 0.08ml of undiluted serum in 1st, 2nd, 3rd, 4th
and 5th circles respectively on the slide.
3. Add one drop of the appropriate antigen suspension which showed agglutination in
screening slide test, to each of the above circles.
4. Mix the contents of each circle with separate applicator stick.
5. Rotate the slide slowly for one minute and observe for agglutination
6. The titer of the antibody is the highest dilution of serum up to which there is clear
agglutination.
7. Repeat steps 1 through 6 with all the antigens, which showed agglutination in screening
slide test.
The serum volumes in the quantitative slide test correspond approximately to the tube test titre
as:
Serum Volume in ml Approximate Tube Test Titre
0.08 1:20
0.04 1:40
0.02 1:80
0.01 1:160
0.005 1:320
Quality Control Procedure
The use of positive control serum, negative control serum and saline control in parallel with
unknown test serum is recommended to assure the laboratory worker that the antigens used in the
test area sensitive as well as specific and to show what results are to be expected in positive and
negative specimens.
Result
The given ……………………. sample is found to be positive / negative for
Interpretation
Agglutinin titre greater than 1:80 is considered significant and usually suggestive of infection,
whereas low titres are often found in normal individuals. A single positive result has less
significance as a definite evidence of infection. A moderate simultaneous rise in the titre of all
three H agglutinins is suggestive of recent TAB vaccination.
