Widal Antigen for Serologic Diagnosis of HumanSalmonella Typhi&Paratyphi(Slide & Tube Tests)


Principle of Widal Test

Bacterial suspension which carry antigen will agglutinate on exposure to antibodies to Salmonella organisms. Patients’ suffering from enteric fever would possess antibodies in their sera, which can react and agglutinate serial doubling dilutions of killed, colored Salmonella antigens in an agglutination test. The main principle of Widal test is that if antibody is present in a patient’s serum, it will react with respective antigen in the reagent and gives visible clumps on the test card and agglutination in the tube. The antigens used in the test are “H” and “O” antigens of S. Typhiand “H” antigen of S. Paratyphi. The paratyphoid “O” antigens are not employed as they cross react with typhoid “O” antigen due to the sharing of factor 12. “O” antigen is a somatic antigen and “H” antigen is flagellar antigen.


Packaging Contents

Each kit contains 6 vials of AO, BO, DO, aH, bH, dH antigens with Widal positive and negative control serum.

Reagent Storage and Stability


1. Store the reagents at 2-8 °C. Do not freeze.

2. Antigens and controls are stable until the expiry date stated on the label.

3. Once opened, the shelf life of the reagent vial is as described on the label provided that it is not contaminated.





          •        Before use, allow reagents to reach room temperature (18–30°C).

          •        Do not use expired reagents.

          •        Use glassware washed and rinsed with distilled water, or preferably use disposables.

          •        Use a new tip for each sample serum.

          •        Bacterial contamination of controls and specimens as well as freezing and thawing of the antigen may lead to false positive results.

          •        The reagents in this kit contain chemical material. Do not allow to contact with skin or mucous membranes. 


Slid-Test Procedure

1. Using a pipette, place 80 µL, 40 µL, 20 µL, 10 µL, 5 µL and 2 µL of patient serum to be tested on 6 different reaction circles on the slide. The corresponding titers obtained will be 1:20, 1:40, 1:80, 1:160, 1:320 & 1:640 respectively.

2. Add one drop of appropriate Widal antigen suspension to the reaction circles containing the patient’s serum.

3. Mix the contents of each circle uniformly over the entire circle with separate mixing sticks.

4. Rock the slide gently back and forth, and macroscopically observe for agglutination at once.

Tube-Test Procedure

1. Take appropriate number (as required; one set for each antigen suspension) of 10 Kahn tubes / test tubes and label them 1 to 10.

2. Pipette 0.9 ml of physiological saline into tube No.1 of all sets.


  1. Add 0.5 ml of physiological saline to each of the remaining tubes (2 to 10).
  2. 4. Add 0.1 ml of serum sample to be tested to tube No.1 of all sets, and mix well.
  3. 5. Transfer 0.5 ml of the diluted serum sample from tube No.1 to tube No.2 and mix well.
  4. 6. Transfer 0.5 ml of the diluted serum sample from tube No.2 to tube No.3 and mix well. Continue this serial dilution till tube No.9 in each set.
  5. 7. Discard 0.5 ml of the diluted serum from tube No.9.
  6. 9. Add 0.5 ml of Widal antigen (the respective well-mixed and diluted 1/20 with normal saline 0.5 %) suspensions from the reagent vials to all the tubes (1 to 9) of each set, and mix well.
  7. 8. Now the dilutions of the tube No.1 to 9 in each set is 1/20, 1/40, 1/80, 1/160, 1/320, 1/640, 1/1280, 1/2560, 1/5120, respectively. Tube No.10 in all the sets serves as a saline control.
  8. 10. Cover and incubate according to table 1 instruction.


Table 1: Time and Temperature of Incubation for Salmonella


11. Dislodge the sediment gently and observe for agglutination.


Limitations of Widal Test

          •        The Widal test is time-consuming (to find antibody titer) and often times when diagnosis is reached it is too late to start an antibiotic regimen.

          •        Because of cross-reactivity with other Salmonella species, the test cannot distinguish between a current infection and a previous infection or vaccination against typhoid.

          •        Widal titers have also been reported in association with the dysgammaglobulinaemia of chronic active hepatitis and other autoimmune diseases.

          •        False positive Widal test results are also known to occur in typhus, acute falciparum malaria (particularly in children), and chronic liver disease associated with raised globulin levels, as well as disorders such as rheumatoid arthritis, myelomatosis and nephrotic syndrome.

          •        False negative results may be associated with early treatment, with “hidden organisms” in bone and joints, and with relapses of typhoid fever. Occasionally the infecting strains are poorly immunogenic.

          •        False negative Widal tests may be due to antibody responses being blocked by early antimicrobial treatment or following a typhoid relapse.

          •        Severe hypoproteinaemia may also prevent a rise in O and H antibody titers.

          •        The antibody levels found in a healthy population, however, may vary from time to time and in different areas, making it difficult to establish a cut off level of baseline antibody in a defined area and community.