Detect the presence of specific antibody and quantitate the amount of antibody in patient’s serum


Table of Contents

Such is the case with the slide and tube Widal test. The agglutinins against the ‘0’ (somatic) and ‘H’ (flagellar) antigens of Salmonella typhi, paratyphi A, and paratyphi B are measured qualitatively (slide test) and quantitatively (Tube test) using a suspension of suitable organisms that have been killed.

Widal-quantitative tube agglutination test

This test aids in the diagnosis of enteric fever by detecting antibodies against Salmonella typhi, S paratyphi A, and S paratyphi B. Specimen: Blood samples of 3-5 mL are collected in sterile, screw-capped, unbreakable, sterile tubes and transferred to the laboratory in an upright position or refrigerated between 2 and 8 degrees Celsius in the event of a delay. If necessary, samples can be spun at 3000 rpm for 10 minutes at room temperature to remove particles prior to testing.


Material and instruments required 

  • S. Typhi ‘o’ antigen suspension. 
  • S. Typhi ‘h’ antigen suspension. 
  • S. Typhi ‘ah’ antigen suspension. 
  • S. Typhi ‘bh’ antigen suspension. 
  • Blood sample 3-5 ml 
  • Plain vacutainer/sst 
  • Normal saline 
  • Micropipettes
  • Glass test tubes


Utilizing commercially available test kits. The kit must be stored between 2 and 80 degrees Celsius. To perform the Widal tube agglutination test, follow the directions provided in the kit package.

Test procedure

  1. For each serum sample being evaluated, put four rows of four tubes in a rack.
  2. Prepare master dilutions by removing four tubes from a separate rack. Place 7 ml of normal saline (0.85% sodium chloride) in one tube and 3.5 ml in each of the other tubes.
  3. Add 0.50 mL of serum to the first tube and thoroughly combine.
  4. Transfer 3.5 mL from the first tube to the second and thoroughly mix.
  5. Transfer 3.5-ml volumes in succession until the final tube is reached.
  6. This will result in final dilutions of 1:30, 60, 120, and 240 upon addition of an equal volume of antigen.
  7. Transfer 0.5 mL from the master dilution tubes to each tube in the corresponding row of the test rack.
  8. As a control, place 0.5 ml of normal saline in each of the tubes in the fifth row.
  9. Add 0.50 ml of S.Typhi “O”, S.Typhi “H”, S.Paratyphi A (H), and S.Paratyphi B(H) antigens to each of the four tubes in the first, second, third, and fourth horizontal rows.
  10. Shake the rack vigorously to combine and incubate at 37 degrees Celsius overnight (16-20 hours).
  11. Note the maximum dilution in which agglutination can be noticed with the naked eye or a hand lens.
  12. With ‘H’ antigens, the agglutination pattern is cottonwool-like and flocculent, whereas with ‘O’ antigen it is granular and appears as a granular mat at the bottom of the tube.
  13. In addition to the pattern of organisms that have sedimented, the decrease in opacity of the supernatant relative to the saline control tube must be noted and considered when determining the degree of agglutination.

Internal quality control 

With each batch of test specimens, the following controls are installed:

  • Negative Regulation: Last row of control tubes with saline
  • Positive Control: pooled serum known to be positive


Normal human sera can agglutinate these antigens in dilutions as high as 1:60. A rise in agglutination titers or repeating the test after a few days will confirm the diagnosis of enteric fever with titers of 1:120 or higher. In most cases of enteric fever, agglutination titers of 1:240 or higher are detected. For interpretation, however, follow the instructions in the kit insert. The causative organism is identified by measuring the ‘H’ agglutinin titre. People who have experienced enteric illnesses in the past or who have received the TAB vaccination may exhibit modest levels of agglutinins when suffering from an unrelated illness. This anamnestic appearance of agglutinins can be distinguished from a real infection by displaying a significant rise/fall in titer when the test is repeated 7-10 days later. A substantial increase in the titers of all three ‘H’ agglutinins simultaneously against all ‘H’ antigens suggests recent TAB vaccination.

All specimens utilised in this test should be regarded as possibly infectious. Gloves should be worn when handling and disposing of hazardous materials during and after usage. For everyday hand washing, use soap.


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