Microbiology Laboratory


Written by MicroDok

AIM: To detect the presence of agglutinins (O and H antibodies) in patient’s serum as an aid in the diagnosis of typhoid and paratyphoid fevers.

PRINCIPLE: The principle is based on the immunological reaction between agglutinins in patient’s serum and febrile antigen suspensions, resulting in visible agglutination within 60 seconds.

MATERIAL/APPARATUS: Blood specimen, centrifuge machine, Pasteur’s pipette, test card, timer, mixing stick, Salmonella typhi O and H antigens, S. paratyphi AO and AH antigens, S. paratyphi BO and BH antigens, S. paratyphi CO and CH antigens.

O = Somatic cell wall antigen (blue in colour).

H = Flagella antigen (red colour).

“The letters A, B, & C” are used to differentiate the 3 strains of S. paratyphi.


  1. Bring the test reagents to room temperature (23oC – 29oC).
  2. Centrifuge the blood specimen in the centrifuge machine at 400rpm for 5 minutes, in order to sediment the red cell component of the blood and thus obtain the serum component of the blood specimen required to undertake this test.
  3. Using the Pasteur’s pipette, place a drop of the serum on 8 different spots of the test card.
  4. Shake the vials containing the antigen reagents and place a drop of the Salmonella typhi O and H antigens, paratyphi AO and AH antigens, S. paratyphi BO and BH antigens, and S. paratyphi CO and CH antigens respectively on the 8 drops of the serum on the test card.
  5. Mix the mixture together using a mixer or the paddle end of the Pasteur’s pipette.
  6. Rock and rotate the test card for 60 seconds.
  7. Observe the test card immediately after rocking, for visible agglutination.


The Widal test is reported by giving the titer values for both the O and H antibodies. This is done by observing any degree of agglutination that is visible within 60 seconds. The highest dilution of serum in which agglutination occurs is taken as the antibody titer. The result is reported using titer values like: 1:20, 1:80, 1:160, 1:30 e.t.c. depending on the degree of visible agglutination observed macroscopically. Titer values less than 80 (e.g. <20) are considered non – reactive or negative, while those above 80 are considered significant. The Widal test result should not be read beyond the 60 seconds time interval, because of the possibility of drying effect which might give a false positive result.

Example of a Widal test result report is thus:

O                 H

Salmonella typhi                                                                                       <20             <20

Salmonella paratyphi A                                                                           <20             <20

Salmonella paratyphi B                                                                           <20             <20

Salmonella paratyphi C                                                                           <20             <20

Significant titer: >80.

Widal test (pronounced as’ Vidal’) is a serological technique which is used to test for the presence of Salmonella antibodies (O and H antibodies) in a patient’s serum. Though good for the investigation of typhoid and paratyphoid fevers caused by S. typhi and S. paratyphi, it is of no value in the investigation of food poisoning caused by S. typhimurium. This test can also be performed by a tube method, as only the slide method is expanded here.

Agglutination is defined as the clumping of blood cells. It is the reaction between a particular antigen and an antibody, which results to the formation of a visible clump.

The ‘O’ antigen is the somatic cell wall of the infecting organism (Salmonella). It is the first to be produced during an infection, and an increase in the titer value of ‘O’ during testing is indicative of the presence of an infection. The ‘O’ antigen suspension used in this test is bleu in colour.

The ‘H’ antigen is the flagella antigen. Its increase in titer value during testing is often not significant as it shows past or old infection. It is also not considered as it can suggest prior immunization of the patient against the infection. The ‘H’ antigen suspension used in this test is red in colour. The difference in colouration helps to differentiate the 2 antigen preparations in order to prevent chaos in reporting the test result.

Salmonella are a group of bacteria in the family Enterobacteriaceae. They cause different forms of infections in humans including: enteric fevers, enterocolitis, and bacteremia. The pathogen gain entrance into the human body via the oral route through contaminated food and water. They can easily spread to susceptible hosts via a direct contact with carriers or infected people, and the best and most adequate way of preventing their spread and contamination is by observing a good personal hygiene. 

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