Laboratory Tests

Widal Test Principle, Procedure, Result, Advantages, Limitation

The Widal test is an agglutination test that determines any antibodies present in the patient's serum that are generated against the causal...

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Sourav Bio
This article writter by Sourav Bio on November 30, 2021

Writer and Founder of I am from India and my main purpose is to provide you a strong understanding of Microbiology.

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Widal Test Principle, Procedure, Result, Advantages, Limitation
Widal Test Principle, Procedure, Result, Advantages, Limitation

Widal Test

  • The Widal test is an agglutination test that determines any antibodies present in the patient’s serum that are generated against the causal agents of the enteric febrile illness (Salmonella Typhi, and Salmonella paratyphi B, A as well as C).
  • It is among the most commonly used methods to determine the serological cause of paratyphoid or typhoid disease particularly in the endemic areas.
  • The test was created by Greembaum and Georges Ferdinand Widal in 1896 to detect enteric fever in humans.
  • The Widal test makes use of antigen-antibody interactions to identify specific antibodies in serum samples of patients suffering from typhoid. The antigens tested are derived from the bacteria of Salmonella Typhi and Salmonella Paratyphi.
  • Salmonella Typhi possesses O antigen on its cell wall, and H antigen on the flagella. Similar to that, Salmonella Paratyphi A and Salmonella Paratyphi B also have O antigens on their cell walls, however, they have AH and BH antigens on their flagella, respectively.
  • In the event of infection, these antigens cause the body to create specific antibodies that release into blood. The specific antibodies that are produced against antigens can be identified in the serum of the patient following 6 days after illness (fever).

The Objective of Widal Test

The test can identify serum antibodies against Salmonella Typhi and Salmonella Paratyphi helping to diagnose enteric fever among patients who are suspected.

Principle of Widal Test

People infected by Salmonella Typhi or S. paratyphi develop antibodies in the form of somatic (O) antigens or flagellar antigens (H). The antibodies that are produced in serum when exposed to bacterial suspensions containing homologous antigens, cause the formation of agglutination. The agglutination process is visible as clustering.

For the testing, patient’s blood is mixed with dead suspensions of bacterial strains from Salmonella that contain specific O H AH as well as BH antigens and analyzed for agglutination reactions. If the patient’s serum has specific antibodies to the antigens, it will show clumping which is a sign of positive results. Agglutination in absence is a sign of a negative test.

Requirements for Widal Test

A fresh, clean serum kit that contains Salmonella antigen (O H, O, AH and BH antigens) Positive control, test card for Widal or slide Applicator stick.

Procedure of Widal Test

The Widal test is conducted in two ways:

  1. Rapid slide test: Rapid slide test can be quantitative or qualitative and is performed on a slide.
  2. Tube test: It’s conducted in tubes, is quantitative, and requires incubation. It is considered to be more accurate than slide tests.


  1. Apply 1 drop of Positive Control onto one of the reaction circles on the slide.
  2. Pipette 1 drop Isotonic salicylate on the next cirlcle of reaction. (Negative Control).
  3. Pipette the drop serum of the patient to be tested on the four circles of reaction.
  4. Make sure to add one drop Widal the TEST Antigen Suspension “H in the two circles of reaction. (PC & NC).
  5. Add one drop of each of O”, ‘H “AH” and “BH” antigens to each of the remaining four reaction circles.
  6. Mix the contents of each circle evenly over the entire circle using different mixing tools.
  7. Slide gently to the left and right, and then look for macroscopically agglutination in less than one minute.


  1. Pipette 1 drop of Isotonic solution into the first reaction circle, and then, place 5, 10 20 40 and 80 ul of the test sample on the other circles.
  2. Add to each circle an amount of antigen that showed agglutination to the test sample using the screening procedure.
  3. Utilizing separate mixing sticks, Mix the ingredients of every circle in a uniform manner across those of the reactions circles.
  4. Slide gently between the two slides; look for macroscopically visible agglutination in less than one minute.


In the Widal Test, two types of tubes were used initially:

(1) Dreyer’s tube (narrow tube with conical bottom) for H agglutination and

(2) Felix tube (short round-bottomed tube) for O agglutination.

These days, 3 x 0.5 milliliter Kahn tubes are used to treat different kinds of Agglutination.

  1. Take four sets of 8 Kahn tubes/test tubes and label them from 1 to 8 to allow for O, H, AH, and the detection of BH antibodies.
  2. Pipette through the tube No.1 of all sets 1.9 Ml of isotonic sodium.
  3. In each of the remaining tubes (2 to 8), add 1.0 mg of isotonic sodium.
  4. To the No.1 tube, in each row, add 0.1 milliliters from the sample of serum to test, and mix thoroughly.
  5. Transfer 1.0 milliliters of dilute serum out of tube no.1 to tube no.2 and mix thoroughly.
  6. Transfer 1.0 milliliters of the diluted amount from the tube no.2 to tube no.3 and mix thoroughly. Repeat this dilution in series until the tube no.7 within each series.
  7. Get rid of 1.0 milliliters of dilute liquid from tubes No.7 from each of the sets.
  8. Tube No.8 in all sets serves as an saline control. The dilution for the serum sample obtained in each set is as follows Tube No. 1 2 3 4 5 , 6 7, 8 (control) Dilutions 1;20 1:40 1:80 1:160 1;320 1:1280 1:1280.
  9. In all tubes (1 through 8) from each set,, add one drop of each WIDALTEST Antigen Suspension (O, H, H, AH, and the BH) from the vials of reagent and mix thoroughly.
  10. Cover the tubes with a lid and let them incubate at 37 deg C over the course of a night (approximately about 18 hours).
  11. Remove the button that has been sucked up and then look for an agglutination.

These protocols can be obtained by a WIDAL TEST. Swemed Diagnostics

Result Interpretation of Widal Test

Positive:  Agglutination occurs within one minute

Negative: No agglutination in the sense that there are no significantly clinically important levels for the relevant antibody in the serum of the patient.

  • The sample that has an amount of 1:100 or greater for O Agglutinations, and 1:200 or higher for H agglutination is taken into consideration as being significantly clinically relevant (active disease).
  • The demonstration of a 4-fold increase between the two cases is an indication of the diagnosis.
  • H agglutination is more stable that O agglutinin.
  • Agglutinin begins to appear in serum towards the end of the first week, with a sharp increase during the 2nd and 3rd weeks and the amount remains steady until the 4th week, following which it decreases.
  • The rising titre of antibodies is more reliable evidence of the presence of infection than just positive tests. It is recommended to test two different samples of sera in a time span between 7 and 10 days to determine a rising antibody level.
  • Low antibody levels are normal in healthy individuals, but are not a sign of an infection.

Advantages of Widal Test

  • It is a quick test for detecting for enteric fever in areas with endemic.
  • If facilities for culture aren’t readily available, Widal tests are useful.
  • It is able to detect infections caused by Salmonella Typhi and Salmonella Paratyph.

Limitations of Widal Test

  • The Widal test is time-consuming (to determine the amount of antibody) and, often, when diagnosis is confirmed, it’s too late to begin an antimicrobial regimen.
  • The Widal test should be considered with reference to the baseline titers for an otherwise healthy local population.
  • The Widal test can not be positive for some patients who have received a previous vaccinations or have contracted S. Typhi.
  • In addition to cross-reactivity with other Salmonella species It is impossible to tell between an ongoing disease and an infection that occurred previously 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 reported in acute falciparum, typhus and malaria
  • (particularly for children) Chronic liver disease is characterized by elevated levels of globulin, and conditions like rheumatoid arthritis myelomatosis, and nephrotic disorder.
  • False negative results could be a result of an early diagnosis “hidden organisms” in joints and bone or with relapses in the typhoid virus. In some cases, the infecting strains aren’t immune-friendly.
  • The false negative Widal tests could be caused by antibody reactions that are blocked by early antimicrobial therapy or after the relapse of typhoid.
  • A severe hypoproteinaemia can cause the growth of the H antibody titres and 0.
  • The levels of antibody present in a healthy population but, the levels can differ between times and across different regions which makes it difficult to determine the cut-off amount of baseline antibody within an area or community.
  • In areas where typhoid is not endemic slow and weak O as well as H antibodies reduce the effectiveness for testing with the Widal test. Also, there are differences among labs in the results and interpretation of Widal tests that compromise the accuracy of the test.
  • The World Health Organization (WHO) has stated that because of the many factors that could affect the outcomes of the Widal test, it’s recommended not to place too much on the results of this test.
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Writer and Founder of I am from India and my main purpose is to provide you a strong understanding of Microbiology.

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