What Is The Widal Test?

The Widal test is an agglutination test used to identify the presence of H and O serum agglutinins in patients with suspected typhoid and paratyphoid fever.

What Is The Widal Test?

The Widal test was named after Georges-Fernand-Isidor Widal, a French physician who invented the test in 1896.

The Widal test is best used when culturing facilities are unavailable and can be used as a possible alternative in diagnosing typhoid fever in endemic regions.

In this article, we will inform you about how scientists perform the Widal test. Find out how to prepare for it and how to identify the results.

However, the Widal test isn’t the only method of assessing typhoid and paratyphoid fever, and several controversies surround it. Let’s begin this article by understanding when the Widal test was first made. 

A Brief History Of The Widal Test

To understand the brief history of the Widal test, we need to understand the period Widal made it.

The inventor of the Widal test, Georges-Fernand-Isidor Widal, was born in 1862 and invented the Widal test in 1896 with the cooperation of Dr. Andre Chantemesse, a French Bacteriologist.

He developed the Widal test to diagnose typhoid fever, understanding that the antibodies in the infected patient’s blood would cause the bacteria to clump together and agglutinate. 

In the same year, the first practical use of a vaccine for typhoid would be developed by Almroth Edward Wright and used by the military.

Back then, many soldiers were more at risk of typhoid than being killed in combat. Due to the widespread effects of typhoid, it was vital that the Widal test was utilized at this time. 

What Is Typhoid Fever?

Typhoid fever is a highly contagious bacterial infection. Typhoid is caused by Salmonella typhi, which is related to the same bacteria that can cause Salmonella in food poisoning.

Typhoid is easily transmitted through feces and urine, so there are higher cases in less-developed areas with poor sanitation and limited access to clean water. 

The symptoms of typhoid fever involve fever, headache, fatigue, pain, coughing, and constipation. You may lose your appetite as typhoid progresses, and you might experience stomach pain and diarrhea.

You must diagnose and treat typhoid as soon as possible, as severe typhoid can be fatal. 

What Is Paratyphoid Fever?

Paratyphoid fever is similar to typhoid fever, with both of these infections caused by different strains of Salmonella. While Salmonella Typhi causes typhoid, Salmonella paratyphi causes paratyphoid.

Paratyphoid fever is less severe than typhoid, and you can become infected similarly. Therefore, it is more likely to occur in countries with poor sanitation available.

The symptoms include fever, headache, abdominal pain, appetite loss, a dry cough, and constipation. These symptoms aren’t as severe as typhoid fever, and you should note that paratyphoid fever is far less fatal than typhoid. 

Where Is The Widal Test Used?

Due to the Widal test being over one hundred years old, it is not the most reliable source to detect enteric fever in developed countries.

For example, the United States doesn’t use the Widal test due to the test having more success in endemic regions in developing nations.

It is therefore beneficial when no culturing facilities are available, and you can use it as a way to test and compare results when a high number of cases emerge.

However, there are issues with this when cases relapse, as they can appear as false negative results. When used in endemic areas, it is often used as a last resort, as doctors should treat typhoid fever as soon as possible, and the Widal test is time-consuming. 

What The Widal Test Assesses

The Widal test assesses the patient by taking specific antibodies from them and mixing them with bacteria which causes typhoid fever.

The combination allows scientists to test for O and H antibodies against eight different stained antigen suspensions. Here is a list of the suspensions which scientists may use. 

Antigen Suspensions Used In The Widal Test

The antigen suspensions used in a Widal test are as follows: 

  • Salmonella typhi 0 antigen suspension, 9, 12
  • Salmonella typhi H antigen suspension, d
  • Salmonella paratyphi A 0 antigen suspension, 1, 2, 12
  • Salmonella paratyphi A H antigen suspension, a
  • Salmonella paratyphi B 0 antigen suspension 1, 4, 5, 12
  • Salmonella paratyphi B H antigen suspension, b, phase 1
  • Salmonella paratyphi C 0 antigen suspension, 6, 7
  • Salmonella paratyphi C H antigen suspension c, phase 1

You can use these antigens via slide and tube techniques. The H and O antigens of S. Typhi and the H antigens of the S. Paratyphi are used.

O antigens of the paratyphoid strain won’t be employed due to their cross-reaction with the typhoid O antigen, as they share a factor of 12. 

When a patient suffers from acute typhoid fever, you will usually detect O antibodies between six to eight days after the fever starts. Scientists will detect H antibodies between ten and twelve days after the onset of typhoid. 

Understanding The Readings Of The Widal Test

Salmonella antibodies usually begin to appear in the serum by the end of the first week. These antibodies then rise sharply during the third week of the patient suffering from an endemic fever. 

Bacteria that carry the antigen will agglutinate or clump upon exposure to the antibodies combined with the Salmonella organisms. When the antibodies react to the Salmonella, the clumping will be visible on the widal test card and in the tube. 

Ways To Do The Widal Test

There are three methods in which you can approach the Widal test, but the first order of business is to prepare your antigens for whichever process you choose. Here is a brief guide on how you should prepare your bacteria.

Preparing The Antigens

To prepare the H suspension of your bacteria, you should add 0.1% formalin to a twenty-four hour broth culture. If you do not have a broth culture available, add your 0.1% of formalin to a saline suspension of agar culture. 

To prepare the O suspension of your bacteria, you should culture them in phenol agar with a ratio of 1:800. By doing this, you will be able to prevent flagella from occurring.

You will then emulsify the growth with a minuscule volume of saline, which should be blended with twenty times its alcohol volume.

Then, heat the growth between 104 degrees Fahrenheit and 122 degrees Fahrenheit for half an hour. Once you complete this step, place it into a centrifuge.

Preserve your antigens using chloroform and add appropriate dyes to enable easy identification of each of your antigens. Once you have prepared your antigens, you should have no difficulty commencing your test.

Widal Slide Test

What Do You Need For The Slide Test?

You will need several items for the slide test, so before you start, here is what you’ll need: 

  • Two slides.
  • One pipette
  • Isotonic Saline
  • Patient’s serum
  • Salmonella typhi H antigen
  • Salmonella typhi O antigen
  • Salmonella paratyphi AH antigen
  • Salmonella paratyphi BH antigen
  • A minimum of twelve wooden sticks 

When you do a slide test, make sure that you place a single drop of positive control on one reaction circle on your slide. Then, you should use your pipette to place a single drop of Isotonic saline on the reaction circle next to it.

You should then pipette a single drop of your patient’s serum onto the four remaining reaction circles on that slide. 

Greb another slide and add a drop of the H antigen suspensions to the first two reaction circles for Positive Control and Negative Control. Then, in the four remaining reaction circles, include a single drop of these antigens: O, H, AH, and BH. 

Using a separate mixing stick for each of your circles, mix the contents of each circle over their entire area.

Then, rock the slide gently back and forth, looking for any agglutination within the first minute of doing this. You don’t need to use a microscope. Just look closely within the first minute to check for any clumping within the circles.


What Do You Need For The Semi-Quantitative Method?

Here’s a list of items that you will need to do the semi-quantitative method: 

  • A new slide
  • A pipette
  • Isotonic saline 
  • Samples of your antigens that you observed agglutination in previously.
  • A minimum of six wooden sticks.

You can do the semi-quantitative method as a secondary method after the slide test to help confirm your results.

To do the semi-quantitative method of the Widal test, you will need to pipette a single drop of isotonic saline into the first reaction circle on your slide. You will then need to place these measurements of a test sample on the free circles on your slides: 

  • 5 ul
  • 10 ul
  • 20 ul
  • 40 ul
  • 80 ul

Once these amounts of your test samples are added to your free circles, add a drop of the agglutinated antigens you found in the slide test to your test samples.

Then get separate mixing sticks to mix the contents of each circle to cover the area. Once you do this, rock the slide gently to check for any agglutination within a minute of doing this. 

Again, you will not need a microscope to check for agglutination. 

Results Of The Slide Test

You will find a positive result in the Widal test if you observe agglutination within 20 ul of your test sample. By watching the level of agglutination here, you will find a clinically significant amount of antibodies in the patient’s serum.

However, a negative result will include no agglutination in the slide test. There will be no clinically significant level of the corresponding antibody in the patient. 

Standard Widal Tube Test

What Do You Need For The Widal Tube Test?

Here is a list of items that you will need to commence your tube test: 

  • Six 0.5 ml Kahn tubes for O and H agglutinations
  • Thirty-two Kahn tubes for analysis
  • One pipette
  • Isotonic saline
  • Salmonella typhi 0 Antigen
  • Salmonella typhi H antigen
  • Salmonella paratyphi AH antigen
  • Salmonella paratyphi BH antigen
  • A minimum of thirty-two wooden sticks

When the Widal test was initially used, scientists used a Dreyer’s tube for H agglutination and a Felix tube for O agglutination. A Dreyer’s tube is a narrow tube with a cone-like shape at the bottom, and a Felix tube is a shorter tube with a more rounded bottom. 

In present-day Widal tests, you should use a trio of 0.5 ml Kahn tubes for H and O agglutinations, respectively. 

Upon starting your test, arrange four sets of eight Kahn tubes and label them 1 to 8. You should label each set for O, H, AH, and BH antigens. 

In the tube labeled 1 in each of your four sets, you should pipette 1.9 ml of isotonic saline into the tube. Then, in tubes, 2 through 8, add 1.0 ml of isotonic saline.

Include 0.1ml of your sample serum in tube 1 of each row so that you can test it. Mix the samples in your tubes well.

Then transfer 1.0 ml of the diluted serum from tube 1 to tube 2 and mix it well. Continue to transfer 1.0ml of your diluted serums until you reach tube 7.

When you get to tube 7, discard 1.0ml of the diluted serum from each set. Then, use tube number 8 for saline control.

You should dilute the serum at the following ratios: 

  • 1:20
  • 1:40
  • 1:80
  • 1:160
  • 1:320
  • 1:640
  • 1:1280

Now that you know the ratio of each tube, you should add a single drop of the antigen suspensions from the reagent vials and mix them.

The antigen suspensions should be O, H, AH, and BH. Once you have done this, cover your Kahn tubes and incubate them at 98.6 degrees Fahrenheit for around eighteen hours.

Then, gently dislodge any sediment and check for any agglutination in the tube.

Results Of The Widal Tube Test

You should assess the tube test results by analyzing the highest dilution of the serum sample with visible agglutination.

A sample that shows a titer of 1:100 or above for O suspensions or 1:200 or above for H suspensions should be considered clinically significant. If this is the case, your patient has an active infection.

A demonstration of a four-fold rise between two is a diagnostic tool to assess whether they are infected. But it is important to remember that H suspensions agglutinated will be a more reliable indicator than if an O suspension agglutinates. 

Any agglutinin should begin to appear in the serum by the end of the first week of incubation. By the second and third week, there should be a sharp rise. After the third week, it should remain at a steady rate and decline in the fourth. 

Controversies Around The Widal Test

Controversies Around The Widal Test

Various factors can influence the results of a Widal test, and because of this, it has been classed as unreliable by the World Health Organization. Here are some explanations why the Widal test may not be used in certain situations.


Analyzing test tubes used in the Widal test takes a significant amount of time. Finding titer and observing the agglutination process takes eighteen hours to get an initial reading, and the agglutination process continues for several weeks.

Once scientists reach a diagnosis, it is often too late to start the patient on antibiotics.

False Negative Results

Widal tests have been classed as unreliable due to the number of factors that can cause a false negative result.

You can get a false negative result from having early treatment or if the patient has a case of typhoid after having been previously infected.

These can also occur because when analyzing the titers in the results, you may find that the titers also react to other illnesses.

False Positive Results

While false negative results have been an issue for Widal test results, so have false positive results.

Many patients who have been vaccinated against typhoid or another infection where they were injected with Salmonella typhi may trigger a positive result in their tests.

They will also occur in patients suffering from typhus, malaria, chronic liver disease, rheumatoid arthritis, myelomatosis, and nephrotic syndrome.

Due to the various illnesses and conditions present here, there are many examples where an unrelated condition could trigger a false positive.


The Widal test is an agglutination test that can identify typhoid and paratyphoid fever. While it is often used in developing countries, it is no longer used in the United States due to the time-consuming process and unreliable factors influencing the results.

Used as a last resort, you may find that a Widal test is the only available method in areas where culturing facilities are unavailable.

This test is over one hundred years old, and since it was invented back in 1896, there have been more rapid response tests that can be used to help identify bacteria that cause typhoid and paratyphoid fever.

Jennifer Dawkins

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