Serological procedure: Typhoid and Paratyphoid Fever

Serological procedure: Typhoid and Paratyphoid Fever

The etiological agent is Salmonella species; it occurs in human only. Sometimes it is termed as enteric fever since they colonize the intestine. Salmonella of medically important species are S.typhi (typhoid fever), S.paratyphi A and B (paratyphoid fever).

Serological procedure: Typhoid and Paratyphoid Fever

Typhoid and paratyphoid fever is transmitted through ingestion of contaminated food or water. They contaminate usually by carriers like rodents, hens, cows, etc. Typhoid or enteric fever is a clinical syndrome characterized by fever, headache, splenomegaly, leucopenia and cough.

Its incubation period ranges from 7 to 14 days. In 5% to10% of untreated patients relapse may occur the symptoms in relapse are milder than the initial illness and begin about two weeks after discontinuation of antimicrobial therapy. The carrier state is as symptomatic and in 1 – 3% of carriers there is continuous excretion of S.typhi for a minimum of one year. The gall bladder is the site of persistent intestinal infection.

Identification of salmonella

Salmonella species can be identified based on their antigenic structure they possess. They have three different antigenic structures.

O- Antigen (somatic antigen): It is lipopolysaccharide of the outer membrane, which is heat and alcohol stable antigen. Salmonella is divided in to five distinct serogroups (A-E) on the basis of somatic antigen.


H-antigen (flagellar antigen): H-antigen is protein, which makes the perithrchous flagella. It is heat and alcohol labile. Salmonella is further subdivided in to more than 1200 serotypes on the basis of flagellar antigens.

Vi- Antigen: This is the antigen that determines the virulence, the ability to cause disease, of the organism.

Preparation of antigen suspension

Salmonella antigen suspension is available commercially and it’s also possible to prepare in the laboratory.

A. Preparation of H antigen (flagellar antigen)


1. Inoculate bacteria from a single colony in to a broth and incubate for 6hrs.

2. View a drop of the culture in a wet film to confirm that most of the bacteria are motile and therefore sufficiently flagellated for the tests.

3. Kill the culture by adding formaldehyde to a concentration of 0.2% and incubate for several hours at 370°C

B. Preparation of O antigen

1. Suspend the bacteria from an agar culture in saline and heat for 30 minute at 1000°C to remove the flagella.

2. Centrifuge to separate the bacteria from the detached flagella.

3. Resuspend the bacteria in saline.


1. Remove the flagella by mixing a dense saline suspension of the bacteria with an equal volume of absolute ethanol

2. Incubate for 20 hr at 370°C

3. Dilute the suspension with saline.

Widal test

Widal test is a serological test, which is commonly used to diagnose typhoid and paratyphoid fever. The patient’s serum is tested for O and H antibodies Rapid slide (Screening) test

1. Clean the glass slides supplied in the kit well and wipe it free of water.

2. Place one drop of undiluted test serum in each of the first circle (1 to 4) and one drop of positive control serum in each of the last two circles.

3. Place one drop of antigen O, H, A (H) and B (H) in circle 1, 2, 3, and 4 respectively and O antigen in circle five and H antigen in circle 6

4. Mix the contents of each circle with separate applicator stick and spread to fill the whole area of the individual circle.

5. Rotate the slide for one minute and observe for agglutination. If agglutination is visible, quantitative estimation of the titer of the appropriate antibodies should be done

Tube agglutination method


1. Take a set of 8 clean dry test tubes for each serum to be tested.

2. Place 1.9ml of saline in tube 1 and 1 ml of saline in other tuber (2-8)

3. Transfer 0.1 ml of undiluted serum to tube 1. Mix thoroughly. The resultant dilution of serum is 1:20.

4. Further dilutions are done in the following

a) Transfer 1ml of the diluted serum from tube 1 and place in tube 2 this leads to 1:40 dilutions in tube2

b) Repeat the transfer process for tube 7 after mixing.

c) Leave 1 ml of saline in tube 8 at the ‘saline control’ Note. Tube 1 has a serum dilution of 1:20, 1:40 (2), 1:80 (3), and 1:160 (4), 1:320 (5), 1:640 (6) and 1:1280 (7).


5. Add one drop of appropriate antigen in each (use only that antigen suspension which has given a positive reaction in the screening test).

Note: Each antigen (O, H, AH, BH) will require a series of 8 tubes for determine the titer of their corresponding antibodies.

6. Mix well and incubate overnight (16-18 hrs) at 370°C.

7. Examine agglutination macroscopically.

8. Two tubes for positive control O and it antigen should be included.


1. Only a titer above 1:80 should be considered as significant.

2. A rise in titer (done each week) is considered to be definite evidence of infection. A single test result is considered of diagnostic value only when it is usually high (above 160).

3. Antibiotic treatment in typhoid fever often prevents a rise in titer,

4. A negative test does not rule out the possibility of infection because of the tine when the blood sample was taken in relation to the stage of the disease.

5. Positive results should always be interpreted with reference to clinical findings.



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