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1. Widal Test & Rose Bengal
Test
Assistant Prof. Dr. Fitua Al-Saedi
Department of Clinical Laboratory Science
College of Pharmacy
Lab training

2. Widal test was devised by Frank Widal in 1896
 Is a serological test which is used for the diagnosis of
enteric fever (typhoid fever &paratyphoid fever).
 Typhoid fever is caused by Salmonella typhi
 Modes of transmission :
Ingestion of contaminated food or water.
Rarely , from person to person – fecal-oral route.
Food handlers/ Carriers.
 Paratyphoid fever is caused by either Salmonella paratyphi A, B, or C.
Widal test

3.  Salmonella possess O antigen on their cell wall and H
antigen on their flagella. On infection, these antigen
stimulates the body to produce specific antibodies
which are released in the blood.
 The Widal test is used to detect these specific
antibodies in the serum sample of patients suffering
from typhoid using antigen-antibody interactions.
 These specific antibodies can be detected in the
patient’s serum after 6 days of infection (fever).

4. Principle of Widal test
 The principle of Widal test is based on a agglutination reaction between patient’s
antibodies and Salmonella sp antigens(KIT).
 Antibody in the serum produced in the response to
Salmonella sp
 The kit contains antigen suspensions that are killed bacteria and are
stained; The blue stained antigens are specific to the somatic
antigens (O-Ag), while the red stained antigens are
specific to the flagella antigens (H-Ag).

5. Requirements for widal test:
i) Serum
ii) The complete kit containing five vials containing stained Salmonella antigen
•S. typhi———-O antigen
•S. typhi———- H antigen
•S. paratyphi —–AH antigen
•S. paratyphi —–BH antigen
iii) Widal positive control

6. Rapid slide test
Using a micropipette dispense one drop (80 µl) of
undiluted serum onto 4 different circles.
Dispense one drop of O, H, AH, and BH antigens on these
circles respectively.
Mix it thoroughly with the aid of the applicator stick and
rotate the slide gently.
Result:
•Agglutination was observed within a minute (+ve test).
•No agglutination (-ve test)
Procedure of Widal Test
Widal test can be done in two ways-
1-Rapid slide test:
2-Tube test:

7. Quantitative slide test (Rapid slide titration)
Rapid slide titration needs to perform for the samples which showed positive titre
during rapid screening.
•Using a micropipette, dispense 80,40, 20, 10 and 5µl of undiluted serum onto a row
of 3 cm diameter circles.
•Shake the reagent bottle and add a drop (30µl) of the undiluted antigen suspension
to each serum aliquot.
•Mix it thoroughly mixed with the aid of a stirring stick and rotate the slide gently.
•Observe the reactions after a minute.
Result:
•Agglutination was observed within a minute (+ve test).
•No agglutination (-ve test)

8. STANDARD TUBE TEST METHOD
1.Take 4 sets of 8 tubes and label them 1 to 8 for O, H, AH and BH antibody detection.
2.Pipette into the tube No.1 of all sets 1.9 ml of isotonic saline.
3.To each of the remaining tubes (2 to 8) add 1.0 ml of isotonic saline.
4.To the tube No.1 tube in each row add 0.1 ml of the serum sample to be tested and mix well.
5.Transfer 1.0 ml of the diluted serum from tube no.1 to tube no.2 and mix well.
6.Transfer 1.0 ml of the diluted sample from tube no.2 to tube no.3 and mix well. Continue this serial dilution till tube no.7 in each set.
7.Discard 1.0 ml of the diluted serum from tube No.7 of each set.
8.Tube No.8 in all the sets, serves as a saline control. Now the dilution of the serum sample achieved 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 1:640 1:1280.
9.To all the tubes (1 to 8) of each set add one drop of the respective WIDALTEST antigen suspension (O, H, AH and BH) from the
reagent vials and mix well.
10.Cover the tubes and incubate at 37° C overnight (approximately 18 hours).
11.Dislodge the sedimented button gently and observe for agglutination.

9. •The titre of the patient
serum using Widal test
antigen suspensions is the
highest dilution of the
serum sample that gives a
visible agglutination.

10. Circle 1 2 3 4 5
Serum
volume(slide
titration)
80 µl 40 µl 20 µl 10 µl 5 µl
Amount of
antigen
1 drop 1 drop 1 drop 1 drop 1 drop
Equivalent tube
titre
1:20 1:40 1:80 1:160 1:320
Reporting Widal test

11. Interpretation of the widal test:

12.  Brucellosis is a widespread zoonosis mainly transmitted from cattle,
sheep, goats, pigs and camels through direct contact with blood,
placenta, fetuses or uterine secretions, or through consumption of
contaminated raw animal products (especially unpasteurized milk and
soft cheese).
 Brucellosis was first diagnosed by Wright and Smith in 1897. Other
names for Brucellosis:
• Undulant fever
• Malta fever
• Gibraltar fever
• Mediterranean fever
Rose Bengal Test
 The rose Bengal test is a rapid test was designed for the diagnosis of brucellosis.

13. Causal agent of Brucellosis
Brucella abortus causing abortion in cattle
Brucella melitensis causing abortion in goats and sheep
Brucella suis causing abortion in pigs
Brucella canis causing abortion in dogs
These microorganisms localize in the reproductive organs of host animals, causing abortions and sterility.
Laboratory diagnosis of Brucellosis
 Rose Bengal test (RBT)
 Isolation of Brucella spp. from blood or other clinical
specimen.
 Enzyme linked immunosorbent assay (ELISA) ELISA test;
 Serum agglutination test,
 2-Mercaptoethanol test,
 Polymerase chain reaction(PCR)

14. PRINCIPLE
The Rose Bengal Test is a rapid slide agglutination
procedure developed for the direct detection of Brucella
antibodies in human and animal sera-
The bacterial suspension(B. abortus strain coloured with Rose Bengal) is
reactive with both immunoglobulin G and immunoglobulin M antibodies
The presence or absence of a visible agglutination, indicates
the presence or absence of antibodies in the samples tested.
Rose Bengal Test

15. Qualitative method
1. Allow the reagents and samples to reach room temperature.
2. Place 1 drop (50 µL) of the serum under test into one of the
circles on the card. Dispense 1 drop of positive control.
3-Add 1 drop of Rose Bengal Antigen to each circle next to the
sample to be tested.
4-Mix the contents of each circle with a disposable stirrer while
spreading over the entire area enclosed by the ring. Use
separate stirrers for each mixture.
Rotate the slide slowly either by hand or by means of a
mechanical rotator (100 r.p.m.) for a period of 4 minutes.
5-Observe immediately under a suitable light source for any
degree of agglutination.

16. The presence of agglutination indicates an antibody anti-
Brucella concentration equal or greater than 25 IU/mL.
Reading the results and interpretation

17. Thanks