For MBBS students

1. WIDAL AND WEIL FELIX TEST
Dr.P.B.PRAVEENKUMAR
FIRST YEAR POSTGRADUATE
DEPARTMENT OF MICROBIOLOGY
THANJAVUR MEDICAL COLLEGE

2. WIDAL TEST
• AIM: This diagnostic test is employed for the
serological diagnosis of enteric fever.

3. PRINCIPLE
• TUBE AGGLUTINATION TEST: Antibodies
against Salmonella typhi, S. Paratyphi A and B
infections are detected in patient serum. Since
two types of agglutinins are generated in the
serum which react with ‘O’ and ‘H’ antigenic
components of the salmonellae, a total of four
antigen suspension namely TO, TH, AH and BH
are employed in the test.

4. PROCEDURE
• Arrange four rows each containing 7 clean and
dry Widal tubes (1:30, 1:60, 1:120, 1:240, 1:480,
1:960, NC) in a Widal rack for each antigen.
• So totally 29 tubes (including Master dilution
tube)
• Mix 0.1 ml of the patient serum with 1.4 ml of
the NS to obtain a 1 in 15 dilution(MASTER
DILUTION)
• Add 0.4 ml of NS from 2nd to 7th tube of each
antigen rows(O, H, AH & BH).

5. PROCEDURE (CONT.)
• Add 0.4 ml of already prepared Master diluted
serum into 1st and 2nd tube of each antigen rows.
• After mixing, transfer 0.4 ml from 2nd tube to 6th
tube and from sixth tube discard 0.4 ml (For all
antigen rows)
• Add 0.4 ml of respective antigen namely TO, TH,
AH and BH in respective rows from 1st to 7th tube.
• Incubate the tubes at 37 degrees celsius in water
bath for 4 hours and read after overnight
refrigeration at 4 degrees celsius.

6. INTERPRETATION
O AGGLUTININ MORE THAN 100 - SIGNIFICANT TITER (Granular chalky clumps)
H AGGLUTININ MORE THAN 200 - SIGNIFICANT TITER (Large loose fluffy cotton woolly clumps)
BUTTON FORMATION – Agglutination doesn’t occur

7. WEIL FELIX TEST
• AIM: This diagnostic test is employed for the
serological diagnosis of rickettsial infections.

8. PRINCIPLE
• HETEROPHILE TUBE AGGLUTINATION TEST:
This test is based upon the sharing of an alkali-
stable carbohydrate antigen between some
rickettsiae and certain non-motile strains of
Proteus, P.vulgaris OX19 and OX2 and
P.mirabilis OXK. The antibodies against the
pathogen appear around 5th day of the onset
of the disease. These can be detected by slide
or tube agglutination.

9. PROCEDURE
• Arrange three rows each containing 7 clean and
dry test tubes (1:20, 1:40, 1:80, 1:160, 1:320,
1:640, NC) in a test tube rack for each antigen.
• So totally 22 tubes (including Master dilution
tube)
• Mix 0.1 ml of the patient serum with 0.9 ml of
the NS to obtain a 1 in 10 dilution(MASTER
DILUTION)
• Add 0.4 ml of NS from 2nd to 7th tube of each
antigen rows(OX19, OX2 & OXK).

10. PROCEDURE (CONT.)
• Add 0.4 ml of already prepared Master diluted
serum into 1st and 2nd tube of each antigen rows.
• After mixing, transfer 0.4 ml from 2nd tube to 6th
tube and from sixth tube discard 0.4 ml (For all
antigen rows)
• Add 0.4 ml of respective proteus antigen namely
OX19, OX2 & OXK in respective rows from 1st to
7th tube.
• Incubate the tubes at 50 degrees celsius in
incubator for 4 hours and read after overnight
incubation at 37 degrees celsius.

11. INTERPRETATION
SINGLE TITRE OF >/=160 – CLINICALLY SIGNIFICANT (GRANULAR CLUMPS)

12. THANK YOU