microbiology ppt

1. WIDAL TEST
MADE BY : VERTIKA AWASTHI
PHARM D 2ND YR
CHITKARA COLLEGE OF PHARMACY

2. INTRODUCTION
• Widal test is a serological method to diagnose enteric or typhoid fever that is cause by the
infection with pathogenic microorganisms like Salmonella typhi, Salmonella paratyphi a, b
and c.
• The method of diagnostic test is based upon a visible to the naked eye agglutination (clumps)
reaction between antibodies of patient serum and antigens specifically prepared from
Salmonella sp (KIT).
• Salmonella possess the following 3 antigens:
 Flagellar antigen or H antigen
 Somatic antigen or O antigen
 Surface antigen or Vi antigen

3. • Widal test, firstly descried by Fernand Widal in 1896.
• The tests measure agglutinating antibodies directed against a Salmonella O
somatic surface antigen and/or a Salmonella H flagella antigen of the
suspected organism.
• The Widal test detects antibodies against O and H antigens.
• Type of techniques is use direct agglutination.
• Principle of Widal test:
Widal test is an agglutination test in which specific typhoid fever
antibodies are detected by mixing the patient's serum with killed
bacterial suspension of Salmonella carrying specific O, H, AH and BH
antigens and observed for clumping ie. Antigen-antibody reaction.

4. • Salmonella are gram negative, peritrichous rod shaped bacilli
• Normal habitat:
S. typhi and S. paratyphi are only found in humans and excreted in the feces
and urine of infected patients and are present in the gall bladder of long term
carriers.
Infection is by the ingestion of the organism in food/water or from
contaminated hands.
S. typhi is mainly spread by water and S. paratyphi by food.

5. Salmonella Antigens:
O Antigens
• Cell wall heat stable antigens.
• Groups are designed A- Z.
• Medically important belong to groups A to G
H Antigens
• Flagella, heat-stable antigens.
• Serotyped by their H antigens.
Vi Antigens
• Surface or capsule (K) antigen can be found in S. typhi, S. paratyphi C and a few other
Salmonella.

6. ANTIBODY APPEARANCE
• Anti-O antibody appears 8 days after the onset of disease & mainly of IgM
• Anti-H antibody appears 12 days after the beginning of disease & mainly of IgG
• Anti-Vi appears in 21 days
• Anti-O antibody disappear 3-6 monthes by treatment, but Anti-H may remind more

7. PROCEDURE OF WIDAL TEST
The Widal test can be conducted in two ways :
•Slide agglutination Widal test
1. Qualitative Slide Test
2. Quantitative Slide Test
•Tube agglutination Widal test
Tube agglutination has more accuracy as compared to the slide agglutination technique.
However, A slide widal test is more popular among diagnostic laboratories as it gives rapid
results

8. QUALITATIVE SLIDE TEST
Procedure :
• Bring all reagents to room temperature and mix well.
• Add 1 drop of test sample (25µl) into each reaction circle labeled as O, H, AH, BH according to given antigen
solution.
• Add 1 drop of positive control (25µl) into the circle marked as PC and 1 drop of negative control (25µl) into the
reaction circle marked as NC.
• Add antigen solutions of Salmonella typhi ‘O’, Salmonella typhi ‘H’, Salmonella paratyphi ‘AH’ and Salmonella
paratyphi ‘BH’ to circles labeled as O, H, AH, BH respectively in which test samples has been added.
• Mix it thoroughly with the aid of applicator stick and rotate the slide gently.
• Observe for agglutination.

9. INTERPRETATION
•Positive Test : Agglutination within a minute
•Negative Test : No agglutination

10. QUANTITATIVE SLIDE TEST
This is performed for the samples which showed positive agglutination during qualitative test.
Procedure :
• Bring all reagents to room temperature and mix well.
• Dispense one drop of saline into the first reaction circle and then place 5, 10, 20, 40, 80 ul of the test sample on the remaining
circles.
• Add a drop of the antigen, which showed agglutination with the test sample in the screening (qualitative) method, to each circle.
• Mix the contents of each circle with the aid of applicator stick and rotate the slide gently.
• Observe for agglutination.
Interpretation :
• The antibody titre of the test sample is its highest dilution that gives a visible agglutination. 80 µl corresponds to 1 in 20 dilution,
40 µl to 1 in 40, 20 µl to 1 in 80, 10 µl to 1 in 160 and 5 µl corresponds to 1 in 320 titre. Agglutinin titre greater than 1:80 is
considered as significant infection and low titres indicate absence of infection Interpretation :
• The antibody titre of the test sample is its highest dilution that gives a visible agglutination. 80 µl corresponds to 1 in 20 dilution,
40 µl to 1 in 40, 20 µl to 1 in 80, 10 µl to 1 in 160 and 5 µl corresponds to 1 in 320 titre. Agglutinin titre greater than 1:80 is
considered as significant infection and low titres indicate absence of infection

11. QUANTITAIVE TUBE TEST
Procedure :
• Bring all reagents to room temperature and mix well.
• Prepare 4 sets of test tubes for individual antigen. Each set contains 1- 8 tubes.
• Add 1.9 ml of 0.85% sterile saline to tube no. 1 of each antigen set.
• To tube no. 2-8 of all sets add 1 ml of physiological saline.
• To tube No. 1 of all sets add 0.1 ml of test sample to be tested and mix well.
• Transfer 1 ml of the diluted serum sample from tube No. 1 to tube No. 2 and mix well.
• Transfer 1 ml of the diluted serum sample from tube No. 2 to tube No. 3 and mix well. Continue this serial dilution till tube No. 7
in each set of antigen.
• Discard 1.0 ml of the diluted serum from tube No.7 of each set.
• So the dilutions of the serum sample from tube No. 1 to 7 respectively in each antigen set are 1:20, 1:40,1:80, 1:160, 1: 320,
1:640, 1: 1280.
• Tube no. 8 is negative control with 0.85% sterile saline.
• To one set i.e. from tube no.1- 8 add 50 µl of Salmonella typhi ‘O’ antigen.
• In second set i.e. from tube no.1- 8 add 50 µl of Salmonella typhi ‘H’ antigen.
• Respectively for third and fourth sets, add Salmonella paratyphi ‘AH’ and Salmonella paratyphi ‘BH’ to all tubes from 1-8.
• Mix well, cover and incubate these tubes overnight at 37 degree Celcius (approximately 18 hours).
• After incubation dislodge the sediment and observe for agglutination.

12. Interpretaton :
The antibody titre of the test sample is its highest dilution that gives a visible agglutination. Agglutinin titre
greater than 1:80 is considered as significant infection and low titres indicate absence of infection.

13. LIMITATIONS OF WIDAL TEST
• Tests done within 7 days of illness and after 4 weeks are usually negative.
• The local titre of the place should be known for the results interpreted correctly.
• This test (Quantitative) is highly time consumable.
• Previous typhoid vaccination may contribute to elevated agglutinins in the non-infected population.
• Other infections of non-enteric salmonella infection such as Typhus, Immunological disorders, chronic
liver disease may cause false positive reaction.
• Cross reaction between malaria parasites and salmonella antigens may cause false positive Widal
agglutination test