Widal Test

Tube method:-

Reagents- Neutralised Typhoid O, Typhoid H, Paratyphoid A H and Paratyphoid B H antigens are

used.
Procedure-

1. All reagents are allowed to come at room temperature.

2. 1 clean dry big test tube is taken.
3. 4750 ul of NS is taken and mixed with 250 ul of patient's serum. It is the master dilution
(Md).
4. Then in a test tube rack 4 sets of small test tubes are taken. Each set contains 7 test tubes.
5. To each and every test tube 1 ml of NS is taken.
6. To the first test tube of one set 1ml of solution from Md Tube is taken and mixed well.
7. Then from that tube 1 ml the mixture is transfer to 2nd test tube. In this way serial dilution
is perfomed up to 6th tube. For each set.
8. Then 1 drop of TO, TH, AH and BH is taken to each tubes of 1st, 2nd, 3rd and 4th set of
tubes respectively. The last tube is for negative control.
9. Then the test tubes are incubated overnight (16-20 hours) at 37°C incubator or water bath.

Or.

1. All reagents are allowed to come at room temperature.

2. 4 sets of small test tubes are taken. Each set contains 7 test tubes.
3. To the first tube 1950 ml normal saline is taken. 50 ml patient’s serum is mixed.
4. To the rest tubes 1 ml of normal saline is added.
5. From the first tube 1 ml of serum saline mixture is taken and mixed with 2 nd tube. Thus serial
dilution is done up to 6th tube.
6. Last tube is for negative control.
7. Then 1 drop of TO, TH, AH and BH is taken to each tubes of 1st, 2nd, 3rd and 4th set of tubes
respectively.
8. the test tubes are incubated overnight (16-20 hours) at 37°C incubator or water bath.
Observation- After incubation presence of cotton wool agglutination is observed at tubes where TO
antigen is added, and granular deposits is observed at tubes where TH, AH & BH antigens are added
after gentle taping.
Interpretation-
1. The highest dilution that shows the agglutination is the titre.
2. Titre of O antigen >1:100 and H antigen >1:200 is clinically significant and signifies presence
of active infection.
3. Rising titre of 4 fold between 2 weeks is more significant and diagnostic.