The Widal test is an in vitro test whereby bacteria causing typhoid fever are mixed with serum containing specific antibodies obtained from an infected individual. It is a presumptive serological test for enteric fever or undulant fever (brucellosis) Brucella melitensis. In case of Salmonella infections, it is a demonstration of the presence of O-somatic and H-flagellar antigens in the blood. For brucellosis, only O-somatic antigen is used.The Widal test is not a very specific test, since patients are often exposed to other bacteria (e.g. Salmonella enteritidis, Salmonella typhimurium and some types of E. coli) that induce cross-reactivity; many people have antibodies against these enteric pathogens, which also react with the antigens in the Widal test, causing a false-positive result. Test results need to be interpreted carefully in the light of past history of enteric fever, typhoid vaccination, and the general level of antibodies in the populations in endemic areas of the world. Typhidot is the other test used to ascertain the diagnosis of typhoid fever. As with all serological tests, the rise in antibody levels needed to perform the diagnosis takes 7-14 days, which limits it applicability in early diagnosis. Other means of diagnosing Salmonella typhi (and paratyphi) include cultures of blood, urine and faeces. These organisms produce H2S from thiosulfate and can be easily identified on differential media such as Bismuth sulfite agar.

WIDAL TEST(QUALITATIVE SLIDE AGGLUTINATION METHOD)AimTo diagnose enteric fever caused by Salmonella species using qualitative slide agglutination test.

PrincipleThe Widal test is applied for the diagnosis of enteric fever that includes typhoid and paratyphoid caused by Salmonella typhi and Salmonella paratyphi respectively. For the slide agglutination test, stained Salmonella antigens are used to detect the presence of specific agglutinin in the patients serum. The slide agglutination test is used as a primary screening procedure. There are some agglutinins that are produced in the patients serum during the fever period, which react with somatic antigen O of Salmonella typhi, A or B of Salmonella paratyphi and then with flagellar antigen H which is common in most of the Salmonella species. In this test four specific antigen suspensions are used viz H, Salmonella typhi (O antigen), Salmonella paratyphi - A and Salmonella paratyphi - B. If agglutination occurs in O antigen added circle, then it is considered positive for Salmonella typhi. If agglutination occurs in A or B antigen added circle, then it is confirmed as positive for paratyphoid. Agglutination will occur in H antigen circle for all the cases of antigens like O, A, and B.

Materials required1. Widal test kit2. Widal test slide3. Suspected serum sample4. Pipette5. Applicator stick

Procedure1. A clean glass marked with six circles was taken.2. The circles were labeled as H, O, A, B, positive control and negative control from the first circle to the last.3. One drop of undiluted serum was placed in the first four circles with the help of a sterile pasture pipette.4. One drop of postive control serum and one drop of negative control serum was placed in the fifth and the last circle respectively.5. 5. One drop of H antigen was added to the first circle and one drop of Salmonella typhi O antigen was added in the second circle. Salmonella paratyphi A antigen and Salmonella paratyphi B antigen were added in the third and fourth circles respectively.6. One drop of H antigen was placed at the fifth circle and sixth circle.7. With separate applicator sticks serum and the antigen was mixed together and was spread well to fill the whole of the individual circle. Then slide was observed for agglutination.

Result:The given test patients serum is positive for typhoid.The given test patients serum is positive for paratyphoid A or BThe given test patients serum is negative for typhoid and paratyphoid.------------------------------------------------------------------------------------------------------------(Draw and write in the left side of your observation note book)Observation:

DRAW THE SLIDE PICTURE HEREThe agglutination has (occurred / not occurred) in H antigen circle.The agglutination has (occurred / not occurred) in O antigen circle.The agglutination has (occurred / not occurred) in A antigen circle.The agglutination has (occurred / not occurred) in B antigen circle.