复旦大学：《医学微生物学 Medical Microbiology（MBBS）》课程讲义_实验课_Experiment 5

Experiment 5 Jungi zhang Department of Medical Microbiology Parasitology Shanghai Medical College, Fudan University

Experiment 5 Junqi Zhang Department of Medical Microbiology & Parasitology Shanghai Medical College, Fudan University

Experiment 5 1. Widal test 2. Ziehl Neelsen Stain(Acid-Fast stain) 3. Identification of Predominant isolates in the Gastrointestinal tract Gram stain Broth inoculation 10/25/2012 Experiment 5/ Junqi Zhang

10/25/2012 Experiment 5/ Junqi Zhang 2 Experiment 5 1.Widal test 2.Ziehl Neelsen Stain (Acid-Fast stain) 3.Identification of Predominant isolates in the Gastrointestinal Tract -Gram Stain -Broth Inoculation

Widal test Patientssuffering from enteric fever would possess antibodies(agglutinins) in their sera Widal test is a tube agglutination test for the detection of agglutinins(antibodies) for H and O antigen for salmonella in patients with enteric fever The test is named after Georges Fernand Isidore Widal, a French physician and bacteriologist, born March 9, 1862, Algeria; died January14, 1929, Paris The result will be used as an indirect signal bf the infection

 Patients’ suffering from enteric fever would possess antibodies (agglutinins) in their sera  Widal test is a tube agglutination test for the detection of agglutinins (antibodies) for H and O antigen for salmonella in patients with enteric fever  The test is named after Georges Fernand Isidore Widal, a French physician and bacteriologist, born March 9, 1862, Algeria; died January14, 1929, Paris  The result will be used as an indirect signal of the infection Widal test

3. Widal test: Tube dilution agglutination Test Principle Patients' suffering from enteric fever would possess antibodies in their sera which can react and agglutinate serial doubling dilutions of killed, coloured Salmonella antigens in a tube agglutination test Widal test is named after georges fernand isidore widal a French physician and bacteriologist, born March 9, 1862 Algeria; died January 14, 1929, Paris

4 3.Widal test: Tube Dilution Agglutination Test Patients’ suffering from enteric fever would possess antibodies in their sera which can react and agglutinate serial doubling dilutions of killed, coloured Salmonella antigens in a tube agglutination test Principle: Widal test is named after Georges Fernand Isidore Widal, a French physician and bacteriologist, born March 9, 1862, Algeria; died January14, 1929, Paris

Widal test Serum agglutinins(antibodies) rise sharply during the second and third weeks of salmonella typhi infection This test demonstrates the presence of somatic(O)and flagellar(H)agglutinins(antibodies) to Salmonella typhi in the patient's serum using suspensions ofO and H antigens Antigens of S paratyphi A, S paratyphi b,s paratyphi C are included in most commercial kits The result will be used as an indirect signal of the infection

5 Serum agglutinins(antibodies) rise sharply during the second and third weeks of Salmonella Typhi infection This test demonstrates the presence of somatic (O) and flagellar (H) agglutinins(antibodies) to Salmonella typhi in the patient's serum using suspensions of O and H antigens. Antigens of S. paratyphi A, S. paratyphi B, S. paratyphi C are included in most commercial kits The result will be used as an indirect signal of the infection Widal test

Antigenic structure ∠O S. typhi flagella

6 Antigenic Structure “Vi” S. typhi “O” “H” flagella

Preparation of antigens a Salmonella typhi is used to prepare S. typhi O and S. typhi H antigens aO antigens for S. paratyphi a and S paratyphi b are not taken as they cross-react with Styphi O antigen a H antigen suspension is prepared by treating overnight broth culture or saline suspension of Salmonella with 0. 1% formalin a For preparing antigen suspension, Salmonella are grown on phenol agar (1: 800)to inhibit flagella. The growth is then emulsified in small volume of saline. mixed with 20 times its volume of alcohol heated at 40C 50C for 30 minutes and centrifuged a The antigens are treated with chloroform (preservative) and appropriate dyes are added for easy identification of antigens Widal test

7  Salmonella typhi is used to prepare S. typhi O and S. typhi H antigens.  O antigens for S. paratyphi A and S.paratyphi B are not taken as they cross-react with S.typhi O antigen.  H antigen suspension is prepared by treating overnight broth culture or saline suspension of Salmonella with 0.1% formalin.  For preparing O antigen suspension, Salmonella are grown on phenol agar (1:800) to inhibit flagella. The growth is then emulsified in small volume of saline, mixed with 20 times its volume of alcohol, heated at 40 ℃ 50 ℃ for 30 minutes and centrifuged  The antigens are treated with chloroform (preservative) and appropriate dyes are added for easy identification of antigens. Preparation of antigens Widal test

Widal test Procedure 1. array forty test tubes into 4 rows, 10 tubes per row. Label the dilution ratio in every tube 2. Add 05ml saline into the first tube. and 0 5ml saline into other tubes in every row 3. Add the diluted serum 0 5ml( the dilution is 1: 5)to the first tube of first row, and beat upon three times. Then transfer 0 5ml to the second tube Repeat these one by one till the ninth tube, and then discard 0.5 ml from the ninth tube Only add 05ml saline in the tenth tube to compare 4. Do the same in the next 3 rows. Then the dilution ratio of every row in turn is 1: 10. 1: 20. 1: 40.1: 80.1: 160 till to 1: 2560. The tenth tube is comparative tube 8

8 Procedure 1.Array forty test tubes into 4 rows, 10 tubes per row. Label the dilution ratio in every tube. 2.Add 0.5ml saline into the first tube, and 0.5ml saline into other tubes, in every row. 3.Add the diluted serum 0.5ml(the dilution is 1:5) to the first tube of first row, and beat upon three times. Then transfer 0.5ml to the second tube. Repeat these one by one till the ninth tube, and then discard 0.5 ml from the ninth tube. Only add 0.5ml saline in the tenth tube to compare. 4.Do the same in the next 3 rows. Then the dilution ratio of every row in turn is 1:10, 1:20, 1:40, 1:80, 1:160 till to 1:2560. The tenth tube is comparative tube. Widal test

Widal test Procedure 5.Add antigen as follow a )add 0 5ml typhoid"H antigen to every tube of the first row b )add 0 5ml typhoid"O antigen to every tube of the second row c)add 05ml a paratyphoid antigen to every tube of the third row d)add 0 5ml B paratyphoid antigen to every tube of the forth row e)At this time, the dilution ratio of every row in turn is 1: 20, 1: 40 1: 80, 1: 160. 1: 320 till to 1: 5120. The tenth tube is comparati tube 6. Incubate at 37C for 18-24 hours Then observe and record the results 7. Firstly observe the comparative tube There should not be any agglutination reaction. secondly observe others tubes one by one. The highest serum dilution ratio of the tube that can be seen the obvious agglutination is the agglutination titer of this serum to specific antigen(next day)

9 5.Add antigen as follow: a)add 0.5ml typhoid “H” antigen to every tube of the first row. b)add 0.5ml typhoid “O” antigen to every tube of the second row. c)add 0.5ml A paratyphoid antigen to every tube of the third row. d)add 0.5ml B paratyphoid antigen to every tube of the forth row. e)At this time, the dilution ratio of every row in turn is 1:20, 1:40, 1:80, 1:160, 1:320 till to 1:5120. The tenth tube is comparative tube . 6.Incubate at 37℃ for 18-24 hours. Then observe and record the results. 7.Firstly observe the comparative tube. There should not be any agglutination reaction. Secondly observe others tubes one by one. The highest serum dilution ratio of the tube that can be seen the obvious agglutination is the agglutination titer of this serum to specific antigen(next day). Procedure Widal test

0.50.50.50.50.50.5 50.50.50.5 Serum dilution1:101:201:401801:1601:3201:6401:12801:2560 Serum 05ml 1:1280 1:5120 dilution 1:40 1:160 1:640 1:2560 control

10 10 Order 1 2 3 4 5 6 7 8 9 10 Serum 0.5ml Discard 0.5ml Saline 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 Serum dilution 1:10 1:20 1:40 180 1:160 1:320 1:640 1:1280 1:2560 Final 1:20 1:80 1:320 1:1280 1:5120 dilution 1:40 1:160 1:640 1:2560 control