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Experiment five Isolation and Identification of influenza virus.

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Presentation on theme: "Experiment five Isolation and Identification of influenza virus."— Presentation transcript:

1 Experiment five Isolation and Identification of influenza virus

2 Isolation and Identification of viruses in lab Specimen Specimen Isolation and identification of virus Isolation and identification of virus Serological diagnosis of viral infection Serological diagnosis of viral infection Rapid diagnosis of virus infections Rapid diagnosis of virus infections

3 Isolation and Identification of viruses in lab Isolation and identification of virus Isolation and identification of virus Isolation of virus Isolation of virus Embryonated Eggs Embryonated Eggs Laboratory Animals Laboratory Animals Tissue Culture Tissue Culture Quantitation of virus Quantitation of virus 50% tissue culture infectious dose (TCID50) 50% tissue culture infectious dose (TCID50) Plaque forming assay Plaque forming assay

4 Isolation and Identification of viruses in lab Serological diagnosis of viral infection Serological diagnosis of viral infection Neutralization test(NT) Neutralization test(NT) Complement fixation test(CF) Complement fixation test(CF) Titration of complement Titration of complement

5 Isolation and Identification of viruses in lab Rapid diagnosis of virus infections Rapid diagnosis of virus infections Identification virus particle Identification virus particle Detection of viral antigens Detection of viral antigens Detection of viral nucleic acid Detection of viral nucleic acid Diagnosis of virus infections by detecting IgM antibodies Diagnosis of virus infections by detecting IgM antibodies Other methods Other methods

6 Isolation and Identification of influenza virus Purpose Purpose To obtain information about microbiological diagnosis methods of influenza virus To obtain information about microbiological diagnosis methods of influenza virus Procedure Procedure Acquiring and managing specimen Acquiring and managing specimen Isolation and Identification of influenza virus Isolation and Identification of influenza virus serological diagnosis of influenza virus serological diagnosis of influenza virus

7 Isolation and Identification of influenza virus Acquiring and managing specimen Acquiring and managing specimen Throat washing or gargling are obtained within 3 days after infection Throat washing or gargling are obtained within 3 days after infection Test at once or stored frozen. Test at once or stored frozen. Centrifuge the washings 15 min at 3000 rpm, Harvest 1 ml of supernatant. Centrifuge the washings 15 min at 3000 rpm, Harvest 1 ml of supernatant. Add antibiotic to prevent bacterial contamination. Add antibiotic to prevent bacterial contamination.

8 Hemagglutination inhibition test complement fixation test Inoculate to fresh embryos after two such passages Treated with penicillin and streptomycin Embryonated egg inoculation (amniotic or allantoic route) (amniotic or allantoic route) Harvest amniotic and allantoic fluids Hemagglutination test the result is negative +- Throat washings or gargling Patients serum Hemagglutination test

9 Isolation and Identification of influenza virus Isolation of influenza virus Isolation of influenza virus MATERIALS MATERIALS 1.Throat washings or garglings 1.Throat washings or garglings 2.Embryos (within 9 to 10-day-old) 2.Embryos (within 9 to 10-day-old) 3.0.5% chicken RBC suspension, physiological 3.0.5% chicken RBC suspension, physiological saline saline 4. Scissors, forceps, drill, syringe, tubes, egg- 4. Scissors, forceps, drill, syringe, tubes, egg- checking box, 75% ethanol, tincture of iodine checking box, 75% ethanol, tincture of iodine

10 Mark the location of embryo air sac under egg-checking box, sterilize the shell with tincture and 75% ethanol, drill a small hole near the marked shell, inoculate 0.2 ml of virus inoculum into the allantoic cavity by pushing the needle just below the surface of the shell, seal the hole with melton wax. Mark the location of embryo air sac under egg-checking box, sterilize the shell with tincture and 75% ethanol, drill a small hole near the marked shell, inoculate 0.2 ml of virus inoculum into the allantoic cavity by pushing the needle just below the surface of the shell, seal the hole with melton wax. Incubate the inoculated embryo at 35C, check it daily. After incubating for 72 h,place the embryo at 4C overnight. If the embryo dead within 72 h, it should be discard. Incubate the inoculated embryo at 35C, check it daily. After incubating for 72 h,place the embryo at 4C overnight. If the embryo dead within 72 h, it should be discard. Harvest allantoic fluid.Assessing HA titer refers to Experiment 25. Harvest allantoic fluid.Assessing HA titer refers to Experiment 25.

11 Hemagglutination test, HT MATERIALS MATERIALS Virus:the allantoic fluid that contains influenza virus; Virus:the allantoic fluid that contains influenza virus; physiological saline; physiological saline; 0.5% chicken RBC suspension. 0.5% chicken RBC suspension. 20-well plastic disposable microtiter plates and glass pipettes. 20-well plastic disposable microtiter plates and glass pipettes.

12 Materials well No. (ml) 1 2 3 4 5 6 7 8 9 10 (ml) 1 2 3 4 5 6 7 8 9 10 Saline 0.45 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 Virus 0.05 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 - Dilution 1:10 1:20 1:40 1:80 1:160 1:3201:6401:12801:2560 con 0.5%RBC 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 incubate the plate at room temperature for 30 to 60 min. Check the agglutination. Hemagglutination test, HT

13 Analysis of the results Analysis of the results

14 Hemagglutination test, HT 1:640 1:320 1:160 1: 80 1: 80 1: 40 1: 20 1: 10 1: 10

15 Hemagglutination test (HT) RESULT RESULT Record the hemagglutination as the followings: ++++ : All the RBC had been agglutinated. ++++ : All the RBC had been agglutinated. +++ : 75% of RBC had been agglutinated +++ : 75% of RBC had been agglutinated ++ : 50% of RBC had been agglutinated. ++ : 50% of RBC had been agglutinated. + : 25% of RBC had been agglutinated. + : 25% of RBC had been agglutinated. - : No RBC was agglutinated. - : No RBC was agglutinated.

16 Hemagglutination inhibition test (HIT) MATERIALS MATERIALS Patient s serum: deactivated 30 min at 56C; Patient s serum: deactivated 30 min at 56C; influenza virus: 4 HA units; influenza virus: 4 HA units; 0.5% chicken RBC suspension; 0.5% chicken RBC suspension; physiological saline physiological saline 20-well microtiter plates, pipettes and tubes. 20-well microtiter plates, pipettes and tubes.

17 Materials well No. (ml) 1 2 3 4 5 6 7 8 9 10 (ml) 1 2 3 4 5 6 7 8 9 10 Saline 0.9 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 Serum 0.1 0.25 0.25 0.25 0.25 0.25 0.25 - 0.25 - Dilution 1:10 1:20 1:40 1:80 1:160 1:320 1:640 - - - Virus 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 - - Mix evenly incubation at 37C for 1h Mix evenly incubation at 37C for 1h 0.5%RBC 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 - 0.25 Mix evenly incubation at 20c for 1 h Mix evenly incubation at 20c for 1 h Result - - - + ++ +++ ++++ ++++ - -

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19 Hemagglutination inhibition test (HIT) RESULT RESULT Assess hemagglutination as above, the hemagglutination inhibition (HI) titer is the reciprocal of the highest dilution of the patient's serum which shows complete inhibition of agglutination.1:40 dilution in Table is the HI titer.

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21 Isolation and Identification of influenza virus Identification of influenza virus Identification of influenza virus Hemagglutination inhibition test (HI) Hemagglutination inhibition test (HI) --- subtype --- subtype Complement fixation test (CF) Complement fixation test (CF) ---type ---type


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