微生物实验：实验7 病毒形态学和培养及血清学English

1、Virus nDemonstrationFungi : morphology, cultureLaboratory diagnosis of viral diseases by morphological method nMorphology（electron microscope）Adenovirus, Herpesvirus nCPE（cytologic examination）uninfected cells, infected cellsInclusion bodies of Rabies virus and Measles virusnOperationHemagglutinatio

2、n assayHemagglutination inhibition testnVideo: Animal experimentInclusion bodiesnInclusion bodies are stainable structures which are either histologic changes in the cells caused by viral components or virus-induced changes in cell structures.nThey may appear within the nucleus or cytoplasm, and the

3、y can facilitate laboratory diagnosisInclusion bodies of Rabies virus （Negri body）Negri body in a neuron Inclusion bodies of Rabies virus （Negri body）Inclusion bodies of Measles virus CPEnCell deathCell rounding, degeneration,aggregation,loss of attachments to the culture dishnCharcteristic histolog

4、ic changesInclusion bodies, margination of chromatinnSyncytianCell surface changesUninfected Vero cellsCPE of HSV infectionHSV-1-infected Vero cellsHARBChemagglutinationVIRUSRBCInfluenza virusMumps virusParainfluenza virusNew castle disease virusAdenovirusEnterovirusMeasle virusReovirusHemagglutinat

5、ion assayHemagglutination inhibition test(HI Test)nprocedure for the measurement of serum antibodies directed against a hemagglutinating virus; the highest dilution of serum that completely inhibits hemagglutination by a standardized viral preparation is reported as the hemagglutination titer.nDetec

6、tion of antibody or serotyping of virusNo. 12345678910Saline（ml）Virus （ml）0.2+0.20.2+0.20.2+0.20.2+0.20.2+0.20.2+0.20.2+0.20.2+0.20.2+0.20.2dilution1 101 201 401 801 1601 3201 6401 12801 2560controlsaline（ml）Each 0.2ml0.5%RBC suspension（ml）Each 0.4mlProcedure of hemagglutination assayLabel the wells

7、 from No.1 to 10.Label the wells from No.1 to 10.Add 0.2ml of saline to each wells.Add 0.2ml of saline to each wells.Dilute the virus according the table below.Dilute the virus according the table below.Add 0.2ml of saline to each wells from No.10 to No.1.Add 0.2ml of saline to each wells from No.10

8、 to No.1.Add 0.4m of 0.5% RBC suspension to each wells from No.10 to No.1.Add 0.4m of 0.5% RBC suspension to each wells from No.10 to No.1.Shake to mixShake to mix and incubate at RT for 45min.and incubate at RT for 45min.0.2discardDetermination of HAU titer4 Hemagglutinating unit(HAU)n1 HAU= the hi

9、ghest dilution of “+”1HAU=1:804HAU=1:20NO.12345678control9control10controlsaline（ml）serum（ml）0.2+0.20.2+0.20.2+0.20.2+0.20.2+0.20.2+0.20.2+0.20.2+0.20.20.4Dilution of serum1 101 201 401 801 1601 3201 6404 HAU virus（ml）Each 0.2ml0.20.5%RBC suspension（ml）Each 0.4ml0.4Label the wells from No.1 to 10.Ad

10、d 0.2ml of saline to each wells from No.1 to No.9, add 0.4ml of saline to No.10.Dilute the serum(antibody) according the table below from No.1 to No.7, add the serum to No 8 well as the control.Add 0.2ml of 4 HAU virus to each wells from No.7 to No.1, add the virus to No 9 well as the control.Shake to mix, then