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Laboratory Manual7-14Copyright 2014 by Mosby, Inc., an affiliate of Elsevier Inc.Copyright 2014 by Mosby, Inc., an affiliate of Elsevier Inc.Tille: Bailey & Scotts Diagnostic Microbiology, 13th Edition

Chapter 7: Exercises in Virology

Laboratory Manual

EXERCISES 32 THROUGH 33

EXERCISE 32

Cultivation of Viruses and Related TechniquesOBJECTIVESOn completion of this exercise, you will be able to do the following:Differentiate between infected and uninfected cells in a cell culture system.Describe the cytopathic effect (CPE) of viruses living within a cell culture system.Determine the preliminary identification of a virus, based on the appearance and selectivity of its CPE and the incubation time required to detect the CPE.Perform and interpret the hemadsorption procedure.Perform and interpret the hemadsorption inhibition procedure.

PREPARATIONBefore attempting this exercise, you should be introduced to the principles and techniques involved in basic diagnostic virology. Before the laboratory session, you should be familiar with the following concepts and topics:Culture systems for virusesVarious forms of the CPEHemadsorption testHemadsorption inhibition test

MATERIALSIn addition to some of the routine supplies mentioned in the introduction to Part I, you will need the following:Test tube holder for microscopeUninoculated tube cell cultureTube cell cultures of the common viruses listed in Study Chart 32-1Sets of primary rhesus monkey kidney (PMK) cells, continuous human epithelial cell (HEP-2), and human diploid fibroblast (HDF) cultures of unknown viruses selected from the list of viruses in Study Chart 32-1PMK culture of influenza A virusTen fresh PMK cell culturesFour 1-ml serologic pipettesThree ml 0.4% guinea pig erythrocyte suspensionTwo slanted cell culture racksRefrigerator200-ml Hanks balanced salt solution0.2 ml of the following receptor-destroying enzymetreated antisera:

Influenza viruses A and BParainfluenza viruses 1, 2, and 3Mumps virus

LABORATORY SESSION 1Examination and Maintenance of Cell Culture System MediaThe inspection and maintenance of cell culture system media is necessary to ensure reliable culture results in the laboratory. Follow the steps given here to examine a tube of cell culture medium:Place a tube holder in the slide holder of the microscope.Obtain an uninoculated tube cell culture, and record the cell type on Worksheet 32-1.Place the cell culture tube in the tube holder on the microscope to ensure that the monolayer of cells is visible with the 10 magnification objective.Scan the monolayer with the 10 magnification objective, and draw a representative section of the monolayer on Worksheet 32-1.Compare the appearance of the monolayer with a description of that particular type of cell monolayer in your text.Based on the appearance of the monolayer, would you consider it adequate or inadequate? Record this evaluation on Worksheet 32-1.Based on the evaluation of the monolayer, record the type of medium that is needed to prepare the cell culture for use in the cultivation of viral agents.

Examination of Cell Cultures for the Detection of the Cytopathic EffectAn examination of cell cultures to detect the CPE of an infecting virus is the first step in the identification of viral agents in culture. Practice recognizing and describing the CPE by following the instructions given here for each virus culture assigned:Record the name of the infecting virus on Worksheet 32-2, and examine the monolayer of the culture as described in the previous section.Review a description of the typical CPE of that virus in your textbook.Find infected cells in the monolayer of the culture that demonstrate the typical CPE of the infecting virus, and draw a few representative cells on Worksheet 32-2.

Cultures of Unknown Viral AgentsThe determination of the type, time required, and selectivity of the CPE is often enough to enable presumptive identification of the infecting virus. For each set of unknown cell cultures assigned, follow the steps given here:Obtain a set of PMK, HEP-2, and HFD cell culture unknowns, and record the unknown number on Worksheet 32-3.Examine each type of monolayer for evidence of the CPE, and describe the appearance of any CPE on Worksheet 32-3. If no CPE is evident, then record that observation.Record on Worksheet 32-3 the number of days from inoculation to the detection of the CPE.For those cultures demonstrating the CPE in one or more of the cell cultures, indicate which type or types of cells demonstrated the CPE by placing a check mark in the column of the affected cell type on Worksheet 32-3.Compare the description of the CPE of the unknown with the descriptions of the CPE of known viral agents.Record the probable identification, if possible, on Worksheet 32-3. If no probable identification is possible, based on the evaluation of the monolayer for CPE alone, then record the further action needed to identify the viral agent or to otherwise make a conclusion about the results of the cell culture.

HemadsorptionSome viral agents insert into the membranes of infected cells surface components that agglutinate erythrocytes from various species of animals. These viruses can be detected in cell cultures by exposing the infected monolayer to the specific type of erythrocyte. Perform the hemadsorption procedure with all appropriate cell culture unknowns from the previous section by following the steps given here:Choose the appropriate cell cultures from the group of culture unknowns, and record the unknown numbers on Worksheet 32-4.Obtain a PMK cell culture of influenza A (positive-control preparation) and an uninfected PMK cell culture (negative-control preparation).With a clean pipette for each tube, aspirate the maintenance medium from all tubes, and discard it into an appropriate container for biohazardous waste.Add 0.2 ml of a 0.4% guinea pig erythrocyte suspension with a sterile pipette to each culture to be tested. To avoid cross-contamination, do not allow the pipette to contact the culture tubes, and add the erythrocyte suspension to the positive-control preparation last.Place the tubes in a slanted rack to ensure that the suspension covers the monolayer, and refrigerate at 4C for 30 minutes.Remove the tubes from the refrigerator and, without delay, invert the cell culture tubes to dislodge loose erythrocytes. Microscopically examine the monolayer in the negative-control tube to ensure that no erythrocytes are adhering to the monolayer. Record the hemadsorption results on Worksheet 32-4.Examine the monolayer in the positive-control tube to ensure that the erythrocytes are adhering to the monolayer. Record the hemadsorption results on Worksheet 32-4.If the positive- and negative-control cultures show acceptable results, then examine the monolayers of the unknown cultures. Record the hemadsorption results on Worksheet 32-4 for each unknown tested.To detect hemagglutination of erythrocytes, place a drop of culture fluid on a glass slide. Add a coverslip to the slide, and microscopically examine the drop of culture fluid for agglutination of erythrocytes. Examine the culture fluid in each unknown culture, and record the hemagglutination results on Worksheet 32-4.Based on the hemadsorption test results, list on Worksheet 32-4 the possible identifications of each unknown viral agent on that worksheet.Answer the questions on Worksheet 32-4.

Hemadsorption InhibitionHemadsorbing viral agents can be positively identified by the hemadsorption inhibition test. Perform the hemadsorption inhibition procedure outlined here with each culture of unknown hemadsorbing viral agent from the previous part of this exercise:Record each unknown number on Worksheet 32-5.Dislodge and suspend the infected cells of the unknown culture by scraping the monolayer off the inside of the culture tube with a sterile Pasteur pipette.Obtain and label nine tubes of fresh PMK culture with the appropriate unknown number.Subculture the hemadsorbing virus by transferring 0.1 ml of the dislodged cell suspension in the unknown culture tube to each of the nine tubes of fresh PMK monolayer.Incubate the PMK subcultures at 36 to 37C for 72 hours.

LABORATORY SESSION 2Hemadsorption InhibitionPerform the following procedure for each culture of hemadsorbing virus from the previous laboratory session:Obtain one of the PMK subcultures from the incubator.With a sterile pipette, add 0.2 ml of a 0.4% guinea pig erythrocyte suspension to the subculture.Place the tube in a slanted rack to ensure that the suspension covers the monolayer, and refrigerate at 4C for 1 hour.Remove the culture from the refrigerator, invert the tube to dislodge any loose erythrocytes, and microscopically examine the monolayer for hemadsorption, as in the previous laboratory session. Scan the monolayer, and estimate the percentage of hemadsorption present.If the hemadsorption is at least 50%, then the titer of the virus should be adequate to perform the hemadsorption inhibition test. Proceed with the hemadsorption inhibition process.Discard the liquid medium in the remaining subculture tubes into a container for biologic waste, and wash the monolayers twice with Hanks balanced salt solution.With a sterile pipette, add 0.6 ml of Hanks balanced salt solution to each washed culture tube.In addition to the unknown number already recorded on the culture tube, label one tube for each antiserum that is to be used in the test. (Refer to Worksheet 32-5 for a list of these antisera.)With a sterile pipette, add 0.2 ml of each antiserum to the appropriately labeled culture tube.The remaining culture should be labeled as the positive-control preparation.Place the cultures in a slanted rack to ensure that the antiserum solution covers the monolayer. Incubate the cultures for 30 minutes at room temperature.With a sterile pipette, add 0.2 ml of a 0.4% guinea pig erythrocyte suspension to each tube.Return the tubes to the slanted rack to ensure that the monolayer is immersed in the suspension. Incubate the cultures at 4C for 30 minutes.Remove the cultures from the refrigerator, and microscopically examine the monolayer of the positive-control preparation for hemadsorption. Record an approximate percentage of monolayer exhibiting adherence of erythrocytes on Worksheet 32-5.

Microscopically examine the monolayer of each test culture for hemadsorption. Record an approximate percentage of monolayer exhibiting adherence of erythrocytes on Worksheet 32-5.For each tested culture, compare its recorded amount of hemadsorption with that recorded for the positive-control preparation. If the result is positive, then the tested culture should show a significant decrease in the amount of hemadsorbing erythrocytes, compared with the control preparation.Based on the comparisons of the percentages of hemadsorption of each test culture with the percentage of hemadsorption of the positive control preparation, record the interpretation (positive or negative) of the hemadsorption inhibition test result on Worksheet 32-5.Based on the results of the hemadsorption inhibition test, record the identification of the virus on Worksheet 32-5.

Cytopathic Effect DescriptionStudy Chart 32-1VirusSpecificity*Days to DetectionDescription of the CPE

PMKHEP-2HDF

Adenovirus

Cytomegalovirus

Enterovirus

Herpes

Inuenza

Mumps

Parainuenza

Respiratory syncytial virus

Rhinovirus

Varicella zoster

Measles

Echovirus

Poliovirus

Coxsackievirus

*Relative sensitivity for recovering a virus: , None recovered; 1+, few; 2+, some; 3+, most.PMK, Primary rhesus monkey kidney; HEP-2, continuous human epithelial; HDF, human diploid broblasts; CPE, cytopathic effect.

Uninoculated Cell Culture MediumWorksheet 32-1Cell type: ____________________________________________________Sketch a few cells of the monolayer here:

Are the cells forming a complete monolayer?Yes: No: Do the cells in the monolayer demonstrate any signs of CPE or other types of abnormalities?Yes: No: Evaluate the cell culture monolayer:Adequate: Inadequate: Type of medium needed:Growth: Maintenance:

Cytopathic EffectWorksheet 32-2Name of the VirusDrawing of a Typical Cytopathic Effect_____________________

Unknown Cultures of Viral AgentsWorksheet 32-3Unknown NumberDescription of the CPEDays to DetectionType of Cells InfectedProbable Identification or Action Needed

PMKHEP-2HDF

PMK, Primary rhesus monkey kidney; HEP-2, continuous human epithelial; HDF, human diploid broblasts.

Hemadsorption TestWorksheet 32-4Culture NumberHemadsorption ResultsHemagglutination ResultsPossible Identification of VirusPositive control

N/AN/ANegative control

N/AN/AUnknown #________

Unknown #________

Unknown #________

A delay in the examination of the monolayer after the removal of the culture from the refrigerator could cause which outcomea false-positive or a false-negative result? Why?__________________________________________________________________________________________________________________________________________________________________________________________________________________________________________The failure to examine the uid phase of the culture medium for hemagglutination could cause which outcomea false-positive or false-negative result? Why?__________________________________________________________________________________________________________________________________________________________________________________________________________________________________________Some uninoculated PMK cell cultures demonstrate a positive hemadsorption test result. State the cause of this false-positive result. What step is taken as part of the hemadsorption procedure to avoid reporting such a false-positive result?__________________________________________________________________________________________________________________________________________________________________________________________________________________________________________

Hemadsorption Inhibition TestWorksheet 32-5Unknown culture number ___________________________AntiseraHemadsorption (%)Interpretation of Hemadsorption Inhibition TestIdentification of VirusNone (positive control)

N/AN/AInuenza A

Inuenza B

Parainuenza 1

Parainuenza 2

Parainuenza 3

Mumps virus

Simian virus

Unknown culture number ___________________________AntiseraHemadsorption (%)Interpretation of Hemadsorption Inhibition TestIdentification of VirusNone (positive control)

N/AN/AInuenza A

Inuenza B

Parainuenza 1

Parainuenza 2

Parainuenza 3

Mumps virus

Simian virus

EXERCISE 33Direct Detection of Viral Agents in Clinical SpecimensOBJECTIVESOn completion of this exercise, you will be able to do the following:Realize the importance of reading the manufacturers package insert and instructions before using a manufacturers test kit to evaluate clinical specimens.Apply the information given in a manufacturers package insert to evaluate the value of the test with respect to quality assurance issues, such as sensitivity, specificity, and false-positive and false-negative results.Following the manufacturers instructions, perform a test to detect the presence of a viral agent in a clinical specimen with a commercially prepared test kit.Interpret the results of a commercially prepared test system according to the manufacturers guidelines.

PREPARATIONBefore attempting this exercise, you should be introduced to the principles and techniques related to the test kits chosen for this exercise. Before laboratory time, you should read the product insert that accompanies each test kit and familiarize yourself with the following concepts that pertain to the particular test kits used:Principle used in the test kit designed to detect the virusType of specimen requiredLevel of specificity of the test systemLevel of sensitivity of the test systemQuality control measures that are included as a part of the test system

MATERIALSIn addition to some of the routine supplies mentioned in the introduction to Part I, you will need the following:Glass slide preparation of material from a suspected herpes simplex virus (HSV) lesionCommercially prepared test kit for the detection of HSV in lesion smears and related materials (Check the product insert for kit-specific materials.)Feces specimenCommercially prepared test kit for the detection of rotavirus in feces and related materials (Check the product insert for kit-specific materials.)

LABORATORY SESSIONDetection of Herpes Simplex Virus in Lesion Specimen SmearsObtain a slide preparation of material from a suspected HSV lesion, and record the specimen identification on Worksheet 33-1.Record the name of the test system that is to be used to detect HSV in the specimen on Worksheet 33-1.Follow the manufacturers instructions to evaluate, prepare, and process the specimen for the detection of HSV in the specimen.Refer to the manufacturers interpretation guidelines, and record the quality control test results, including the evaluation of the quality of the specimen, on Worksheet 33-1.If the quality control test results are acceptable, record the specimen test result on Worksheet 33-1.

Detection of Rotavirus in FecesObtain a specimen of feces, and record the specimen identification on Worksheet 33-1.Record the name of the test system that is to be used to detect rotavirus in the specimen on Worksheet 33-1.Follow the manufacturers instructions to evaluate, prepare, and process the specimen for the detection of the rotavirus.Refer to the manufacturers interpretation guidelines, and record the quality control test results, including the evaluation of the quality of the specimen.If the quality control test results are acceptable, record the specimen test result on Worksheet 33-1.

DETECTION OF VIRAL AGENTS IN CLINICAL SPECIMENSWorksheet 33-1SUSPECTED HERPES SIMPLEX VIRAL LESION SMEARSpecimen identication: __________________________________________________________Test system: ___________________________________________________________________Results:Quality control test results:Positive: Negative: Specimen quality evaluation:Positive: Negative: ______________________________Specimen:Positive: Negative: for the presence of herpes simplex virusFECES SPECIMENSpecimen identication: __________________________________________________________Test system: ___________________________________________________________________Results:Specimen quality evaluation: ______________________________________________________Quality control test results:Positive: Negative: Specimen quality evaluation:Positive: Negative: ______________________________Specimen:Positive: Negative: for the presence of rotavirus