Tille: Bailey & Scotts Diagnostic Microbiology, 13th Edition

Chapter 7: Eercises in !irology

"aboratory Man#al
E$E%C&SES 3' T(%)*+( 33
E$E%C&SE 3'
Cultivation of Viruses and Related Techniques
)B,ECT&!ES
On completion of this exercise, you will be able to do the following:
1. ifferentiate between infected and uninfected cells in a cell culture system!
2. escribe the cytopathic effect "C#$% of viruses living within a cell culture system!
3. etermine the preliminary identification of a virus, based on the appearance and selectivity
of its C#$ and the incubation time required to detect the C#$!
4. #erform and interpret the hemadsorption procedure!
5. #erform and interpret the hemadsorption inhibition procedure!
-%E-.%.T&)/
&efore attempting this exercise, you should be introduced to the principles and techniques
involved in basic diagnostic virology! &efore the laboratory session, you should be familiar with
the following concepts and topics:
Culture systems for viruses
Various forms of the C#$
'emadsorption test
'emadsorption inhibition test
M.TE%&."S
(n addition to some of the routine supplies mentioned in the introduction to #art (, you will need
the following:
Test tube holder for microscope
)ninoculated tube cell culture
Tube cell cultures of the common viruses listed in *tudy Chart +,-.
*ets of primary rhesus mon/ey /idney "#01% cells, continuous human epithelial cell "'$#-
,%, and human diploid fibroblast "'2% cultures of un/nown viruses selected from the list of
viruses in *tudy Chart +,-.
#01 culture of influen3a 4 virus
Ten fresh #01 cell cultures
2our .-ml serologic pipettes
Three ml 5!67 guinea pig erythrocyte suspension
Two slanted cell culture rac/s
Refrigerator
,55-ml 'an/s8 balanced salt solution
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5!, ml of the following receptor-destroying en3yme<treated antisera:
(nfluen3a viruses 4 and &
#arainfluen3a viruses ., ,, and +
0umps virus
".B)%.T)%0 SESS&)/ 1
Examination and Maintenance of Cell Culture System Media
The inspection and maintenance of cell culture system media is necessary to ensure reliable
culture results in the laboratory! 2ollow the steps given here to examine a tube of cell culture
medium:
1. #lace a tube holder in the slide holder of the microscope!
2. Obtain an uninoculated tube cell culture, and record the cell type on =or/sheet +,-.!
3. #lace the cell culture tube in the tube holder on the microscope to ensure that the monolayer
of cells is visible with the .5> magnification ob?ective!
4. *can the monolayer with the .5> magnification ob?ective, and draw a representative section
of the monolayer on =or/sheet +,-.!
5. Compare the appearance of the monolayer with a description of that particular type of cell
monolayer in your text!
6. &ased on the appearance of the monolayer, would you consider it adequate or inadequate@
Record this evaluation on =or/sheet +,-.!
7. &ased on the evaluation of the monolayer, record the type of medium that is needed to
prepare the cell culture for use in the cultivation of viral agents!
Examination of Cell Cultures for the Detection of the Cytopathic Effect
4n examination of cell cultures to detect the C#$ of an infecting virus is the first step in the
identification of viral agents in culture! #ractice recogni3ing and describing the C#$ by
following the instructions given here for each virus culture assigned:
1. Record the name of the infecting virus on =or/sheet +,-,, and examine the monolayer of the
culture as described in the previous section!
2. Review a description of the typical C#$ of that virus in your textboo/!
3. 2ind infected cells in the monolayer of the culture that demonstrate the typical C#$ of the
infecting virus, and draw a few representative cells on =or/sheet +,-,!
Cultures of Unknown Viral Agents
The determination of the type, time required, and selectivity of the C#$ is often enough to enable
presumptive identification of the infecting virus! 2or each set of un/nown cell cultures assigned,
follow the steps given here:
1. Obtain a set of #01, '$#-,, and '2 cell culture un/nowns, and record the un/nown
number on =or/sheet +,-+!
2. $xamine each type of monolayer for evidence of the C#$, and describe the appearance of
any C#$ on =or/sheet +,-+! (f no C#$ is evident, then record that observation!
3. Record on =or/sheet +,-+ the number of days from inoculation to the detection of the C#$!
4. 2or those cultures demonstrating the C#$ in one or more of the cell cultures, indicate which
type or types of cells demonstrated the C#$ by placing a chec/ mar/ in the column of the
affected cell type on =or/sheet +,-+.
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5. Compare the description of the C#$ of the un/nown with the descriptions of the C#$ of
/nown viral agents!
6. Record the probable identification, if possible, on =or/sheet +,-+! (f no probable
identification is possible, based on the evaluation of the monolayer for C#$ alone, then
record the further action needed to identify the viral agent or to otherwise ma/e a conclusion
about the results of the cell culture!
Hemadsorption
*ome 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! #erform the
hemadsorption procedure with all appropriate cell culture un/nowns from the previous section
by following the steps given here:
1. Choose the appropriate cell cultures from the group of culture un/nowns, and record the
un/nown numbers on =or/sheet +,-6!
2. Obtain a #01 cell culture of influen3a 4 "positive-control preparation% and an uninfected
#01 cell culture "negative-control preparation%!
3. =ith a clean pipette for each tube, aspirate the maintenance medium from all tubes, and
discard it into an appropriate container for bioha3ardous waste!
4. 4dd 5!, ml of a 5!67 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!
5. #lace the tubes in a slanted rac/ to ensure that the suspension covers the monolayer, and
refrigerate at 6AC for +5 minutes!
6. Remove the tubes from the refrigerator and, without delay, invert the cell culture tubes to
dislodge loose erythrocytes! 0icroscopically examine the monolayer in the negative-control
tube to ensure that no erythrocytes are adhering to the monolayer! Record the hemadsorption
results on =or/sheet +,-6!
7. $xamine the monolayer in the positive-control tube to ensure that the erythrocytes are
adhering to the monolayer! Record the hemadsorption results on =or/sheet +,-6!
8. (f the positive- and negative-control cultures show acceptable results, then examine the
monolayers of the un/nown cultures. Record the hemadsorption results on =or/sheet +,-6
for each un/nown tested!
9. To detect hemagglutination of erythrocytes, place a drop of culture fluid on a glass slide! 4dd
a coverslip to the slide, and microscopically examine the drop of culture fluid for
agglutination of erythrocytes! $xamine the culture fluid in each un/nown culture, and record
the hemagglutination results on =or/sheet +,-6!
10. &ased on the hemadsorption test results, list on =or/sheet +,-6 the possible identifications of
each un/nown viral agent on that wor/sheet!
11. 4nswer the questions on =or/sheet +,-6!
Hemadsorption nhi!ition
'emadsorbing viral agents can be positively identified by the hemadsorption inhibition test!
#erform the hemadsorption inhibition procedure outlined here with each culture of un/nown
hemadsorbing viral agent from the previous part of this exercise:
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1. Record each un/nown number on =or/sheet +,-B!
2. islodge and suspend the infected cells of the un/nown culture by scraping the monolayer
off the inside of the culture tube with a sterile #asteur pipette!
3. Obtain and label nine tubes of fresh #01 culture with the appropriate un/nown number!
4. *ubculture the hemadsorbing virus by transferring 5!. ml of the dislodged cell suspension in
the un/nown culture tube to each of the nine tubes of fresh #01 monolayer!
5. (ncubate the #01 subcultures at +CA to +;AC for ;, hours!
".B)%.T)%0 SESS&)/ '
Hemadsorption nhi!ition
#erform the following procedure for each culture of hemadsorbing virus from the previous
laboratory session:
1. Obtain one of the #01 subcultures from the incubator.
2. =ith a sterile pipette, add 5!, ml of a 5!67 guinea pig erythrocyte suspension to the
subculture!
3. #lace the tube in a slanted rac/ to ensure that the suspension covers the monolayer, and
refrigerate at 6AC for . hour!
4. 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! *can the monolayer, and estimate the percentage of hemadsorption present!
5. (f the hemadsorption is at least B57, then the titer of the virus should be adequate to perform
the hemadsorption inhibition test! #roceed with the hemadsorption inhibition process!
6. iscard the liquid medium in the remaining subculture tubes into a container for biologic
waste, and wash the monolayers twice with 'an/s8 balanced salt solution!
7. =ith a sterile pipette, add 5!C ml of 'an/s8 balanced salt solution to each washed culture
tube!
8. (n addition to the un/nown number already recorded on the culture tube, label one tube for
each antiserum that is to be used in the test! "Refer to =or/sheet +,-B for a list of these
antisera!%
9. =ith a sterile pipette, add 5!, ml of each antiserum to the appropriately labeled culture tube!
10. The remaining culture should be labeled as the positive-control preparation!
11. #lace the cultures in a slanted rac/ to ensure that the antiserum solution covers the
monolayer! (ncubate the cultures for +5 minutes at room temperature!
12. =ith a sterile pipette, add 5!, ml of a 5!67 guinea pig erythrocyte suspension to each tube!
13. Return the tubes to the slanted rac/ to ensure that the monolayer is immersed in the
suspension! (ncubate the cultures at 6AC for +5 minutes!
14. 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 =or/sheet +,-B.
15. 0icroscopically examine the monolayer of each test culture for hemadsorption! Record an
approximate percentage of monolayer exhibiting adherence of erythrocytes on =or/sheet +,-
B.
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16. 2or each tested culture, compare its recorded amount of hemadsorption with that recorded for
the positive-control preparation! (f the result is positive, then the tested culture should show a
significant decrease in the amount of hemadsorbing erythrocytes, compared with the control
preparation!
17. &ased 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 =or/sheet +,-B.
18. &ased on the results of the hemadsorption inhibition test, record the identification of the virus
on =or/sheet +,-B.
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C0T)-.T(&C E11ECT DESC%&-T&)/
St#dy Chart 3'21
!ir#s
Speci3icity4
Days
to Detection
Description o3
the C-E -M5 (E-2' (D1
4denovirus
Cytomegalovirus
$nterovirus
'erpes
(nDuen3a
0umps
#arainDuen3a
Respiratory
syncytial virus
Rhinovirus
Varicella 3oster
0easles
$chovirus
#oliovirus
Coxsac/ievirus
E
Relative sensitivity for recovering a virus: F, Gone recoveredH 1I, fewH 2I, someH 3I, most!
PMK, #rimary rhesus mon/ey /idneyH HEP-2, continuous human epithelialH HDF, human diploid
JbroblastsH CPE, cytopathic effect.
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*/&/)C*".TED CE"" C*"T*%E MED&*M
6or7sheet 3'21
Cell type: KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK
*/etch a few cells of the monolayer here:
4re the cells forming a complete monolayer@
Les: Go:
o the cells in the monolayer demonstrate any signs of C#$ or other types of abnormalities@
Les: Go:
$valuate the cell culture monolayer:
4dequate: (nadequate:
Type of medium needed:
Mrowth: 0aintenance:
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C0T)-.T(&C E11ECT
6or7sheet 3'2'
/a8e o3 the !ir#s Dra9ing o3 a Typical Cytopathic E33ect
KKKKKKKKKKKKKKKKKKKKK
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*/5/)6/ C*"T*%ES )1 !&%." .+E/TS
6or7sheet 3'23
*n7no9n
/#8ber
Description o3
the C-E
Days to
Detection
Type o3 Cells &n3ected
-robable &denti3ication
or .ction /eeded -M5 (E-2' (D1
PMK, #rimary rhesus mon/ey /idneyH HEP-2, continuous human epithelialH HDF, human diploid
Jbroblasts!
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(EM.DS)%-T&)/ TEST
6or7sheet 3'2:
C#lt#re /#8ber
(e8adsorption
%es#lts
(e8aggl#tination
%es#lts
-ossible &denti3ication o3
!ir#s
#ositive control GP4 GP4
Gegative control GP4 GP4
)n/nown QKKKKKKKK
)n/nown QKKKKKKKK
)n/nown QKKKKKKKK
4 delay in the examination of the monolayer after the removal of the culture from the refrigerator
could cause which outcomeFa false-positive or a false-negative result@ =hy@
KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK
KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK
KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK
The failure to examine the Duid phase of the culture medium for hemagglutination could cause
which outcomeFa false-positive or false-negative result@ =hy@
KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK
KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK
KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK
*ome uninoculated #01 cell cultures demonstrate a positive hemadsorption test result! *tate the
cause of this false-positive result! =hat step is ta/en as part of the hemadsorption procedure to
avoid reporting such a false-positive result@
KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK
KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK
KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK
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(EM.DS)%-T&)/ &/(&B&T&)/ TEST
6or7sheet 3'2;
)n/nown culture number KKKKKKKKKKKKKKKKKKKKKKKKKKK
.ntisera
(e8adsorption
<=>
&nterpretation
o3 (e8adsorption
&nhibition Test &denti3ication o3 !ir#s
Gone "positive control% GP4 GP4
(nDuen3a 4
(nDuen3a &
#arainDuen3a .
#arainDuen3a ,
#arainDuen3a +
0umps virus
*imian virus
)n/nown culture number KKKKKKKKKKKKKKKKKKKKKKKKKKK
.ntisera
(e8adsorption
<=>
&nterpretation
o3 (e8adsorption
&nhibition Test &denti3ication o3 !ir#s
Gone "positive control% GP4 GP4
(nDuen3a 4
(nDuen3a &
#arainDuen3a .
#arainDuen3a ,
#arainDuen3a +
0umps virus
*imian virus
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E$E%C&SE 33
irect etection of Viral 4gents in Clinical *pecimens
)B,ECT&!ES
On completion of this exercise, you will be able to do the following:
1. Reali3e the importance of reading the manufacturer8s pac/age insert and instructions before
using a manufacturer8s test /it to evaluate clinical specimens!
2. 4pply the information given in a manufacturer8s pac/age 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!
3. 2ollowing the manufacturer8s instructions, perform a test to detect the presence of a viral
agent in a clinical specimen with a commercially prepared test /it!
4. (nterpret the results of a commercially prepared test system according to the manufacturer8s
guidelines!
-%E-.%.T&)/
&efore attempting this exercise, you should be introduced to the principles and techniques related
to the test /its chosen for this exercise! &efore laboratory time, you should read the product insert
that accompanies each test /it and familiari3e yourself with the following concepts that pertain to
the particular test /its used:
#rinciple used in the test /it designed to detect the virus
Type of specimen required
:evel of specificity of the test system
:evel of sensitivity of the test system
Ruality control measures that are included as a part of the test system
M.TE%&."S
(n addition to some of the routine supplies mentioned in the introduction to #art (, you will need
the following:
Mlass slide preparation of material from a suspected herpes simplex virus "'*V% lesion
Commercially prepared test /it for the detection of '*V in lesion smears and related
materials "Chec/ the product insert for /it-specific materials.%
2eces specimen
Commercially prepared test /it for the detection of rotavirus in feces and related materials
"Chec/ the product insert for /it-specific materials.%
".B)%.T)%0 SESS&)/
Detection of Herpes Simplex Virus in "esion Specimen Smears
1. Obtain a slide preparation of material from a suspected '*V lesion, and record the specimen
identification on =or/sheet ++-.!
2. Record the name of the test system that is to be used to detect '*V in the specimen on
=or/sheet ++-..
3. 2ollow the manufacturer8s instructions to evaluate, prepare, and process the specimen for the
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detection of '*V in the specimen!
4. Refer to the manufacturer8s interpretation guidelines, and record the quality control test
results, including the evaluation of the quality of the specimen, on =or/sheet ++-..
5. (f the quality control test results are acceptable, record the specimen test result on =or/sheet
++-.!
Detection of #ota$irus in %eces
1. Obtain a specimen of feces, and record the specimen identification on =or/sheet ++-.!
2. Record the name of the test system that is to be used to detect rotavirus in the specimen on
=or/sheet ++-..
3. 2ollow the manufacturer8s instructions to evaluate, prepare, and process the specimen for the
detection of the rotavirus!
4. Refer to the manufacturer8s interpretation guidelines, and record the quality control test
results, including the evaluation of the quality of the specimen!
5. (f the quality control test results are acceptable, record the specimen test result on =or/sheet
++-.!
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DETECT&)/ )1 !&%." .+E/TS &/ C"&/&C." S-EC&ME/S
6or7sheet 3321
S*S-ECTED (E%-ES S&M-"E$ !&%." "ES&)/ SME.%
*pecimen identiJcation: KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK
Test system: KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK
%es#lts:
Ruality control test results: #ositive: Gegative:
*pecimen quality evaluation: #ositive: Gegative: KKKKKKKKKKKKKKKKKKKKKKKKKKKKKK
*pecimen: #ositive: Gegative: for the presence of herpes simplex virus
1ECES S-EC&ME/
*pecimen identiJcation: KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK
Test system: KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK
%es#lts:
*pecimen quality evaluation: KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK
Ruality control test results: #ositive: Gegative:
*pecimen quality evaluation: #ositive: Gegative: KKKKKKKKKKKKKKKKKKKKKKKKKKKKKK
*pecimen: #ositive: Gegative: for the presence of rotavirus
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