The Clinical Virology Laboratory Flashcards
Specimen Collection:
- Samples may be collected from the site of _____ _____ or from a site where the virus is being ______ (asymptomatically)
viral pathology
shed
When should specimen be collected after onset of symptoms?
as soon as possible after onset of symptoms
What is the viral transport medium used for?
Used to prevent drying, maintain viability of virus, inhibit growth of contaminants
What specimen are placed in transport medium and what specimens are not?
Are: specimen from contaminated body sites
Are not: specimen from sites that are free from usual flora
Controls pH and keeps sample moist; prevents drying
Hanks Balanced salt solution or buffered broth
stabilizes or “cushions” virus; use fetal bovine serum, albumin, and gelatin
Proteinaceous Supplement
inhibits growth of bacteria and fungi
Antibiotics
signals the pH and indicates if pH is “off”; red/orange color change
pH indicator
collect in a sterile screw-capped container; do NOT use transport medium
spinal fluid
Rub on surface to collect cells and place in transport medium
Swab collection
collect in a sterile container; do NOT use transport medium
Urine collection
collect in anticoagulant such as heparin, EDTA, or sodium citrate; do NOT use transport medium
whole blood collection
If a sample will be processed within 3-5 days use ________ to store and ship on _____ ice.
refrigerator; wet
If sample will be processed after 3-5 days, use ________ at ____‘C or lower. Ship on ______ ice.
freezer; -70’C; dry
If respiratory syncytial virus (RSV) or cytomegalovirus (CMV) is suspected what don’t you do?
DO NOT Freeze
Two general steps in processing most clinical samples for viral culture
centrifugation and addition of antibiotics
following centrifugation of transport medium, the _______ is used to inoculate the cell culture tubes
supernatant
following centrifugation of transport medium, the _______ is used to prepare antigen smears for immunofluorecence testing
sediment
In whole blood samples, the ________ are separted out and used to inoculate cell cultures
leukocytes
In urine samples, _____-_______ urine is used to inoculate cell cultures
well-mixed
cell culture freshly explanted from tissue of origin; may have indigenous contaminants; ex: rhesus monkey kidney
primary cell culture
Cell cultures that have diploid chromosome number; can be passed 50 times so can’t survive long term; ex: MRC-5, MRHF
Diploid (Normal)
Cell cultures that have heteroploid chromosome number; originate from cancer tissue; can be passed indefinitely; Ex: A549
Established (heteroploid)
In cell culture tubes how do viruses grow?
cells grow in a single layer on one side of the tube called a “monolayer” and are on their side to ensure they are bathed in medium
inoculation performed by adding processed fluid to the cell culture tube; 2/10-3/10 of fluid
standard inoculation
Inoculation procedure performed where the cell culture medium is decanted from the tube, processed material is applied to the monolayer, incubated in a slant rack (1hr at 35’C), excess inoculum is removed and idscarded, and fresh cell culture medium is added
Adsorption inoculation
Incubation temperature of inoculated cell culture tubes
36-37’C on rotating and stationary racks (to keep the specimen out of the lid)
____ _______ media contains the same components as transport media but have added enrichments such as amino acids, vitamins, and other nutrients.
cell culture
color of cell culture medium in healthy cell culture tube
peach and clear
cell culture medium color in a tube that is contaminated with bacteria or fungi
bright yellow and turbid
cell culture medium color in which the pH is too basic
bright cherry pink
how are cell culture tubes examined under a light microscope?
- 10x with reduced light
- railroad track to keep media in bottom of tube
- look for changes in cell morphology
changes in the cell morphology of viruses including swelling, shrinking, and detachment from the vessel
cytopathogenic effect (CPE)
When are cell culture tubes are routinely examined?
on alternate days for 14 days
the only culture that is examined for only 7 days
Herpes Simplex Virus (HSV)
When are CMV cultures examined?
30 days , slowest grower
Viral identity is PREDICTED by what 4 factors?
1 characteristic appearance of CPE
2 Time to appearance of the CPE
3 Cell line in which the CPE appears
4 source of the clinical sample
Viral identity is CONFIRMED by what 2 factors?
1 Virus-infected cells are reacted with antibodies of known specificity and when they bind, identity confirmed
2 most common confirmatory method is immunofluorescence
Procedure that can help detect viruses by replacing cell culture medium with a dilute suspension of erythrocytes (guinea pig) and refrigerating in a slant rack at 4’C for 30 min
Hemadsorption
Describe a positive HAD test
RBCs stick to monolayer
Describe a negative HAD test
the erythrocytes do not stick to the monolayer and float free in the tube
Shell vial system procedure
- clinical sample placed directly on monolayer
- centrifuge at low speed for 1 hour
- add medium and incubate
- decant and add fixative
- add stain to coverslip in vial
- coverslips removed and mounted on a slide
- fluroescence microscope to examine
Shell vials are not evaluated for ______
CPE
Advantages of shell vial
quicker detection, shorter turnaround time, no CPE to read
Disadvantages to shell vial system
tedious to read, virus-specific (rather than “open” system)
WHy are shell vials flexible?
specificity can be changed by changing the type of cells on the monolayer and the monoclonal antibodies used
Monolayers of cells that include two different types of cells grown together
co-cultivated cell lines
What are some marketed names for co-cultivated cell lines?
R-Mix (Respiratory viruses), E-Mix (for enteric viruses), H&V-Mix (for HSV, CMV, and VZV)
Advantages of co-cultivated cell lines?
- Cell lines retain usual characteristics of susceptibility to viruses
- virology labs do not need to stock as many types of cell cultures
- same as shell vial advantages
Disadvantages of co-cultivated cell lines?
same as shell vial; tedious to read, virus specific
Explain the meaning behind the picomaviridae family
small RNA viruses
have fecal/oral route of transmission, replicate in the bowel but produce pathology at sites outside the bowel, most grow in standard cell cultures, difficult to differentiate from each other and from rhinoviruses, molecular methods are best for detection, ESPECIALLY for CSF samples
enteroviruses
replicates in the bowel, moves to CNS to produce pathology, recent change from oral to injection vaccine, extinct in Western Hemisphere
Poliovirus
trasmission and replication in the bowel, pathology may be CNS but more often in the heart
coxsackievirus A and B
transmission and replication same as polio, may be isolated when disease is not present “orphan”
echovirus also called parechoviruses
What does E. C. H. O stand for?
E-Enteric
C-cytopathogenic
H-human
O-orphan
member of Picomaviridae family but is NOT an enterovirus, proliferate in upper respiratory tract and causes common cold, diagnosed clinically
Rhinovirus
