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; uses 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 w/in 3-5 days, use ____ to store and ship on ____ ice
Refrigerator; wet
If a sample will be processed after 3-5 days, use ____ at ____C or lower. Ship on ____ ice
Freezer; -70C; 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 Ag smears for immunofluorescence testing
Sediment
In whole blood samples, the ____ are spearated out and used to inoculate cell cultures
Leukocytes
In urine samples, ____-____ urine is used to inoculate cell cultures
Well-mixed
Cell cultures freshly explanted from tissue of origin; may have indigenous contaminants (e.g., rhesus monkkey kidney)
Primary cell culture
Cell cultures have diploid c’some number; can be passed 50x so it can’t survive long term (e.g., MRC-5, MRHF)
Diploid (normal)
Cell cultures that have heteroploid c’some number; originate from cancer tissue; can be passed indefinitely (e.g., A549)
Established (heteroploid)
In a cell culture tube, how do tissues grow?
Cells grow in a single layer on one side of the tube called a “monolayer” and are on their side to ensure they’re bathed in medium
Inoculation performed by adding processed fluid to the cell culture tube
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 35C), excess inoculum is removed and discarded, and fresh cell culture medium is added
Adsorption inoculation
Incubation temperature of inoculated cell culture tubes
36-37C 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 w/ 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 w/ 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 routinely examined?
On alternate days for 14 days
The only cell 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?
- Characteristic appearance of CPE
- TIme to appearance of the CPE
- Cell line in which teh CPE appears
- Source of the clinical sample
Viral identity is CONFIRMED by what 2 factors?
- Virus-infected cells are reacted w/ Abs of known specificity and when they bind, identity confirmed
- Most common confirmatory method is immunofluorescence
Procedure that can help detect viruses by replacing the cell culture medium w/ a dilute suspension of erythrocytes (from a guinea pig) and refrigerating in a slant rack at 4C for 30 min
Hemadsorption (HAD)
Describe a positive HAD test
RBCs stick to monolayer
Describe a negative HAD test
RBCs don’t 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
- Fluorescence 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 Abs 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 (enteric viruses)
- H&V-mix (HSV, CMV, 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 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
- Transmission 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 (aka 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
Why is processing of most clinical samples required prior to their inoculation into cell cultures?
Must be treated to remove contaminating organisms and other agents taht may be toxic or infective for cell cultures
When specimens are collected on swabs and sent to the lab in viral transport medium, what steps are performed on the sample before inoculation of cell culture tubes?
- If swab is received in transport medium, medium is vortexed and then removed
- Liquid transport medium, which now contains specimen material, is centrifuged (if antibiotics aren’t included in transport medium, add them)
Why is more than one type of cell culture tube used in most viral cultures?
Virologists select an array of cell cultures that’re susceptible to a variety of human viral pathogens, especially those frequently associated w/ infections of anatomical site from which specimen was collected
What does the insignia or label on cell culture tubes tell you?
The insignia/label is located on the opposite side of the monolayer
