The Clinical Virology Laboratory Flashcards by Liz Haywood

Specimen collection:

- Samples may be collected from the site of ____ ____ or from a site where the virus is being ____ (asymptomatically)

Viral pathology; shed

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When should specimen be collected after onset of symptoms?

As soon as possible after onset of symptoms

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What is the viral transport medium used for?

Used to prevent drying, maintain viability of virus, inhibit growth of contaminants

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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

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Controls pH and keeps sample moist; prevents drying

Hanks Balanced salt solution or buffered broth

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Stabilizes or “cushions” virus; uses fetal bovine serum, albumin, and gelatin

Proteinaceous supplement

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Inhibits growth of bacteria and fungi

Antibiotics

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Signals the pH and indicates if pH is “off”; red/orange color change

pH indicator

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Collect in a sterile screw-capped container; do NOT use transport medium

Spinal fluid

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Rub on surface to collect cells and place in transport medium

Swab collection

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Collect in a sterile container; do NOT use transport medium

Urine collection

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Collect in anticoagulant such as heparin, EDTA, or sodium citrate; do NOT use transport medium

Whole blood collection

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If a sample will be processed w/in 3-5 days, use ____ to store and ship on ____ ice

Refrigerator; wet

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If a sample will be processed after 3-5 days, use ____ at ____C or lower. Ship on ____ ice

Freezer; -70C; dry

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If respiratory syncytial virus (RSV) or cytomegalovirus (CMV) is suspected, what DON’T you do?

DO NOT FREEZE!

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Two general steps in processing most clinical samples for viral culture

Centrifugation and addition of antibiotics

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Following centrifugation of transport medium, the ____ is used to inoculate the cell culture tubes

Supernatant

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Following centrifugation of transport medium, the ____ is used to prepare Ag smears for immunofluorescence testing

Sediment

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In whole blood samples, the ____ are spearated out and used to inoculate cell cultures

Leukocytes

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In urine samples, ____-____ urine is used to inoculate cell cultures

Well-mixed

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Cell cultures freshly explanted from tissue of origin; may have indigenous contaminants (e.g., rhesus monkkey kidney)

Primary cell culture

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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)

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Cell cultures that have heteroploid c’some number; originate from cancer tissue; can be passed indefinitely (e.g., A549)

Established (heteroploid)

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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

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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