06: Sample Collection

Very general practices to pay attention to when submitting a sample

Keep it clean and cold

Why might a green top tube not be great for viral PCR

Heparin can interfere with the polymerase in a PCR

Why might a purple top tube not be great for virus isolation

EDTA can block virus-cell binding

Type of tube that can be used for some viral tests, but check with the lab first

Citrate tube

Type of sample container for a fecal

Anything sterile or clean

Common type of sample collection for viral testing

Swabs in media/solution

Why can’t you submit a bacterial sample in viral media

Viral media often has antimicrobials!

Sample typically used for Ab testing

Serum

Why do we not freeze blood we are sending off

Will cause hemolysis

Why does serum need to be separated or removed from blood before sending off

RBCs continue to metabolize, and that will mess up your serum chemistry

Ways to send off tissue for testing

Fresh (never frozen) or fixed with 1:10 formalin solution

How do you ensure the lab gets the best possible sample for testing

Keep sample cold and expedite shipping

Methods for virus isolation

• Tissue culture

• Embryonated eggs

• Animal inoculation

Historic use for virus isolation and amplification

Used to be used for diagnosis

Present use for virus isolation and amplification

Research and vaccine production

Types of tissue culture

• Explant: grows fragments of tissues

• Cell culture: develops a monolayer

Most common virus isolation mechanism

Cell culture

Cell culture mechanism

Trypsin dissociates cells, which are then transferred to flasks and plates to make a monolayer

Primary cell culture

Culture of cells directly from the original organ

Why can’t we use a primary cell culture forever

The cells run out of space on the plate, and they also only divide a limited number of times before losing their ability to proliferate

How do we then continue to utilize the cells from a primary culture

A cell line is subcultured, and then it is transformed into an immortal, continuous cell line using tumor cells

Why can’t we use continuous cell lines for everything

They don’t always look enough like the original primary cell culture, and some viruses are very cell specific

Cytopathic effects

Visible morphologic changes to cells as a result of come viral infections

How do CPEs assist in virus identification

CPEs are characteristic of a specific group of viruses; not all viruses cause CPEs

CPE characterized by rounding of cells and cell death

Plaques

CPE characterized by multinucleated giant cells

Syncytia

Inclusion bodies

Accumulation of viral proteins and virions

How do inclusion bodies help with viral identification

Inclusion bodies usually show up where the virus replicates (cytoplasm v nucleus)

Virus type that normally produces intracytoplasmic inclusion bodies (if any)

RNA virus, usually

Virus type that normally produces intranuclear inclusion bodies (if any)

DNA virus, usually

Purpose of inoculating embryonated eggs

Grows viruses that won’t grow in cell cultures

Purpose of animal inoculation

• Pathogenesis studies

• Vaccine research

• Ab production

Units for measuring virus concentration

• PFU: plaque forming units

• TCID₅₀: median tissue culture infective dose

Virus titration

Serial dilution and inoculation of wells to determine viral concentration

Plaque assay

Counts number of plaques formed per inoculated well