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Infectious Agents 2 > Collection and Preparation of Clinical Specimens > Flashcards

Collection and Preparation of Clinical Specimens Flashcards

1
Q

You want to collect a specimen in the _____ phase of disease

A

Acute, before antibody formation

- can not detect a virus if it is bound to an antibody

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2
Q

What are examples of specimens commonly used?

A
  • blood
  • nasal swabs
  • feces
  • urine
  • pus
  • vesicular fluid
  • skin lesions
  • spinal fluid
  • biopsy and necropsy tissue
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3
Q

It is important to collect specimens immediately _______

A

After death

- autolytic changes are detrimental to viruses

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4
Q

Specimens should be taken with ______ instruments, using ______ technique

A

Sterile; aseptic

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5
Q

What 3 methods are used for isolation of viruses?

A
  • embryonated eggs (most common)
  • cell cultures
  • live animals
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6
Q

Fluid specimens

A

Must be bacteria free

- inoculated directly, or after dilution

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7
Q

Solid tissue preparation

A

10-20% solution in 0.15M PBS, pH 7.2

  • centrifuged at 2,000 xg for 10 minutes to remove tissue and cellular debris
  • bacteria must be eliminated prior to inoculation using 10-15% ether for 1-2 hours
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8
Q

Antibiotics

A

Penicillin at 1,000 U/ml +/- 100 ug/ml streptomycin

- used to eliminate contaminating bacteria

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9
Q

Sedimentation

A

Centrifugation at 40,000 xg for 1 hr will concentrate most viruses
- pellet will be resuspended in a small volume of PBS prior to filtration and inoculation

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10
Q

_____ filters will trap most bacteria

A
  1. 22 micron

- does not trap myoplasma!! (lack a cell wall)

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11
Q

Storage

A

Fresh tissues are frozen to -60 C after collection

  • specimens may be placed at -20 C until dry ice is obtained
  • vials must be air tight due to CO2 gaseous phase of dry ice that will lower pH and inactivate certain viruses
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12
Q

Are live animals used for isolation of viruses?

A

NO

  • specifically not used in diagnostics
  • exception: rabies virus in neonatal mice
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13
Q

What are the 2 methods for detection of viruses in clinical specimens?

A
  • direct

- indirect

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14
Q

Direct method

A

The virus itself, or component antigens or viral molecules, are detected

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15
Q

Indirect methods

A

Serologic evidence, in the form of antibodies, is taken as a sign of viral infection

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16
Q

Are all test methods applicable to all viruses?

A

No, some test methods work well for some viruses, but not others
- due to variations between individual viruses and standardizes results among different labs for consistency

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17
Q

T/F: Only avian viruses will replicate in embryonated chicken eggs

A

False!

- cells and extra-embryonic membranes of chickens lack a high degree of specialization

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18
Q

Why are chicken embryos used for viral isolation?

A
  • availability
  • economy
  • convenient size
  • relative freedom from letent infection and extraneous contamination
  • lack of production of antibodies against viral inoculum
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19
Q

Incubation process

A

Preliminary incubation at 100.4-102.2F with incubation of incoulated embryos at 98.9-99.5 F (can stay at this temp throughout the entire period)
- lower temps may be required under certain cirumstances

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20
Q

When are eggs obtained?

A

Get eggs at 9 days old, and they will hatch at 21 days

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21
Q

Yolk sac inoculation

A

Done on 5-7 day old eggs

  • used with smaller viruses, that will invade the embryo and multiply in the body tissues
  • 0.2-1.0 ml volume
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22
Q

Chorioallantoic cavity inoculation

A

Used for viruses that cause respiratory infections and encephalomyelitis viruses that readily multiply in the entodermal cells of the chorioallantoic sac wall

  • 8-11 day old eggs
  • 0.1-0.2 ml volume
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23
Q

Chorioallantoic membrane inoculation

A

Largest, cleanest, easiest to harvest

  • 10-12 day old eggs
  • 0.1-0.5 ml volume
24
Q

Amniotic sac inoculation

A

Surrounds the embryo itself, is used primarily for isolation of influenza viruses from throat washings

  • 7-15 day old eggs
  • 0.1-0.2 ml volume
25
Q

Intravenous inoculation

A

Not used often

  • 10-15 day old eggs
  • used for adaptation and replication of foot and mouth disease
  • 0.02 - 0.05 ml volume
26
Q

Intracerebral inoculations

A

Done on 8-14 day old eggs

  • rabies and herpes simplex cultivated by this route
  • 0.01 - 0.02 ml volume
27
Q

What are factors influencing the growth of viruses in chicken embryos?

A
  • age of the embryo
  • route of inoculation
  • concentration of virus and volume of inoculum
  • temperature of incubation
  • time of incubation following inoculation
28
Q

Methods determining replication of a virus in embryos

A
  • sampling of the virus in extraembryonic fluids and membranes
  • pathologic alterations
  • serologic tests
  • hemagglutination
  • antigenicity
  • immunogenecity
29
Q

_____ are the most widely used method for isolation of viruses from clinical material

A

Cell cultures

  • denotes growing of cells in vitro
  • use from homologous species (canine cells to propagate canine distemper)
30
Q

Primary cell culture

A

Tissue culture started from material taken directly from an animal

  • preferred due to increased susceptibility to viruses
  • can become immortalized and are then referred to as cell lines, capable of indefinite subculturing
31
Q

Established cell lines have advantage in _____ and ______

A

Consistency and uniformity

32
Q

What allows for differentiation and preservation of architecture and function?

A

Growth of whole organs, or parts of an organ

- difficult

33
Q

At ____ cuts, the cell starts to mutate and adapt to the culture

A

40

34
Q

________ inoculation in mice is used to isolate neurotropic viruses

A

Intracerebral

35
Q

Electron microscopy

A

Use negative contrast transmission to morphologically characterize viral particles (direct method)

  • commonly used with enteric viruses
  • examination of excretions, secretions, solid tissues
36
Q

What is the preferred concentration of viral particles for electron microscopy?

A

10^8/ml permits rapid detection

- 10^6/ml is good, 10^5/ml is barely detectable

37
Q

Knowledge of ____ and _____ is necessary to identify viruses with electron microscopy

A
  • viral sizes

- morphology

38
Q

Direct fluorescent antibody test

A

Quick, easy, need a special microscope to view results

  • use a specimen from the host that contains the virus and attach commercially fluorescent labelled antibodies to the virus
  • used to track multiple viruses within a host at a single time
  • doe not work with all viruses
39
Q

Indirect fluorescent antibody test

A

Specimen contains the virus and an unlabelled antibody (made from the host) and the commercial fluorescent labelled antiglobulin attaches to the antigen-antibody complex
- all antibodies are of known specificity and concentration

40
Q

Enzyme linked immunosorbent assay

A

Used for detection of virus or viral antigen, that involves virus capture by specific antibody attached (absorbed or covalently bound) to a solid substrate

  • captured antigen is detected by an enzyme labeled antiviral secondary antibody
  • final washing step reveals result based on a visible color change following the addition of a substrate
41
Q

What is the enzyme used in ELISA?

A

Usually horesradish peroxidase or alkaline phosphatase

42
Q

ELISA can be designed to detect _____ or _____

A

Antigen (direct) or antibody (indirect)

43
Q

Radioimmunoassay

A

Not commonly used

- similar to ELISA, but indicator system is isotope labeled antibody instead of enzyme labeled antibody

44
Q

PCR

A

Common for labs, not clinic use

- nucleic amplification assays are universally applicable with the appropriate primers

45
Q

PCR primer

A

Short, oligonucleotide sequence that attaches to a segment flanking the gene targeted for amplification
- after attachment, primers are extended into target gene region by the enzymatic addition of new nucleotides, added sequentially to the primer

46
Q

What gene regions do primers attach to?

A

One serves as the upstream (forward) primer, the other as the downstream (reverse) primer
- bracket the target gene so that both opposing DNA strands are amplified = numerous double stranded copies

47
Q

How are the double stranded copies turned into single strands?

A

Double strands are dissociated (melted) into single strands by increasing the reaction temperature

  • temp is then decreased to reintroduce the primers
  • cycle will repeat
48
Q

General cycle of PCR

A

Dissociation –> annealing –> polymerase activity

- cycle will repeat 30-40 times

49
Q

Taq polymerase

A

Heat-stable DNA polymerase commercially purified from the thermophilic organism Thermus aquaticus

  • most expensive commercial ingredient
  • high active temperature of this enzyme shortens temperature swings between strand “melting” and polymerase activity = quicker cycling
  • extend primers between 2 fixed points on a strand of DNA
50
Q

Thermocycler

A

Takes reaction mixture to 94 C for 60 sec to allow for melting –> 55 C for 60 sec to permit primer attachment –> 72 C for 120 sec to allow for polymerase addition of nucleotides to the primer
- final cycle of 72 C for 300 sec allows completion of new strands

51
Q

What is the final step of PCR?

A

Detection and identification of amplified DNA

  • PCR product, or amplicon
  • done with agarose-gel electrophoresis, or gene probes
52
Q

Gene probes

A

Species-specific oligonucleotide segments that incorporate markers to facilitate detection
- binding of probe to target gene demonstrates sequence homology, establishing identity

53
Q

______ is critical to successful use of PCR

A

Primer selection

54
Q

Restriction fragment length polymorphism

A

Electrophoretically analyze PCR products

- used to compare related organisms or characterize

55
Q

Nested primers

A

Increase sensitivity and specificity in the detection of organisms

56
Q

RT-PCR

A

Frequently applied in the detection and analysis of certain viruses, many of which utilize RNA in their genetic code
- requires an extra cycle to precede PCR to allow RT to convert viral RNA into DNA

57
Q

PCR can be applied to _____ or _______

A

Viral DNA or RNA

Infectious Agents 2 (29 decks)

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