3 - Virological Techniques I. Collect of Specimen A....

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Virological Techniques I. Collect of Specimen A. General rules: - Collect as early as possible (first three days after the onset of the disease). - Proper labeling (Patient’s name, age, sex, date of onset of disease and date of collection, clinical diagnostic, etc) - Immediately ship to laboratory. - Store at 4C for no more than 3 days, -70 C for longer period. Never at –20C. B. Choice of Specimen - depend on the clinical disease and the virus suspected.
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Virological Techniques
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Virological Techniques C. Procedures for collection -to obtain adequate amount of viruses for studies For body fluids , place in leakproof, screw cap, sterile containers. Serum/Plasma: No dilution is needed. Stored at 4C until inoculation into culture. Whole blood with anticoagulant: No dilution is needed. Stored at R.T. until inoculation into culture within 2-5 hours. To search for viruses harboured in the leukocyte fraction, higher isolation rate can be obtained from purified leukocytes. Leukocytes can be isolated by Ficoll-Hypaque before inoculation. Urine: 1:1 (v/v) dilution in VTM (Virus transport medium). For swabs (throat, eye, lesion, genital, etc) , place them into VTM to prevent drying. VTM: balanced salt solution with 0.5% gelatin, serum or BSA + antibiotics + Phenol Red. For stools: make a 20%(w/v) stool suspension in PBS containing antibiotics. Vortex and centrifuge at 1400 x g for 20 mins. Collect supernatant for inoculation. For tissue samples (Autopsy or Biopsy): The tissues are either minced or homogenized in VTM to break cells and release intracellular viruses. Intact cells after mincing are allowed for co-culture. II. Virus isolation and identification. In general, the first step in virus identification is to propagate the virus. Unlike bacteria, virus can only propagate in living cells. Before 1949, viruses were only propagated in living animals. The discovery that poliovirus can be propagated in cultured cells of non-neuronal origin leaded to a Nobel Prize in 1954. In modern virology, three kinds of hosts are commonly used: 1. Cell culture (primary cell culture or cell lines). 2. SPF embryonic eggs. 3. Specific-pathogen-free animals (SPF): chicken, mouse or even pig. The use of cell culture is more convenient than SPF eggs or animals. However, not all viruses can be propagated in cell culture. For example, hepatitis C can only be propagated in Chimpanzees. 1. Cell culture (a) Primary cell culture: Cells are prepared from tissues or organs dissected from the susceptible animals. E.g. Chicken embryo fibroblast, Monkey kidney cells, mouse embryo cells. - Contains several cell types and have a limited life span (5-10 passages) (b) Continuous cell lines: a single cell type that can be propagated indefinitely in culture.
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Virological Techniques Propagation of Virus in cell culture General protocol: Seed cells and add virus (Optional: remove the added virus after 1 hour of adsorption). When CPE is obvious (2-4 d.p.i), freeze and thaw the cells thrice to release the intracellular virus
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This note was uploaded on 10/28/2010 for the course SCI biol3214 taught by Professor Lam during the Spring '10 term at CUHK.

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3 - Virological Techniques I. Collect of Specimen A....

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