Medium ConstituentsBalance salt solution : Phosphate buffer, Mg , CaInorganic ions and trace elements : for membrane potential and osmotic pressure : bufferEnergy source : glucose, glutamineAmino acid : metabolism and biological synthesis
Role of Serum• Buffer, Chelator, Carrier proteins• Bind to toxin• Protease inhibitor• Promotes attachment of cell to substratum• Source of Intermediate metabolites, hormone and growth factor
Primary cell culture and Establishment cell line • Preparation of cell suspension from intact tissue . Single cell preparation : use mechanical, Chemical, and/or enzymatic method . Disaggregate or dissociate cell : cutting, homogenizing, rotary shaker, vortex, pipette
• Preparation of cell suspension from intact tissue (cont.) Enzymes • Trypsin (crude) : from cattle and pig’s pancrease
• Preparation of cell suspension from intact tissue (cont.) . Removal of tissue and place in Isotonic (or growth medium with antibiotic) . Trim off unwanted parts and cut into smaller pieces . Dissociate cells using enzyme . Remove large debris and wash cells
• Preparation of cell suspension from intact tissue (cont.) . Suspend cells in growth medium accordingly . Pipette into culture vessel and incubate
Common Cell line (cont.)• HeLa cancer tissue:cervical carcinoma : harbors HPV type genome• Vero : from African green monkey kidney : preparation of Poliovirus vaccine
Work in Safety cabinet Class-IIUVethanol for decontaminationCulture medium and Reagent 1.PBS 2. Growth medium : %FCS-DMEM 3. Trypsin-Versene
Subpassage Remove growth medium Wash with PBSDetach cell with Trypsin-Versene Remove TV
Refresh with growth medium Mix cell by pipette Count and calculate number of cell: Hemacytometer
Detection of virus in cell culture:•Cytopathic effect -CPE:changed monolayerarchitecture, the entire cell/ nucleus thickening andshrinking (pycnosis), inclusion bodies formation, giant cell(syncytia)formation, nucleolar displacement,rearangementand margination of the nuclearchromatin,vacuolization,the type of CPE depends on virusspecifity and the kind of cells, polio virus, adenovirus, HSV, CMV;•Viral hemagglutination–direct reaction with redcells, influenza;•Hemadsorption –adsorption of erythrocytes to infectedcells;parainfluenza, influenza•Plaques effect –clear areas appear in culture (PFU)due tocell lysis;
Hemagglutination test: method 1:8 1:2 1:2 1:2 1:2 1:2 virusserial dilution 8 16 32 64 128 256 mix with red blood cells side view top view Titer = 32 HA units/ml
Hemagglutination assay: influenza virusHemagglutination assay. Seven different samples of influenza virus, numbered 1 through 7 at the left, were serially diluted as indicated at thetop, mixed with chicken red blood cells (RBC), and incubated on ice for 1 to 2 hours. Wells in the bottom row contain no virus. Agglutinated RBCs coatwells evenly, in contrast to nonagglutinated cells, which form a distinct button at the bottom of the well. The HA titer, shown at the right, is the lastdilution that shows complete hemagglutination activity. (From Fields Vriology (2007) 5th edition, Knipe, DM & Howley, PM, eds, WoltersKluwer/Lippincott Williams & Wilkins, Philadelphia Fig. 2.9)
Quantitation of viruses a. TCID50(50% Tissue culture infected dose): Infections dose of 50% CPE of tissue culturescaused by viral minimum infected dose is calledTCID50 b. PFU : under controlled condition, singleplague can arise from a single infection virusparticle, termed a plague-forming unit.
Virus cultivation 2. Inoculation of embryonated chick eggs• USE OF FERTILE EGGS Largely replaced by use of TC• Still useful in cultivation and detection of some viruses, flu• Three parts of the egg are of use – A The amniotic cavity: • Surrounds the embryo & lined by a single layer of epithelial cells. • The amniotic fluid bathes the external surface of the embryo and comes into contact with the respiratory and alimentary tracts. – B The allantoic cavity: • Comprises an outgrowth of the hind-gut of the embryo and is lined with endoderm.• Both are useful for the cultivation of viruses, particularly • orthomyxoviruses (e.g., influenza) and paramyxoviruses (e.g., mumps).Membranes major source of cells in which virus growth occurs, but the embryo may also become infected.• The allantoic cavity is routinely used because it is technically much simpler to inoculate.• However, some viruses (e.g., human influenza isolates) may need to be egg-adapted by growth in the amniotic cavity before they will grow efficiently in the allantoic cavity; the reason for this is not known.C The chorio-allantoic membrane : The membrane consists of an outer layer of stratified epithelium which constitutes the respiratory surface of the egg, and an inner layer of endoderm (the lining of the allantoic cavity). The membrane may be used as a cell sheet provided it is first dropped away from the shell membrane. Dermatropic viruses (poxviruses and some herpes viruses) will grow on this membrane, and at low concentrations, will give discrete foci of infection which consist of centres of cell proliferation and necrosis (pocks). The membrane may therefore be used to assay these viruses. In addition, different viruses cause pocks of different colour and morphology, and this is of diagnostic value for distinguishing between different poxviruses.
Growth of virus on embryonated eggs Davis, Duylbecco, Eisen, Ginsberg “Microbiology” 4th ed, J.B. Lippincott 1990, Fig. 48-1
•production of pocks or plaques on chorioallantoicmembrane(herpes, smallpox, vaccinia);•development of hemagglutinins in embryonicfluid(influenza);•death of embryo -encephalitis viruses;Convenient, inexpensive, the viral suspension injected into the fluid of the egg, viral growth is detected by death of the embryo, or lesion on the membrane of the egg. It is the most common method for viral growth and isolation , and used for preparation of viral vaccine.