Review Article Open Access Cell Culture Cytopathic Effect

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Journal of Microbiology and Modern Techniques 2,3 Antigen detection immunofluorescence ELISA etc. 4 Molecular techniques for the direct detection of viral genomes. Indirect Examination, 1 Cell Culture CPE haemadsorption confirmation by neutralization immunofluorescence etc. 2 Eggs pocks on CAM haemagglutination inclusion bodies. Detection of rising titers of antibody between acute and convalescent stages of infection or the detection of IgM in primary. infection can be done by the following methods,1 Complement fixation tests CFT. 2 Enzyme linked immunosorbent assay ELISA,3 Haemagglutination inhibition tests HAI. 4 Immunofluorescence techniques IF,5 Recombinant immunoblot assay RIA.
Viral Diagnosis Methods, Direct examination methods are often also called rapid diagnostic methods because they can usually give a result either within the. same or the next day This is extremely useful in cases when the clinical management of the patient depends greatly on the rapid. availability of laboratory results e g diagnosis of RSV infection in neonates or severe CMV infections in immunocompromised. patients However it is important to realize that not all direct examination methods are rapid and conversely virus isolation and. serological methods may sometimes give a rapid result With the advent of effective antiviral chemotherapy rapid diagnostic. methods are expected to play an increasingly important role in the diagnosis of viral infections 1 2. Virus Isolation, Cell culture is an important component of the clinical virology laboratory to isolate virus Several laboratories have been successful. in the primary culture of adult rat hepatocytes Cultured cells eggs and laboratory animals may be used for virus isolation Although. embroyonated eggs and laboratory animals are very useful for the isolation of certain viruses cell cultures are the sole system for. virus isolation in most laboratories The development of cultivating animal cells has been essential for progress to animal virology. Isolation of viruses in cell culture has been the standard procedure in diagnostic laboratory for a long time Viruses that can. be commonly isolated in standard cell cultures include adenovirus cytomegalovirus many of the enteroviruses coxsackie B. virus echovirus poliovirus types 1 2 and 3 herpes simplex virus types 1 and 2 influenza virus A and B measles virus mumps. virus parainfluenza virus types 1 2 3 and 4 respiratory syncytial virus varicella virus To prepare cell cultures tissue fragments. are first dissociated usually with the aid of trypsin or collagenase The cell suspension is then placed in a flat bottomed glass or. plastic container together with a suitable liquid medium After a variable lag the cells will attach and spread on the bottom of the. container and then start dividing giving rise to a primary culture Attachment to a solid support is essential for the growth of. normal cells 2 3,Cell Cultures, In the 1900s embryonated eggs and laboratory animals were used for isolation of viruses Typically cell cultures are developed. from tissue samples and then disaggregated by mechanical chemical and enzymatic methods to extract cells suitable for isolation. of viruses With the utilization of cell culture technique use of laboratory animals in experiments has decreased significantly. 3 Cell culture is the complex process by which cells are grown under controlled conditions generally outside of their natural. environment In practice the term cell culture now refers to the culturing of cells derived from multi cellular eukaryotes. especially animal cells The historical development and methods of cell culture are closely interrelated to those of tissue culture. and organ culture 4 5, Cells can be isolated from tissues for ex vivo culture in several ways Cells can be easily purified from blood however only the. white cells are capable of growth in culture Mononuclear cells can be released from soft tissues by enzymatic digestion with. enzymes such as collagenase trypsin or pronase which break down the extracellular matrix Alternatively pieces of tissue can be. placed in growth media and the cells that grow out are available for culture Cells are grown and maintained at an appropriate. temperature and gas mixture typically 37 degrees Celsius 5 CO2 for mammalian cells in a cell incubator Culture conditions. vary widely for each cell type and variation of conditions for a particular cell type can result in different phenotypes 3 4 There. are three types of cell cultures, Primary Cells These are essentially normal cells obtained from freshly killed adult animals These cells can only be passaged sub.
cultured once or twice e g primary Monkey Kidney cells MK primary rabbit kidney cells RK and mice fibroblasts. Annex Publishers www annexpublishers com Volume 2 Issue 1. 3 Journal of Microbiology and Modern Techniques, Semi Continuous Cells Semi continuous cells have diploid number of chromosomes and can be passaged for several times e g. Human embryonic kidney HEK and skin fibroblast cells MRC 5 and WI 38 These are cells taken from embryonic tissue and. may be passaged up to 50 times, Continuous Cells Continuous or transformed cell lines are immortalized cells that can be passaged without limit e g HeLa. Henrietta Lacks Vero Hep 2 LLC MK2 and BGM These are immortalized cells i e tumor cell lines and may be passaged. indefinitely,Preparation of Cell Culture,1 Take a piece of tissue organ. 2 Subject to the action of certain proteolytic enzymes plus mechanical shock. 3 Isolate wash and count cells, 4 Suspend in growth media which contains buffered isotonic solution amino acid fatty acids carbohydrates vitamins precursor. of nucleic acids penicillin streptomycin and keep pH between 7 1 7 4. 5 Place in tube bottle or petri dish, 6 Multiply a single monolayer of cells then it is ready to be inoculated with virus of interest to cultivate.
Primary Cultures, Primary cultures are derived directly from excised normal animal tissue healthy tissue and cultures either as an explant culture. or following dissociation into a single cell suspension by enzyme digestion Such cultures are initially heterogeneous but later. become dominated by fibroblasts Primary cell cultures are widely acknowledged as the best cell culture systems available since. they support the widest range of viruses Primary cells are cells straight from the tissue with no passages Primary cultures by. definition have not been passaged as soon as they are passaged they become a cell line and are no longer primary Cell lines have. at least one passage 1 6,Steps to prepare a primary cell culture. Mince organ and treat with a protease to separate cells. Wash count and dilute cells in growth medium let settle on flat surface. Incubate under 5 CO2 and about 37 C, Cell cultures vary greatly in their susceptibility to different viruses It is of utmost importance that the most sensitive cell cultures. are used for a particular suspected virus Specimens for cell culture should be transported to the laboratory as soon as possible. upon being taken Swabs should be put in a vial containing virus transport medium Bodily fluids and tissues should be placed in. a sterile container 7 Figure 1,Adenovirus Source CDC. Figure 1 a Uninfected epithelial cell b Early infected epithelial cell c Late infected epithelial cell. Continuous Cultures, Continuous cultures are comprised of a single cell type that can be serially propagated in culture either for a limited number of cell.
divisions approximately thirty or otherwise indefinitely Cell lines of a finite life are usually diploid and maintain some degree. of differentiation The fact that such cell lines senesce after approximately thirty cycles of division means it is essential to establish. a system of master and working banks in order to maintain such lines for long periods Continuous cell lines can be propagated. indefinitely because they have been transformed into tumor cells 3 Tumor cell lines are often derived from actual clinical tumors. but transformation may also be induced using viral oncogenes or by chemical treatments Transformed cell lines present the. advantage of almost limitless availability but the disadvantage of having retained very little of the original in vivo characteristics. It is good to grow fastidious types of viruses 8, Annex Publishers www annexpublishers com Volume 2 Issue 1. Journal of Microbiology and Modern Techniques 4, Several different types of cell cultures are inoculated with each viral specimen and are cultivated in tubes or Roux flasks made of. a special glass or plastics mostly in stationary way at 37 degrees Celsius Cultivation media contain proteins vitamins minerals. and amino acids and can be enriched with fetal calf serum Bacterial contamination is suppressed with combination of antibiotics. added to the medium pH is set up with solution of NaHCO3 Cell passaging or splitting is a technique that enables an individual. to keep cells alive and growing under cultured conditions for extended periods of time Cells should be passed when they are 90. 100 confluent 7 9, Commonly used monkey kidney cells are used for recovery of myxoviruses paramyxoviruses and many enteroviruses Human fetal. diploid cells are fibroblastic cells that support the growth of herpesviruses cytomegalovirus varicella zoster virus adenoviruses. and picornaviruses Hep2 cell derived from a human cancer are used for isolation of respiratory syncytial virus and adenoviruses. Some other viruses as HIV coxsackie A togaviruses require special procedures either co cultivation with mononuclear blood. cells or inoculating to a suckling mouse or special tissue cultures 9. Specimen Processing and Inoculation, Performing a viral culture requires the following steps processing of the specimen and inoculation onto the cell culture maintenance. of the inoculated cell culture and detection of viral growth Normally sterile body fluids such as CSF may be inoculated directly. onto cell cultures Urine specimens should have the pH adjusted toward neutrality before inoculation Specimens from body sites. typically contaminated with bacteria such as respiratory or genital specimens are treated with antibiotics before inoculation to. prevent bacterial or fungal overgrowth After inoculation cultures are incubated at 35 to 37 C and inspected periodically for. example once daily or every other day Respiratory cultures directed at detection of influenza or rhinoviruses may be incubated. at 33 C 10,Storage Temperature, Proper storage of specimens is important as it can take several days before some specimens are finally inoculated onto isolation.
systems Most viruses can be stored at 4 to 8 C for several days before there is an excessive loss of infectivity Freezing specimens. to 70 C and bellow will preserve residual viral infectivity of most specimens although a significant loss of infectivity may occur. with repeating freeze thaw cycle Many viruses lose their infectivity rapidly when stored at 15 to 20 C For a long time storing of. viral strains the liquid oxygen system 196 C is used 9. The inoculated tubes should be read at least every other day for the presence of cytopathic effect Certain specimens such as. urine and feces may be toxic to cell cultures that may produce a CPE like effect If toxic effects are extensive it may be necessary. to passage the inoculated cells Cell cultures that are contaminated by bacteria should either be put up again or passed through. a bacterial filter Cell cultures should be kept for at least one to two weeks longer in the case of CMV Cell cultures should be. reefed with fresh maintenance medium at regular intervals or if required should the culture medium become too acidic or alkaline. When CPE is seen it may be advisable to passage infected culture fluid into a fresh culture of the same cell type For cell associated. viruses such as CMV and VZV it is necessary to trypsinize and passage intact infected cells Other viruses such as adenovirus can. be sub cultured after freezing and thawing infected cells 3 11 12. Identification of Growing Virus, Viral growth in cell cultures is most often detected based on the development of microscopically visible cytopathic effect CPE. and haemadsorption changes Some viruses however can grow to high titer without producing visible CPE and must be detected. by other means 10, Cytopathic Effect CPE The term cytopathic effect is frequently applied to virus induced cellular changes that are visible by. light microscopy Cytopathic effect is noticed as the monolayer cells deteriorate as a result of the viral infection These changes. include swelling or shrinkage of cells the formation of multinucleated giant cells syncytia and the production of inclusions. made visible by staining in the nucleus or cytoplasm of the infected cell It may be specific or non specific e g HSV and CMV. produces a specific CPE whereas enteroviruses do not 13 Figure 2 Cytopathic effects include. Detachment plaques,Ballooning Giant cell,Fusion syncytium formation. Inclusion body formation, Haemadsorption Cells acquire the ability to stick to mammalian red blood cells Haemadsorption is mainly used for the. detection of influenza and parainfluenza viruses Confirmation of the identity of the virus may be carried out using neutralization. haemadsorption inhibition immunofluorescence or molecular tests 13. Annex Publishers www annexpublishers com Volume 2 Issue 1. 5 Journal of Microbiology and Modern Techniques,Source Courtesy .
Citation Dilnessa T Zeleke H 2017 Cell Culture Cytopathic Effect and Immunofluorescence Diagnosis of Viral Infection J Microbiol Modern Tech 2 1 102 Review Article Open Access Viruses are obligate intracellular parasites that require living cells in order to replicate Cell culture for propagation and identification of

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