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Chapter 10.2 : Selection, Maintenance, and Observation of Uninoculated Monolayer Cell Cultures

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Abstract:

Viruses are obligate intracellular parasites requiring metabolically active cells to support their replication. While many viruses of diagnostic interest can be cultured in readily available monolayer cell cultures ( Table 10.2-1 ), there are several agents that can be isolated only using specialized systems ( Table 10.2-2 ) such as organ culture (e.g., coronaviruses), leukocyte culture (e.g., the human immunodeficiency viruses and Epstein-Barr virus), or animals (e.g., rabies virus, several coxsackie A viruses, and arboviruses). In addition, there are several important viral agents for which an in vitro system has not been identified (e.g., hepatitis B and C viruses, human papillomaviruses, Norwalk virus, and parvovirus B19).

Citation: Garcia L. 2010. Selection, Maintenance, and Observation of Uninoculated Monolayer Cell Cultures, p 20-30. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch10.2
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Figures

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Figure 10.2-1

Assessment, incubation, and maintenance of uninoculated cell cultures.

Citation: Garcia L. 2010. Selection, Maintenance, and Observation of Uninoculated Monolayer Cell Cultures, p 20-30. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch10.2
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Image of Figure 10.2-2
Figure 10.2-2

Focal area of replicating A-549 cells that have inadvertently crosscontaminated an MRC-5 monolayer.

Citation: Garcia L. 2010. Selection, Maintenance, and Observation of Uninoculated Monolayer Cell Cultures, p 20-30. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch10.2
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References

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1. Albert, M. J. 1986. Enteric adenoviruses. Arch. Virol. 88:117.
2. Arens, M. Q.,, E. M. Swierkosz,, R. R. Schmidt,, T. Armstrong,, and K. A. Rivetna. 1986. Enhanced isolation of respiratory syncytial virus in cell culture. J. Clin. Microbiol. 23:800802.
2a.. Boivin, G.,, Y. Abed,, G. Pelletier,, L. Ruel,, D. Moisan,, S. Cote,, T. C. T. Peret,, D. D. Erdman,, and L. J. Anderson. 2002. Virological features and clinical manifestations associated with human metapneumovirus: a new paramyxovirus responsible for acute respiratory-tract infections in all age groups. J. Infect. Dis. 186:13301334.
2b.. Chan, P. K. S.,, J. S. Tam,, C. W. Lam,, E. Chan,, A. Wu, et al. 2003. Human metapneumovirus detection in patients with severe acute respiratory syndrome. Emerg. Infect. Dis. 9:10581063.
3. Espy, M. J.,, C. Hierholzer,, and T. F. Smith. 1987. The effect of centrifugation on the rapid detection of adenovirus in shell vials. J. Clin. Pathol. 88:358360.
4. Espy, M. J.,, T. F. Smith,, M. W. Haromon,, and A. P. Kendal. 1986. Rapid detection of influenza virus by shell vial assay with monoclonal antibodies. J. Clin. Microbiol. 24:677679.
4a.. Fouchier, R. A.,, N. G. Hartwig,, T. M. Bestebroer,, B. Niemeyer,, J. C. de Jong,, J. H. Simon,, and A. D. Osterhaus. 2004. A previously undescribed coronavirus associated with respiratory disease in humans. Proc. Natl. Acad. Sci. USA 101:62126216.
5. Frank, A. L.,, R. B. Couch,, C. A. Griffis,, and B. D. Baxter. 1979. Comparison of different tissue cultures for isolation and quantitation of influenza and parainfluenza viruses. J. Clin. Microbiol. 10:3236.
5a.. Gillim-Ross, L.,, J. Taylor,, D. R. Scholl,, J. Ridenour,, P. S. Masters,, and D. E. Wentworth. 2004. Discovery of novel human and animal cells infected by the severe acute respiratory syndrome coronavirus by replication- specific multiplex reverse transcription- PCR. J. Clin. Microbiol. 42:31963206.
6. Gleaves, C. A.,, C. F. Lee,, C. I. Bustamante,, and J. D. Meyers. 1988. Use of murine monoclonal antibodies for laboratory diagnosis of varicella-zoster infection. J. Clin. Microbiol. 26:16231625.
7. Gleaves, C. A.,, T. F. Smith,, E. A. Shuster,, and G. R. Pearson. 1984. Rapid detection of cytomegalovirus in MRC-5 cells inoculated with urine specimens by using low-speed centrifugation and monoclonal antibody to an early antigen. J. Clin. Microbiol. 19:917919.
8. Gleaves, C. A.,, D. J. Wilson,, A. D. Wold,, and T. F. Smith. 1985. Detection and serotyping of herpes simplex virus in MRC-5 cells by use of centrifugation and monoclonal antibodies 16 h postinoculation. J. Clin. Microbiol. 21:2932.
8a.. Hamelin, M. E.,, and G. Boivin. 2005. Human metapneumovirus: a ubiquitous and long-standing respiratory pathogen. Pediatr. Infect. Dis. J. 24:S203S207.
9. Johnston, S. L. G.,, and C. S. Siegel. 1990. Evaluation of direct immunofluorescence, enzyme immunoassay, centrifugation culture, and conventional culture for the detection of respiratory syncytial virus. J. Clin. Microbiol. 28:23942397.
9a.. Ksiazek, T. G.,, D. Erdman,, C. S. Goldsmith, et al. 2003. A novel coronavirus associated with severe acute respiratory syndrome. N. Engl. J. Med. 348:19531966.
10. Landry, M. L.,, and D. Ferguson. 1993. Comparison of quantitative cytomegalovirus antigenemia assay with culture methods and correlation with clinical disease. J. Clin. Microbiol. 31:28512856.
11. Lipson, S. M.,, R. Walderman,, P. Costello,, and K. Szabo. 1988. Sensitivity of rhabdomyosarcoma and guinea pig embryo cell cultures to field isolates of difficult-to-cultivate group A coxsackieviruses. J. Clin. Microbiol. 26:12981303.
11a.. MacPhail, M.,, J. H. Schickli,, R. S. Tang,, J. Kaur,, C. Robinson, et al. 2004. Identification of small-animal and primate models for evaluation of vaccine candidates for human metapneumovirus (hMPV) and implications for hMPV vaccine design. J. Gen. Virol. 85:18551863.
12. Matthey, S.,, D. Nicholson,, S. Ruhs,, B. Alden,, M. Knock,, K. Schultz,, and A. Schmuecker. 1992. Rapid detection of respiratory viruses by shell vial culture and direct staining by using pooled and individual monoclonal antibodies. J. Clin. Microbiol. 30:540544.
13. Minnich, L. L.,, F. Goodenough,, and C. G. Ray. 1991. Use of immunofluorescence to identify measles virus infections. J. Clin. Microbiol. 29:11481150.
14. Olsen, M. A.,, K. M. Shuck,, A. R. Sambol,, S. M. Flor,, J. O’Brien,, and B. J. Cabrera. 1993. Isolation of seven respiratory viruses in shell vials: a practical and highly sensitive method. J. Clin. Microbiol. 31:422425.
15. Paya, C. V.,, A. D. Wold,, and T. F. Smith. 1987. Detection of cytomegalovirus infections in specimens other than urine by the shell vial assay and conventional tube cell cultures. J. Clin. Microbiol. 25:755757.
15a.. Peret, T. C.,, G. Boivin,, Y. Li,, M. Couillard,, C. Humphrey, et al. 2002. Characterization of human metapneumoviruses isolated from patients in North America. J. Infect. Dis. 185:16601663.
16. Proffitt, M. R.,, and S. A. Schindler. 1995. Rapid detection of HSV with an enzymelinked virus inducible system (ELVIS™) employing a genetically modified cell line. Clin. Diagn. Virol. 4:175182.
16a.. Rogers, J.,, S. Rohal,, D. Carrigan,, S. Kusne,, K. Knox,, T. Gayowski,, M. Wagener,, J. J. Fung,, and N. Singh. 2000. Human herpesvirus 6 in liver transplant recipients: role in pathogenesis of fungal infections, neurologic complications, and outcome. Transplantation 69:25662573.
17. Schirm, J.,, J. M. Janneke,, G. W. Pastoor,, P. C. vanVoorst Vader,, and F. P. Schroder. 1989. Rapid detection of varicella-zoster virus in clinical specimens using monoclonal antibodies on shell vials and smears. J. Med. Virol. 28:16.
18. Schmidt, N. J.,, H. H. Ho,, and E. H. Lennette. 1975. Propagation and isolation of group A coxsackieviruses in RD cells. J. Clin. Microbiol. 2:183185.
19. Smith, M. C.,, C. Creutz,, and Y. T. Huang. 1991. Detection of respiratory syncytial virus in nasopharyngeal secretions by shell vial technique. J. Clin. Microbiol. 29:463465.
20. Stabell, E. C.,, S. R. O’Rourke,, G. A. Storch,, and P. D. Olivo. 1993. Evaluation of a genetically engineered cell line and a histochemical b-galactosidase assay to detect herpes simplex virus in clinical specimens. J. Clin. Microbiol. 31:27962798.
21. Takiff, H. E.,, S. E. Straus,, and C. F. Garon. 1981. Propagation and in vitro studies of previously noncultivatable enteral adenoviruses in 293 cells. Lancet ii:832834.
22. Tobita, K.,, A. Sugiura,, C. Enomoto,, and M. Furuyama. 1975. Plaque assay and primary isolation of influenza A viruses in an established line of canine kidney cells (MDCK) in the presence of trypsin. Med. Microbiol. Immunol. 162:914.
22a.. Van den Hoogen, B. G.,, J. C. de Jong,, J. Groen,, T. Kuiken,, R. de Groot, et al. 2001. A newly discovered human pneumovirus isolated from young children with respiratory tract disease. Nat. Med. 7:719724.
23. Waris, M.,, T. Aiegler,, M. Kivivirta,, and O. Ruuskananen. 1990. Rapid detection of respiratory syncytial virus and influenza A virus in cell cultures by immunoperoxidase staining with monoclonal antibodies. J. Clin. Microbiol. 28:11591162.
24. West, P. G.,, B. Aldrich,, R. Hartwig,, and G. J. Haller. 1988. Increased detection rate for varicella-zoster virus with combination of two techniques. J. Clin. Microbiol. 26:26802681.
1. Frank, A. L.,, R. B. Couch,, C. A. Griffis,, and B. D. Baxter. 1979. Comparison of different tissue cultures for isolation and quantitation of influenza and parainfluenza viruses. J. Clin. Microbiol. 10:3236.
2. Tobita, K.,, A. Sugiura,, C. Enomoto,, and M. Furuyama. 1975. Plaque assay and primary isolation of influenza A viruses in an established line of canine kidney cells (MDCK) in the presence of trypsin. Med. Microbiol. Immunol. 162:914.

Tables

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Table 10.2-1

Monolayer cell cultures used for viral culture

Several viruses (e.g., SARS-CoV, variola virus, highly pathogenic avian influenza virus) that are categorized as BSL 3 or BSL 4 agents can infect commonly used cell cultures. Do not perform viral culture in a BSL 2 setting on specimens collected from individuals suspected of having such infections. Contact your Laboratory Response Network (LRN) reference laboratory or local public health department for testing information regarding these agents. Information for sentinel laboratories can be obtained at http://www.cdc.gov and http://www.asm.org.

Primary or low-passage cell cultures: AGMK, African green monkey kidney; CMK, cynomolgus monkey kidney; HNK, human neonatal kidney; PMK, monkey kidney; RK, rabbit kidney. Semicontinuous (diploid) cell cultures: HLF, MRC-5, WI-38, human embryonic lung fibroblasts. Continuous cell lines: A-549, human epidermoid lung carcinoma; BGMK, buffalo green monkey kidney; CV-1, African green monkey kidney; Graham-293, adenovirustransformed human kidney; H292, human pulmonary epidermoid carcinoma; HEp-2, human laryngeal carcinoma; LLC-MK2, rhesus monkey kidney; MDCK, Madin-Darby canine kidney; ML, mink lung; RD, rhabdomyosarcoma; Vero, African green monkey kidney.

Viral antigen can be detected in the absence of viral replication.

Members of the genus are now classified among five species, including polioviruses and human enteroviruses (HEV) A, B, C, and D. HEV-A includes coxsackieviruses A2 to A8, A10, A12, A14, and A16 and enterovirus 71. HEV-B includes coxsackieviruses A9 and B1 to B6; echoviruses 1 to 7, 9, 11 to 21, 24 to 27, and 29 to 33; and enteroviruses 69 and 73. HEV-C includes coxsackieviruses A1, A11, A13, A15, A17 to A22, and A24. HEV-D includes enteroviruses 68 and 70. Several coxsackie A viruses cannot be cultivated in cell culture.

Citation: Garcia L. 2010. Selection, Maintenance, and Observation of Uninoculated Monolayer Cell Cultures, p 20-30. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch10.2
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Table 10.2-2

Viral culture systems and availability

Has been cultivated in human lung fibroblast shell vial cultures ( ).

Need to consider vaccination and BSL 3/BSL 4 recommendations.

Animal inoculation may supplement cell culture for isolation of some agents and may be more sensitive than cell culture inoculation in some cases.

Citation: Garcia L. 2010. Selection, Maintenance, and Observation of Uninoculated Monolayer Cell Cultures, p 20-30. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch10.2
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Table 10.2-3

Assessment of monolayered cell cultures and troubleshooting

Should be clear and orange-red to cherry pink (pH 6.8 to 7.2).

Perform Gram stain of sediment obtained from centrifuged culture supernatant; inoculate broth medium (e.g., THIO, TSB) or agar (e.g., blood, Sabouraud dextrose) with sediment and culture medium, and observe for bacterial or fungal growth for 10 to 14 days.

Monolayered cell cultures should consist of a single layer of adherent cells exhibiting normal morphology at or near confluency (100% monolayer). Monolayers that are slightly subconfluent are preferred for viral culture.

Mycoplasmal contamination can have a profoundly detrimental effect on viral isolation. Contamination with these organisms frequently does not result in obvious changes of the cell monolayer but may present as a generalized deterioration of the monolayer; occasionally a CPE mimicking viral growth may be observed. Obtain written certification from the vendor that cultures are mycoplasma free.

Citation: Garcia L. 2010. Selection, Maintenance, and Observation of Uninoculated Monolayer Cell Cultures, p 20-30. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch10.2
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Untitled

Composition of balanced salt solutions and EMEM

PBS, phosphate-buffered saline.

Citation: Garcia L. 2010. Selection, Maintenance, and Observation of Uninoculated Monolayer Cell Cultures, p 20-30. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch10.2
Generic image for table
Untitled

Cell culture medium

Citation: Garcia L. 2010. Selection, Maintenance, and Observation of Uninoculated Monolayer Cell Cultures, p 20-30. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch10.2
Generic image for table
Untitled

Cell culture medium

Citation: Garcia L. 2010. Selection, Maintenance, and Observation of Uninoculated Monolayer Cell Cultures, p 20-30. In Clinical Microbiology Procedures Handbook, 3rd Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817435.ch10.2

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