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Blind passaging

S. Saengamnatdej, Ph.D.

Isolation of a virus in a clinical sample is the goal standard of laboratory diagnosis of viral
diseases. It is the most sensitive and confident method, yet it is difficult, laborious, time-consuming,
and most expensive. Generally, a virus is isolated by inoculation of the sample into a susceptible host
which can be animals, embryonated eggs, or cell cultures. Blind passaging is the best practice in the
isolation of viruses. It is used when the presence of the viral growth is not apparent as being noticed
from observable changes (death, abnormalities, paralysis, cytopathic effects, etc). The unnoticeable
changes are possible, even though a virus is actually present, because viruses do not always multiply to
sufficient numbers in the few days that some systems1, the system which are used to indicate the
presence of virus, can be maintained (As sometimes happens with continuous cell lines, at the end of
one week “ageing” or degeneration of cultures becomes evident also in the inoculated control
cultures.2).
In principle, the first attempted tissues were collected and repassed. The detailed example was
performed as following; freeze the tubes at –20°C, thaw and passage 0.2 ml of culture fluid to tubes
containing fresh monolayers of the same cell type and examine daily for a further 7–10 days (or at least
5 days, as in the supplement polio lab manual) . If cultures show no CPE by this stage, the result is
regarded as negative2 . In the case that the viruses do not grow well in the first type of cell culture and,
therefore, are inoculated to another more permissive cell culture, the process is rather called re-
passaging. For instance, the L20B cells were found to be more sensitive for propagation of poliovirus
than either RD or Hep2 cells3.
The numbers of blind passage are commonly at least three before the absence of virus is
reported1,4.

References

1. Rovozzo, GC. and Burke, CN. (1973) A manual of basic virological techniques. Prentice-Hall,
Inc., USA.
2. World Health Organization: Polio Laboratory Manual WHO/IVB/0410 and Supplement to the
WHO Polio Laboratory Manual, World Health Organization. 4th edition. 2004.
3. Wood DJ, and Hull B. (1999) L20B cells simplify culture of polioviruses from clinical samples.
J Med Virol. Jun;58(2):188-92. (Abstract).
4. Public Heath Agency of Canada (PHAC). Appendix D - Measles Surveillance: Guidelines for
Laboratory Support. : MEASLES VIRUS ISOLATION. Available at http://www.phac-
aspc.gc.ca/publicat/ccdr-rmtc/99vol25/dr2524ee.html, accessed on Aug. 16, 2010.

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