1993 Fiscal Year Final Research Report Summary
Elimination of mycoplasmas contaminating cell cultures.
| Project/Area Number |
04557076
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| Research Category |
Grant-in-Aid for Developmental Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Morphological basic dentistry
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| Research Institution | Hokkaido University |
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Principal Investigator |
WATANABE Tsuguo Hokkaido University School of Dentistry Professor, 歯学部, 教授 (10064362)
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| Co-Investigator(Kenkyū-buntansha) |
SHIBATA Ken-ichiro Hokkaido University School of Dentistry Instructor, 歯学部, 助手 (50145265)
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| Project Period (FY) |
1992 – 1993
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| Keywords | Cell culture / Mycoplasma contamination / Elimination of mycoplasmas / Mycoplasma orale / Mycoplasma fermentans / HeLa cell / Incubation temperature |
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| Research Abstract |
Effects of detergents, trypsin, MnCl_2, heating and incubation temperature on viability of mycoplasmas (Mycoplasma orale and Mycoplasma fermentans) most frequently contaminating cell cultures were examined. Based on the results obtained, experiments were carried out to eliminate mycoplasmas from HeLa cells artificially infected with M.orale. As a result, incubation of the contaminated cells at 40。C was suggested to be most effective of all tested, and then the following procedures were established.
1. The contaminated cells (5x10^4) are suspended in 10 ml of DME medium supplemented with 10% FCS (FCS-DME) and incubated at 40。C for 24 hours with vigorous shaking.
2. The cells are sedimented by centrifugation at 1, 000 rpm for 5 min and washed 3 times with 10 ml of FCS-DME.For each washing, a centrifuge tube (15 ml) is changed for a new one.
3. Repeat steps 1 and 2 at least 3 more times.
In the process of studying a variety of properties of M.fermentans, acid phosphatase, purified from cell membranes of the organism, were found to exhibit protein tyrosine phosphatase activity. As the organism penetrates mammalian cells, the enzyme may disturb the tyrosine phosphorylation-dephosphorylation pathway of the cells.
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