Detection of Mycoplasma hominis and Mycoplasma orale in cell cultures by immunofluorescence

Mycoplasma contaminants of animal and human cell cultures were rapidly detected and identified by an indirect immunofluorescent technique. Cells suspected of being contaminated by mycoplasmas were grown as monolayers on chamber slides in a culture medium selected to promote mycoplasmal growth. Before fixation by acetone, the monolayers were subjected to a hypotonic treatment to cause swelling of the mycoplasmas. Detection and identification were then performed by indirect immunofluorescence using rabbit antisera to various mycoplasma species. The correlation between results obtained by the standard isolation procedure and those obtained by this method was very close.

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PRESENT KNOWLEDGE AND EXPERIENCE ON THE STRATEGIES EMPLOYED BY MYCOPLASMA CONTAMINATION OF THE HUMAN CELL CULTURES

CBU International Conference Proceedings ◽

10.12955/cbup.v4.846 ◽

2016 ◽

Vol 4 ◽

pp. 763-766

Author(s):

Nevenka Velickova ◽

Misko Milev ◽

Gorgi Sumanov ◽

Biljana Petrova

Keyword(s):

Cell Culture ◽

Cell Cultures ◽

External Surface ◽

Micronucleus Assay ◽

Mycoplasma Species ◽

Mycoplasma Contamination ◽

Growth Morphology ◽

Aerosol Droplet ◽

Human Cell Cultures ◽

A Cell

Introduction: Mycoplasma species often contaminate cell cultures and other cell-derived biological substances, leading to detrimental effects on the host that include changes in growth, morphology, metabolism and protein synthesis. In cell cultures, mycoplasma are extracellular parasites, usually attached to the external surface of a cell membrane. Many researchers use a mixture of penicillin and streptomycin in the cell culture to prevent contamination. Material and methods: We prepared cell cultures of lymphocytes from peripheral blood of 12 subjects and used micronucleus, assay which is the standard method, for detection of micronuclei in binuclear lymphocytes.Results: Use of standard antibiotics does not protect cell cultures against mycoplasma contamination. Penicillin has no effect on mycoplasma since mycoplasma lack cell wall. Streptomycin inhibits about half the mycoplasma strains but is ineffective against others. In fact, mycoplasma is generally resistant to most antibiotic mixtures commonly used in cell culture. We didn’t find any mycoplasma contamination in the cell culture where penicillin-streptomycin mixture was absent, but confirmed infection in the culture containing mixture of antibiotics.Conclusion: Antibiotics and mixture of antibiotics like penicillin-streptomycin mixture does not protect the cell culture against mycoplasma contamination. Hence, contamination can spread rapidly to other cell lines through aerosol droplet dispersion.

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Treatment of Mycoplasma Contamination in Cell Cultures with Plasmocin

Journal of Biomedicine and Biotechnology ◽

10.1155/2012/267678 ◽

2012 ◽

Vol 2012 ◽

pp. 1-8 ◽

Cited By ~ 23

Author(s):

Cord C. Uphoff ◽

Sabine-A. Denkmann ◽

Hans G. Drexler

Keyword(s):

Cell Lines ◽

Cell Cultures ◽

Culture Medium ◽

Detection Method ◽

Mycoplasma Species ◽

Mycoplasma Contamination ◽

Cure Rate ◽

Mycoplasma Detection ◽

Additional Cell ◽

Limited Period

A high percentage of cell lines are chronically infected with various mycoplasma species. The addition of antibiotics that are particularly effective against these contaminants to the culture medium during a limited period of time is a simple, inexpensive, and very practical approach for decontaminating cell cultures. Here, we examined the effectiveness of the new antimycoplasma compound Plasmocin that has been employed routinely to cleanse chronically infected cell lines. In a first round of treatment 45 out of 58 (78%) mycoplasma-positive cell lines could be cured. In a second attempt using back-up cryopreserved original cells, four additional cell lines were cured; thus, the overall cure rate was 84%. Even if the mycoplasma contamination was not eradicated by Plasmocin, the parallel treatment with several other antibiotics (Baytril, BM-Cyclin, Ciprobay, MRA, or MycoZap) led to the cure of all 58 cell lines. The successful decontamination was permanent as mycoplasmas were no longer detected at day +14 posttreatment and at later time points as examined by PCR which is the most sensitive and specific mycoplasma detection method. Collectively, our results highlight certain antibiotics as effective antimycoplasma reagents and support the therapeutic rationale for their use in the eradication of this notorious cell culture contaminant.

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BIOSYNTHESIS AND SECRETION OF β-THROMBOGLOBULIN BY A HUMAN MEGAKARYOBLASTIC CELL LINE ( MEG-01 )

10.1055/s-0038-1644618 ◽

1987 ◽

Author(s):

M Ogura ◽

N Tanabe ◽

M Hamaguchi ◽

T Hotta ◽

H Saito

Keyword(s):

Cell Line ◽

Culture Medium ◽

De Novo ◽

Immunoblot Analysis ◽

De Novo Synthesis ◽

Serum Free ◽

Cell Lysates ◽

Specific Radioimmunoassay ◽

Immunofluorescent Technique ◽

Indirect Immunofluorescent

β-Thromboglobulin ( βTG ) is a well known platelet specific a-granular protein, but synthesis of βTG by human megakaryocytes has not been fully proved. A human megakaryoblastic cell line ( MEG-01 ) was investigated for the presence of βJG in the culture medium and cell lysates using a specific radioimmunoassay ( RIA ). The concentration of βTG increased with time in the serum-free culture medium as well as in the cell lysates as shown in the following table.By an indirect immunofluorescent technique using a monospesific rabbit anti serum against human βTG, βTG antigen was detected in MEG-01 cells. Immunoblot analysis of culture medium revealed a single band ( mol wt 8,900 ) that is identical to the band of human plasma βTG. De novo synthesis of βTG was demonstrated by the presence of specific immunoprecipitable radioactivity in the medium after 5 h of labeling of the cells with [35S]-methionine as a 8,900 mol wt protein. These results indicate that human megakaryocytes produce βTG, The production of βTG by MEG-01 cells may be useful for the study of megakaryocyte maturation and differentiation.

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Identification of hepatitis a virus in cell cultures by solid phase immune electron microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100142815 ◽

1986 ◽

Vol 44 ◽

pp. 238-239

Author(s):

C.D. Humphrey ◽

T.L. Cromeans ◽

E.H. Cook ◽

D.W. Bradley

Keyword(s):

Electron Microscopy ◽

Liquid Phase ◽

Cell Cultures ◽

Rapid Detection ◽

Hepatitis A ◽

Hepatitis A Virus ◽

Solid Phase ◽

Immune Electron Microscopy ◽

Detection And Identification

There is a variety of methods available for the rapid detection and identification of viruses by electron microscopy as described in several reviews. The predominant techniques are classified as direct electron microscopy (DEM), immune electron microscopy (IEM), liquid phase immune electron microscopy (LPIEM) and solid phase immune electron microscopy (SPIEM). Each technique has inherent strengths and weaknesses. However, in recent years, the most progress for identifying viruses has been realized by the utilization of SPIEM.

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