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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/24—Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
  • G01N2333/295—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Chlamydiales (o)

Definitions

• the present invention is concerned with a method for the isolation of chlamydia for diagnosis of chlamydial infection, in which method a clinical sample is inoculated in the cells of a culture cell line, incubated, and the inclusions formed are detected most appropriately photo icroscopically after colouring.
• Chlamydia trachomatis is a prokaryote orga- nism which infects eukaryote cells as an obligatory cell-internal parasite. Chlamydia trachomatis causes several diseases in man, and awareness of its signifi ⁇ cance for the public health is rapidly increasing (Paavonen, J.,1979, "Chlamydial infections. Micro- biological, diagnostic and clinical aspects", Med.Biol. 54:135-151; Paavonen, J., P.
• the McCoy cell line originally used is closely related to the fibroblastic cancer cell line L929 of mice (Blyth, W.A., and J. Taverene, 1972, "Cultivation of TRIC agents: a comparison between the use of BHK-21 and irradiated McCoy cells", J.Hyg. 72: 121-128) .
• McCoy cell line it has been established that many other cell lines are also suscep ⁇ tible to chlamydia (Croy, T.R. , C.C. Kuo, and S.P. Wang, 1975, "Comparative susceptibility of eleven mammalian cell lines to infection with trachoma organ ⁇ isms", J.Clin. Microbiol.
• the cells are usually pre-treated or after-treated so as to prevent their division and to increase the cell-internal division of chlamydia.
• the practical problems related to irradiation have resulted in the use of alternative methods in the . treatment of the cells.
• E cobalt source for the purpose of irradiation.
• all compounds that inhibit the metabolism of cells and all cytotoxic compounds may have some effects of health risk on the laboratory personnel.
• the employment of different cell lines and treatments will establish that the best technique for the isolation of chlamydia has not been discovered as yet.
• the method in accordance with the present invention is mainly characterized in that the sample is inoculated in the cells of a cultured cell line grown out of epithelial cells of an embryo of the mouse species Mus musculus castaneous.
• An epithelial cell is the primary object of a chlamydial infection in man. Since it is difficult to produce non-transformed, pure epithelial cell cul- tures, experimental infection of epithelial cells has been difficult to produce.
• Successful culture of a stable epithelial cell line isolated from the embryo of the mouse species Mus musculus castaneous, MMC-E (Rapp, U.R., J. Keski-Oja, and U.I. Heine, 1979, "Establishment and characterization of the epithelial mouse embryo cell line MMC-E", Cancer Res. 39:4111-4118) and character ⁇ ization of the cell line have permitted a study in vitro of various aspects of such diseases as usually produce al ⁇ terations in the epithelium.
• the MMC-E cell line was originally isolated from an embryo of Mus musculus castaneous and was classified as epithelial on the basis of its morphology. In a MMC-E culture, dense joints and desmosomic struc ⁇ tures are seen when examined as highly enlarged. In imm ⁇ nofluorescence, little quantities of the fibronec- tine of the cell surface are seen and, moreover, out of the cultures, by means of virus transformation, REA
• the cells also contain keratin filaments, which are a specific feature of the epithelial cells (Keski-Oja, J., Lehto, V.-P., and Virtanen I., 1981, "Keratin filaments of cultured mouse epithelial cells are rapidly affected by epidermal growth factor", J. Cell Biol. 90:531-541).
• the MMC-E cells are not tumorigenic in "nude-mice", and therein large quantities of receptors of "epidermal growth factor” (EGF) appear.
• the EGF may stimulate the cells to divide themselves, and produces morphological alterations resembling differen ⁇ tiation (Keski-Oja, J., I.U. Heine, U.R. Rapp, arid B. Wetzel, 1980, "Epidermal growth factor-induced alterations in proliferating mouse epithelial cells", Exp. Cell Res. 128:279-290.
• the "murine sarcoma" growth factors produce a transformed phenotype in these cells (Keski-Oja, J. , J.E. DeLarco, U.R. Rapp, and G.J. Todaro, 1980, "Murine sarcoma growth factors affect the growth and morphology of cultured mouse epithelial cells", J. Cell Physiol. 104:41-46).
• the untreated embryo epithelial cell line MMC-E of Mus musculus castaneous is equally susceptible to infection with Chlamydia trachomatis as the standard McCoy method in which irradiated cells are used, and it possesses remarkable advantages both in clinical routine diagnosis and in_ vitro when studying an experi ⁇ mental chlamydial infection of epithelial cells.
• MMC-E cells are susceptible to infection with
• Chlamydia trachomatis without any pre- or after-treat ⁇ ment are comparable with irradiated McCoy cells, which are usually used for the isolation.
• MMC-E cells treated with cycloheximide permit routine dia ⁇ gnoses of chlamydia infections in ever more and more laboratories and are useful in studies in vitro of the mechanisms related to chlamydial infections of epi ⁇ thelial cells.
• MMC-E cells for the isolation of chlamydia is useful for routine diagnoses of chlamydial infections, at least in such laboratories in which the pre-treatments of McCoy cells are too difficult to perform.
• the use of MMC-E cells thereby permits the performance of chlamydia isolation tests in ever more and more laboratories.
• the MMC-E cells were grown in plastic flasks treated for cell culture (Tissue culture plastic petri dish) in BHK-liquid to which 10 % of calf embryo serum and 50 ⁇ g/mg of gentamycin had been added.
• the origi ⁇ nal cells were detached for passaging with trypsin-EDTA on a weekly basis in the ratio of 1:5.
• About 3 x 10 ⁇ cells were grown in 1.0 ml of nutrient liquid in a plane-bottom plastic tube, which contained a round, 13 mm diameter sterilized covering glass to which the cultured cells adhered.
• the strain used was ____ trachomatis serotype L2 (434Bu) , which had been originally obtained from an
• the cells were incubated for two days at 37°C.
• iodine colouring the nutrient liquid was removed from the tube and 1 ml of methanol was added, and the mix was allowed to stand for 10 minutes at the room temperature.
• the methanol was removed and 1 ml of colouring liquid sold under the trade mark Lugol was added (manufacturer: Merck, Darmstadt) , and the mix was allowed to stand for 20 minutes at the room temperature.
• the colouring liquid was removed and the covering glass was removed out of the tube.
• the counting of the iodine-coloured inclu ⁇ sions was performed by means of the standard technique under microscope (Paavonen, J. , M. Kousa, P. Saikku, E. Vesterinen, E.
• MMC-E cells In stead of untreated MMC-E cells, it is also possible to use, e.g., MMC-E cells treated with cyclo- heximide. The treatment then takes place in the same way as the treatment of McCoy cells.

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• 1981
  • 1981-12-15 FI FI814023A patent/FI63778C/fi not_active IP Right Cessation
• 1982
  • 1982-12-15 WO PCT/FI1982/000063 patent/WO1983002122A1/en not_active Application Discontinuation
  • 1982-12-15 EP EP19830900038 patent/EP0097196A1/en not_active Withdrawn
  • 1982-12-15 JP JP83500105A patent/JPS58502083A/ja active Pending
• 1983
  • 1983-08-12 SU SU833637905A patent/SU1296011A3/ru active

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