Jpn.J.Leprosy 67, (1998)

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1 Jpn.J.Leprosy 67, (1998)

2 Fig.1. Macroscopic appearance of the cultured slides stained by the Ziehl-Neelsen method. Up to bottom: kept at 4 C, 18 weeks, cultured for 6 weeks, 8 weeks, 10 weeks, 12 weeks and 18 weeks, respectively Fig.2. Light microscopic features of the peripheral area of a smear of M. leprae cells. Top: held at 4 `C for 12 weeks. Bottom cultured at 30 C for 12 weeks ( Magnification x 40). Fig.3. Scanning electron micrographs of the central area of the smear. Top: held at 4 `C for 12 weeks. Bottom: cultured at 30 C for 12 weeks (Magnification x 2,000) 288

3 Jpn.J.Leprosy 67, (1998) Fig.4. Estimation of the growth by PCR products. (1) Smear kept at 4 'C: 2 weeks, undiluted, (2) 2-fold diluted Lane 1-sample, (3) Smear cultured at 30 `C 2 weeks, undiluted, (4) 2-fold diluted Lane 3-sample, (5) Smear cultured at 30 t: 4 weeks, undiluted, (6) 2-fold diluted Lane 5 sample, (7) Smear cultured at 30 t: 6 weeks, undiluted, (8) 2-fold diluted Lane 7 sample, (9) Smear cultured at 30 `C: 6 weeks, undiluted, (10) 2-fold diluted Lane 9 sample, Samples in Lane 7 and 9 were from duplicate slides cultured under the same condition. Fig.5. The amount of ATP extracted from the cells of M. Ieprae smeared on a silicon-slide at periodic inter vals during cultivation (n=7).

4 1) Nakamura,M.: Optimal ph for preserving the activity of M.leprae during incubation of cells in a cell-free liquid medium. Intl. J. Lepr., 61: (1995). 4) Nakmaura, M. : Multiplication of M.lepraemurium in cell-free medium contain ing a-ketoglutaric acid and cytochrome c. J.Gen. Microiol., 73: (1972). 6) Woods,S.A. and Cole, S.T.: A rapid method for the detection of potentially viable Myco bacterium leprae in human biopsis: a novel application of PCR. FEMS Microbiol. Lett. 65: (1980).

5 Jpn.J1eprosy 67, (1998) Morphological features to be considered as the growth of Mycobacterium leprae Thai-53 strain on a silicon coated slide in a cell-free liquid medium Masahiro Nakamura * and Masanod Matsuoka** ' Koga Hospital Medical Research Institute " National Institute of Infectious Diseases, Leprosy Research Center (Received 1 June 1998/ Accepted 7 Jul 1998) Key words: Cultivation of Mleprae, Cell-free liquid medium, Slide glass culture method Morphological findings of the cells of Mycobacterium leprae Thai-53 strain smeared on a silicon-coated slide cultured in Kirchner liquid medium,ph 7.0, enriched with adenosine, egg yolk, folinic acid, vitamin K3, lecithin, and N-acethylglucosamine at 30 t were demonstrated. On the basis of the results with exquisite morphological growth patterns and the increase in the amount of tempelate DNA prepared from the cultured cells, it is evident that the cells of Mleprae are capable of multiplication under cell free in vitro conditions. The reason why the ATP content did not increase in parallel with morpho logical features and the increase in the DNA is presumably that the multiplication of Mleprae in this culture system was supported only by consuming the energy derived from the infected host cells.

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