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1.
Nihon Hansenbyo Gakkai Zasshi ; 70(3): 127-33, 2001 Aug.
Artigo em Japonês | MEDLINE | ID: mdl-11579510

RESUMO

Our previous paper reported that the intracellular ATP content in cells of M. leprae consistently increased in the medium containing adenosine after 4-6 weeks of cultivation and decreased thereafter. The reason why ATP generation ceased 4-6 weeks after cultivation is not clear, but it was determined that the termination in ATP generation was not a result of deterioration in the culture medium during cultivation because a renewal trial of the old culture medium by freshly prepared culture medium had no effect further maintenance or progressive increase in ATP generation. From the results obtained in a renewal trial of the culture medium, I would like to speculate that the reason why M. leprae cells do not multiply in vitro might be due to the characteristic property of the cell wall of M. leprae, i.e., fragility.


Assuntos
Mycobacterium leprae/crescimento & desenvolvimento , Trifosfato de Adenosina/metabolismo , Parede Celular/fisiologia , Meios de Cultura , Mycobacterium leprae/citologia , Mycobacterium leprae/metabolismo
2.
Int J Lepr Other Mycobact Dis ; 69(1): 13-20, 2001 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11480311

RESUMO

The ATP generation in cells of Mycobacterium leprae Thai-53 strain takes place in vitro when the cells are cultivated in Kirchner liquid medium, pH 7.0, enriched with egg-yolk solution, pyruvate, transferrin, and adenosine at 30 degrees C. Among the supplements, adenosine was key and critical for the ATP generation. The optimal concentration of adenosine was 50 micrograms/ml of the medium. ATP generation, however, was limited; the rates of increase in ATP content extracted from the cells were approximately two- to threefold compared to that of the starting samples, and the increase reached a maximum at 4 or 6 weeks after incubation. No significant ATP generation in M. leprae cells was demonstrated in medium at pH 6.2 or pH 6.6, in the original Kirchner medium with or without adenosine, or when cultured at 37 degrees C, or when containing an antileprosy drug. No detectable increase in the number of M. leprae cells was observed with the increase in intracellular ATP content and DNA replication. No effect was seen with renewal of the cultured medium by freshly prepared medium at 6 weeks' cultivation on the progressive ATP generation in M. leprae.


Assuntos
Trifosfato de Adenosina/biossíntese , Adenosina/metabolismo , Mycobacterium leprae/metabolismo , Animais , Contagem de Colônia Microbiana , Meios de Cultura , DNA Bacteriano/análise , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mycobacterium leprae/genética , Mycobacterium leprae/crescimento & desenvolvimento , Reação em Cadeia da Polimerase , Temperatura , Fatores de Tempo
3.
Nihon Hansenbyo Gakkai Zasshi ; 68(3): 157-63, 1999 Nov.
Artigo em Japonês | MEDLINE | ID: mdl-10659611

RESUMO

Since the success of the multiplication of Mycobacterium lepraemurium in cell-free liquid medium by the author in 1972, various factors affecting its growth have been reported. In particular, it was emphasized that the key essential factor for the growth was alpha-ketoglutaric acid(alpha-kg). However, recent data now indicate that the critical factor for the growth of M. lepraemurium is not alpha-kg, but the optimal pH of the liquid culture medium, quite similar to the condition of egg-yolk solid medium. The recent experimental results clearly indicated that an abundant multiplication of M. lepraemurium took place in an acid liquid medium containing egg-yolk extract, without alpha-kg. This finding means that the cells of M. lepraemurium can multiply in a liquid medium, if the pH of the medium is adjusted to 6.0-6.2. The reason why the cells of M. lepraemurium multiplied in NC medium, and NC-5 medium, containing alpha-kg was that the pH of neutral liquid base medium of the NC or NC-5 medium, was adjusted to 6.0-6.2, optimal for the growth of M. lepraemurium by addition of the 10% alpha-kg (where 10% alpha-kg is strongly acidic; pH:1.22). Other supplements, such as cytochrome c, l-cysteine, and hemin, which are routinely added to the base medium of NC and NC-5 medium, have shown stimulatory effects on multiplication of M. lepraemurium, rather than a key essential effect. In addition, recent experimental data have suggested a second key essential component for growth present in egg-yolk extract.


Assuntos
Meios de Cultura/química , Mycobacterium lepraemurium/crescimento & desenvolvimento , Animais , Sistema Livre de Células , Concentração de Íons de Hidrogênio , Ácidos Cetoglutáricos , Camundongos
4.
Nihon Hansenbyo Gakkai Zasshi ; 67(2): 287-91, 1998 Jul.
Artigo em Japonês | MEDLINE | ID: mdl-9785844

RESUMO

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-acetylglucosamine at 30 degrees C 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 M.leprae are capable of multiplication under cell-free in vitro conditions. The reason why the ATP content did not increase in parallel with morphological features and the increase in the DNA is presumably that the multiplication of M.leprae in this culture system was supported only by consuming the energy derived from the infected host cells.


Assuntos
Meios de Cultura , Mycobacterium leprae/crescimento & desenvolvimento , Silício , Animais , DNA Bacteriano/análise , DNA Bacteriano/biossíntese , Camundongos
5.
Nihon Rai Gakkai Zasshi ; 65(2): 94-9, 1996 Jul.
Artigo em Japonês | MEDLINE | ID: mdl-8810561

RESUMO

In the previous paper, it was indicated that the activity (ATP values extracted from collected cells) of Mycobacterium leprae could be maintained in the phosphate buffer (pH 7.0) containing fetal calf serum (10%) for more than 4 weeks under the incubation at 30 degrees C. The present paper describes that the activity of cells of M.leprae is prolonged and somewhat stimulated when glycerin and dextran are added to the buffer serum system. The optimal concentrations for glycerin and dextran are 2% and 1%, respectively. In addition, it is found that the dextran of MW. 200,000-300,000 is much more effective than that of MW. 100,000-200,000.


Assuntos
Dextranos/farmacologia , Glicerol/farmacologia , Mycobacterium leprae/crescimento & desenvolvimento , Meios de Cultura , Relação Dose-Resposta a Droga , Peso Molecular
6.
Nihon Rai Gakkai Zasshi ; 64(2): 119-23, 1995 Jul.
Artigo em Japonês | MEDLINE | ID: mdl-7592160

RESUMO

Evidence was presented in the previous paper that the activity of cells of Mycobacterium leprae was maintained in the phosphate buffer(pH 7) containing fetal calf serum (10%) with/without glycerin (2%) for approximately 4 weeks during incubation of cells at 30 degrees C, when the inoculum having more than 3,000 pg ATP was used. In the present paper, it is confirmed that the definite inoculum sizes are essential for obtaining the reproducible results that are described above, by using the inocula containing more and less than 3,000 pg ATP.


Assuntos
Mycobacterium leprae/crescimento & desenvolvimento , Trifosfato de Adenosina/análise , Animais , Bovinos , Contagem de Colônia Microbiana , Meios de Cultura , Concentração de Íons de Hidrogênio , Mycobacterium leprae/metabolismo
7.
Int J Lepr Other Mycobact Dis ; 63(1): 28-34, 1995 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-7730716

RESUMO

The effect of the pH of a cell-free liquid medium on the activity of Mycobacterium leprae during incubation of the cells was investigated. As a parameter for evaluating the activity, the amount of adenosine triphosphate (ATP) extracted from the incubated cells collected by centrifugation was measured. The results demonstrate that the activity of M. leprae cells was maintained at a significant level for approximately 4 weeks at 30 degrees C in 0.05 M phosphate buffer containing 10% fetal calf serum at pH 7.0 compared to cells at other pHs tested, but activity was not preserved in phosphate buffer at pH 7.0 without serum and incubated at 37 degrees C. The maintenance of the activity under these conditions was prolonged somewhat by the addition of glycerin (2%) to the medium, and was definitely inhibited by rifampin but not by either penicillin or isoniazid. From the results reported here, it could be postulated that the optimal pH of cell-free media for the study of cultivation of M. leprae is 7.0.


Assuntos
Mycobacterium leprae/fisiologia , Trifosfato de Adenosina/análise , Animais , Antibióticos Antituberculose/farmacologia , Meios de Cultura , Glicerol/farmacologia , Concentração de Íons de Hidrogênio , Camundongos , Camundongos Endogâmicos C3H , Camundongos Nus , Técnicas Microbiológicas , Mycobacterium leprae/efeitos dos fármacos , Temperatura
8.
Nihon Rai Gakkai Zasshi ; 63(3): 99-104, 1994 Nov.
Artigo em Japonês | MEDLINE | ID: mdl-7730213

RESUMO

Effect of chloroform on the extraction of ATP from the cells of Mycobacterium leprae was investigated. The results demonstrate that the yields of ATP from the resting cells of M. leprae were much more higher when 100mM Tris-EDTA was added to the procedure than that of the routine method. The amounts of ATP extracted from the cells of M. leprae incubated either in 10% calf serum-buffer (pH 7) or 10% calf serum-2% glycerin-Dubos medium at 30 degrees C gradually increased with time of incubation when chloroform and 100mM-Tris-EDTA were used for extraction of ATP, whereas no increase and periodical decrease of ATP were observed when Tris-EDTA only without chloroform was used in the procedure of ATP extraction. Therefore, it is noted that the use of chloroform and 100mM-Tris EDTA is indispensable for extraction of ATP from the cells of M. leprae.


Assuntos
Trifosfato de Adenosina/isolamento & purificação , Clorofórmio , Mycobacterium leprae/química , Ácido Edético
9.
Nihon Rai Gakkai Zasshi ; 63(2): 47-50, 1994 Jul.
Artigo em Japonês | MEDLINE | ID: mdl-7844062

RESUMO

For elimination of contaminants in a homogenate of nude-mouse footpad infected with M.leprae, the pretreatment with trypsin was tested. The results obtained indicated that the final sterile product was satisfactorily obtained, if the pretreatment with trypsin 0.05%, 37 degrees C, 60min to the homogenate was carried out before the treatment with sodium hydroxide 1%, 37 degrees C, 15min. This procedure might contribute to make a suspension of partially purified M.leprae from the infected tissues, because both the treatments with trypsin and sodium hydroxide could eliminate the infected tissues surrounding M.leprae as well as any contaminants, except other mycobacteria, present in a tissue homogenate.


Assuntos
Hanseníase/microbiologia , Mycobacterium leprae/isolamento & purificação , Animais , Técnicas Bacteriológicas , , Camundongos , Camundongos Nus , Hidróxido de Sódio , Tripsina
10.
Int J Lepr Other Mycobact Dis ; 61(3): 415-20, 1993 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-8228440

RESUMO

Although the viability of Mycobacterium leprae suspended in distilled water with or without 10% fetal calf serum was reduced approximately 10(-2) to 10(-4) from that of the starting material during the process of lyophilization, bacilli capable of multiplication in nude mouse foot pads were found in the lyophilized samples stored for 4 years at 4 degrees C. The multiplication rate of the lyophilized bacilli which were suspended in 10% serum-water was much higher than that of the bacilli suspended in water only. On the other hand, no reduction of the viability of M. leprae suspended in 10% skim milk-water was demonstrated during the process of lyophilization as well as storage for 2 years at 4 degrees C. From the results obtained here, it could be suggested that M. leprae might be preserved in vitro by means of lyophilized M. leprae was extremely stable during cryopreservation when the bacilli were suspended in 10% skim milk-water. Therefore, the composition of the solution for suspending the bacilli is definitely critical for the maintenance of M. leprae viability by means of lyophilization.


Assuntos
Hanseníase/microbiologia , Mycobacterium leprae/crescimento & desenvolvimento , Preservação Biológica/métodos , Animais , Feminino , Liofilização , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Leite , Água
11.
Nihon Saikingaku Zasshi ; 46(2): 533-9, 1991 Mar.
Artigo em Japonês | MEDLINE | ID: mdl-1712050

RESUMO

Since the establishment of Gram stain by H.C.Y. Gram in 1884, it has been widely and routinely used as an aid for differentiation of bacteria. The bacteria are divided into three categories by the staining properties; Gram-positive, -negative, and -indefinite. All the text books in the world describe that mycobacteria such as M. tuberculosis are Gram-positive. By the merest chance, however, it was found that M. lepraemurium grown in tissues was not stained by the routinely used Gram staining method. Therefore, we tried to stain some of the mycobacteria by the Gram staining procedure which is widely used at present. The results obtained indicated that the mycobacteria tested were divided into three groups; the unstainable group such as M. leprae and M. lepraemurium, the Gram-positive and difficult-to-stain group which involves such slow growing mycobacteria as M. tuberculosis, M. avium, and M. intracellulare, and the Gram-indefinite group which contains such rapid growing mycobacteria as M. phlei, M. smegmatis, and M. chelonae. However, if Gram stain is carried out by the heating procedure at the first staining step, all the mycobacteria would become Gram-positive. Therefore, we emphasize that Gram staining of mycobacteria should be performed by the heating procedure.


Assuntos
Temperatura Alta , Mycobacterium/fisiologia , Coloração e Rotulagem
12.
Acta Leprol ; 7 Suppl 1: 36-8, 1989.
Artigo em Inglês | MEDLINE | ID: mdl-2504002

RESUMO

In an effort to preserve Mycobacterium leprae in vitro, the effect of freezing and drying, i.e., lyophilization, on viability of M. leprae was studied. The viability of the bacilli was quantitatively measured with foot-pad inoculation method using nude mouse. The results obtained demonstrate that the viability of M. leprae was reduced approximately 10(-2) to 10(-3) from that of the starting material, during the process of lyophilization; no viable bacilli were detected in the lyophilized sample containing less than 1.8 X 10(3) bacilli. On the other hand, the bacilli capable of multiplication in nude mouse foot-pads were found in the lyophilized sample with more than 10(5) bacilli. From the results obtained here, it could be suggested that there might be a possibility to preserve M. leprae in vitro by means of lyophilization.


Assuntos
Mycobacterium leprae/crescimento & desenvolvimento , Preservação Biológica/métodos , Animais , Liofilização , Camundongos , Camundongos Nus , Mycobacterium leprae/citologia , Mycobacterium leprae/isolamento & purificação
14.
Int J Lepr Other Mycobact Dis ; 53(1): 52-5, 1985 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-3889191

RESUMO

It was demonstrated that the virulence of Mycobacterium lepraemurium could be maintained in vitro for 30 years when the bacilli from the infected subcutaneous mouse tissue were suspended in 10% bovine serum-water, frozen, dried, and stored in a refrigerator. However, it was noted that a complete loss of virulence occurred when the bacilli were suspended in saline. Thus, the selection of the suspending solution is of the utmost importance in maintaining bacterial virulence by lyophilization.


Assuntos
Liofilização , Mycobacterium lepraemurium/fisiologia , Animais , Liofilização/métodos , Camundongos , Mycobacterium lepraemurium/patogenicidade , Virulência
17.
Int J Lepr Other Mycobact Dis ; 50(4): 480-7, 1982 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-6763005

RESUMO

Confirmation experiments on colony formation of M. leprae on the M-Y 14b agar medium developed by Murohashi and Yoshida were carried out for two years independently by individual members of an organized research group, according to the method described by Murohashi and Yoshida. The results obtained can be summarized as follows: a) No colony production by M. leprae on M-Y 14b agar medium was seen. b) No increase in the number of cells of M. leprae on M-Y 14b agar medium during cultivation was seen. c) Light and electron microscopic observation indicated that there was an increase in the number of non-solid bacterial cells and ghost cells with time of cultivation. d) It was found by mouse foot pad inoculation that four of six samples of M. leprae used as inocula were definitely viable. e) By means of mouse foot pad inoculation, it was shown that viability of M. leprae inoculated onto M-Y 14b agar medium was lost within approximately seven weeks of cultivation. From these results, can be definitely concluded that there is no evidence indicating that multiplication of M. leprae took place on M-Y 14b agar medium.


Assuntos
Ágar , Mycobacterium leprae/crescimento & desenvolvimento
20.
Int J Lepr Other Mycobact Dis ; 50(2): 193-9, 1982 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-6180993

RESUMO

The growth of M. lepraemurium in cell-free liquid medium was strongly stimulated by addition of DL-aspartic acid at a final concentration of 0.01% to 0.02% to ND and ND-5 media containing dextran and liposome. No effect of DL-aspartic acid was observed when it was added to ND and ND-5 media without dextran and liposome. Optimal pH of the culture medium is critical for the stimulating effect of DL-aspartic acid, and it varies with the composition of the medium; the optimal pH was 6.0 in ND medium containing dextran and liposome (NDLA), and was 6.6 in NDLA medium supplemented with alpha-ketoglutarate, 1-cysteine HCl, hemin, and cytochrome c (NDLA-5). A possible mechanism of the effect of aspartic acid is discussed.


Assuntos
Ácido Aspártico/metabolismo , Meios de Cultura , Mycobacterium lepraemurium/fisiologia , Dextranos/metabolismo , Concentração de Íons de Hidrogênio , Lipossomos/metabolismo
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