Since the demonstration of Mycoba Abstract terium leprae murium and its recognition as the causative agent of rat leprosy, this intracellular parasite has been an object of unusual interest in connection with the problem of human leprosy. Like the. human leprosy bacillus, it has thus far defied all efforts at cultivation in vitro, whether in bacteriological media or in cell cultures.
Many microbiologists have attemped to do artificial culture of Mycobacterium leprae.
Hansen in 1872, after this finding of Mycobacterium leprae, was the first person to discover that this micro-organism could cause an infectious disease in the human body. This was 10 years before the discovery of Mycobacterium tuberculosis by Ko. . It is generally known that there are no any succcssful artificial culture meth-
ods of Mycobacterium leprae at the present time.
In the 1940´s, Hanks tried to cultivate the leprae bacilli by means of the tissue culture method extensively and systemically but in 1950, according to his report it was impossible to cultivate the Mycobacterium leprae.
However, Chang i t the National Institute of Health, U.i.A., attempted to culture Mycobacterium laprae murium with the tissue culture method without regarding the report of Hanks. In 1960, the reported the multiplication of Mycobactium leprae murium in the abdominal macrophages of mice.
In 1963, Rees and his associates reported that Mycobacterium leprae were cultured in the fibroblasts of mice and rats. Japanese microbiologist, Nakamura, reported the multiplication of Mycobacterium leprae murium in the testicular fibroblasts in mice.
To date there is no validity of
Ch-Yang´s report, therefore we are trying F_ to do similar experiments to test the
validity of the contribution. At the
same time attempts were made to cul-
tivate Mycobacterium leprae murium ;r
and Mycobacterium leprae with vairous cell cultures of cold blooded animals.
Materials and Methods
Media ;
NCTC-109
YLA (Yeast extract lactoalbumin hydrolysate)
Hanks´s balanced salt solution Medium 199
Beef embryo extract
Horse serum & calf serum Penicillin & streptomycin
Heparin
Glass ware ;
Pyrex Leighton-type culture glass tube
Coverslips
Pasteur pipettes
M air mixture;
Expired air
Host cells;
.Abdominal macrophages from mice and frogs, kidney cells from frogs, Ophicephalus argus and Cyprinus carpio. These cell cultures were used as host cells to cultivate Mycobacterium leprae murium and Mycobacteriumleprae.
Bacteria ;
Mycobacterium leprae murium; Hawaiian strain of murine leprosy was preinoculated in rat testis and fresh murine leprosy granuloma was emulsified in medium 109.
Mycobacterium leprae; a fresh leproma from a untreated lepromatous type of leprosy case was removed and emulsified in medium 109.
Preparation of cell cultures ;
Abdominal macrophages from mice and cold blooded animals. Animals were killed by cervical fracture. The skin of the whole animal was sterilized with alcohol and tincture of iodine, and an opening was made just below the tip of the.xiphoid cartilage. With a bent-tip Pasteur pipette and rubber bulb, the peritoneal cavity was irrigated at first with 4 to 6 ml of Hanks´s balanced salt solution and later NCTC 109 medium containing heparin, diluted 1:20,000. To avoir injury and hemorrhage, the pipette was cautiously inserted and pushed gently into the pelvis with the tip facing the abdominal wall. The intestines were moved from side to side with the pipette while the xiphoid was lifted with forceps to prevent overflow of the irrigating solution. This made it possible to obtain a well-distributed cell suspSnsion. The pipette was inserted in the right side
and the suspension aspirated from the area between the liver and the abdominal wall. Three -to 5 ml of suspension, containing approximately 2x 106 cultivable cells, was generaly obtained from each animal. The suspensions were not centrifuged or washed. Sometimes pooled suspensions were made from several animals.
Kidney cells from frog, Ophicephalus argus and Cyprinus carpio were prepared by removing kidnies aseptic-ally minsed with sissors and trypsinized. These trypsinized cells were resuspended in medium 109.
Methods of implanting culture;
Emulsions of Mycobacterium leprae murium and Mycobacterium leprae in medium 109 were introduced into those cell suspension. 1 ml of cell suspension inoculated with the organ-isms was placed in a Leighton tube containing the coverslip.
After 1 to 3 hours at 37C in a CO2 condition, the supernatant fluid was replaced with fresh medium. During
this time the viable cells attached themselves to the glass while the lymphocytes, which generaly do not stick to the glass, were almost entirely removed. The medium was changed twice a week.
The cell cultures were carefully observed twice a week under weak magnification and a coverslip was taken out each week for a stained specimen.
The coverslip was stained in fuchsia ´ hematoxylin stain and the multiplication of the organisms was examined under microscopy.
Results;
Mice macrophages, obtained froi t the peritoneal exudate, can be maintained in good condition for 26 days . in a medium consisting of 90 per cent
horse serum, 10 per cent Medium NCTC-109 and 10 per cent beef embryo extract, provided that the pH is carefully maintained at 7.2.
The peritoneal exudate cells usually become active within 1 to 3 hours and most of the bacilli are phagocytized within this time. The macrophages inoculated with Mycobacterium leprae murium began to elongate gradually, the process being completed in about ten days and there was a definite sign of multiplication thereafter. The first signs of the growth of the Mycobacterium leprae murium in the culture is elongation, which is obtained as early as in 10th day; maximum length
usually is seen after 2 weeks. The length of the bacilli is sometimes remarkably long.
Mouse macrophages are found to be suitable host cells for Mycobacterium leprae murium. In orie experiment, the cells were harvested at the 30th day and shaken throughly until they were homogenized. Intraperitoneal injection of the homogenized material
in. mice at this point resulted in proirressive murine leprosy within 2 to months.
Mycobacterium, leprae inoculated in macrophages from the abdominal cavity of mice did not show any signs of elongation neither multiplication which was observed in Mycobacterium leprae murium.
Cells from frogs, Ophicephalus argus and Cyprinus carpio have not been successfully cultured in tissue culture long enough to offer suitable conditions to those organisms. The cells ´from those cold blooded animals can maintain their life- span only about, a week in tissue culture.
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