Cultivation of mycobacterium leprae murium and mycobacterium leprae by the tissue culture method
[영문]Since the demonstration of Mycobacterium 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 attempted to do artificial culture of Mycobacterium leprae.
Hansen in 1872, after his finding of Mycobacterium leprae, was the first person to discover that this microorganism could cause an infectious disease in the human body. This was 10 years before the discovery of Mycobacterium tuberculosis by Koch. It is generally known that there are no any successful artificial culture methods 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 at the National Institute of Health, U.S.A., attempted to culture Mycobacterium leprae murium with the tissue culture method without regarding the report of Hanks. In 1960, he reported the multiplication of Mycobacterium 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 Chang's report, therefore we are trying to do similar experiments to test the validity of the contribution. At the same time attempts were made to cultivate Mycobacterium leprae murium and Mycobacterium leprae with various cell cultures of cold blooded animals.
Materials and Methods
YLA(Yeast extract lactoalbumin hydrolysate)
Hanks' balanced salt solution
Beef embryo extract
Horse serum & calf serum
Penicillin & streptomycin
Pyrex Leighton-type culture glass tube
CO^^2 air mixture;
Abdominal macrophages from ice 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 Mycobacterium leprae.
Mycobacterium leprae murium; Hawaian 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 ice 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' balanced salt solution and later NCTC-109 medium containing heparin, diluted 1:20,000. To avoid 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 suspension. 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 2 x 10**6 cultivable cells, was generally 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 Ciprinus carpio were prepared by removing kidnies asepticaly 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 suspensions. 1 ml of cell suspension inoculated with the organisms was placed in a Leighton tube containing the coverslip.
After 1 to 3 hours at 37℃ in a CO^^2 condition, the supernatant fluid was replaced with fresh medium. During this time the viable cells attached themselves to the glass while the lymphocytes, which generally 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 fuchsin-hematoxylin stain and the multiplication of the organisms was examined under microscopy.
Mice macrophages, obtained from 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 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 days; 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 one experiment the cells were harvested at the 30th day and shaken throughly until they were homogenized. Intrapsritoncal injection of the homogenized material in mice at this pint resulted in progresaive murine
leprosy within 2 to 3 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.