Cultivation of the Douglas strain of Mycobacterium lepraemurium in continuous culture*

Bull. Org. mond. Sante 11971, 45, 209-212 Bull. Wld Hith Org f Cultivation of the Douglas Strain of Mycobacterium lepraemurium in Continuous Culture* L. gULA 1 & J. DUBINA 2 Cultivation of Mycobacterium lepraemurium in cell-free media has not yet been successful, perhaps because of deterioration of the nutritional quality of the medium with prolonged incubation at 370C. Trials with a continuous culture technique, involving regular change ofa simple liquid medium preparedfrom human placenta, demonstrated activefission of the Myco. Iepraemurium cells. Abundant growth in the form by macroscopically visible colonies has not been achieved even after prolonged incubation up to 8 months. The results of attempts to cultivate Mycobacterium leprae and Myco. kepraemurium in cell-free media are not yet very encouraging. Only Rees & Wong (1958) and Hart & Valentine (1960) have succeeded in ob- taining elongation of the acid-fast rods, using a spe- cial liquid medium with low pH, but without obtain- ing active multiplication of the strain. These experi- ments were repeated by Nakamura (1968) who con- firmed Hart & Valentine's observations and stressed the necessity for acidity of the medium, freshness of the inoculum, and low oxygen tension. It is well known that the metabolism of Myco. lepraemurium is very slow and that it requires on an average 14-21 days for cell division. During this period, a medium incubated at 37°C may deteriorate and eventually become completely unsuitable for promoting growth. One way of overcoming this difficulty would be to maintain fresh nutritional medium throughout the whole period of extended incubation, perhaps for 6-12 months. The techniques used in our previous successful studies of the cultivation of Mycobacterium tubercu- losis in continuous liquid culture were applied to the growth of Myco. lepraemurium. MATERIALS AND METHODS Culture vessel The same culture vessel was employed as had been used previously for growing Myco. tuberculosis. The * Supported by a grant from the World Health Organi- zation. 1 Chief, WHO International Reference Centre for the Diag- nosis of Tuberculosis, Prague, Czechoslovakia. 2 Research Assistant, Tuberculosis Research Institute, Prague, Czechoslovakia. design of the vessel is shown schematically in Fig. 1. The device consists of a cultivation chamber of approximately 800-ml capacity with a convex bot- tom, enabling the medium to be changed without the loss of many bacteria. On one side a tube supplies medium to the flask, and on the other side a dropping device regulates the discharge of the medium. Media The medium used was a simple liquid medium prepared from the human placenta as recommended by Weiszfeiler (1969) for growing mycobacteria. This medium was prepared as follows: 1 kg of placenta tissue without membrane was ground and 4 000 ml of 5% H2SO4 were added. The hydrolysis was carried out at 130°C for 3 hours. After filtration, the placenta digest was stored at 4°C. Using the placenta digest, the medium was made up as follows: Acid digest of placenta 100 ml Distilled water 900 ml Sodium chloride 5 g Glycerol 50 ml The pH was adjusted to 7.4 with 30% NaOH and the mixture was autoclaved for 30 min at 120°C. After cooling, the medium was again filtered and sterilized. For some experiments the original Hart's liquid medium (Hart & Valentine, 1960) was also used but elongation of the rods was not always achieved. Strain of inoculum The " Douglas " strain of Myco. kepraemurium, in the form of mouse liver suspension, was used.' The 1 Kindly supplied by Dr Rees, National Institute for Medical Research, Mill Hill, London, England. 2718 209 L. SULA & J. DUBINA Fig. 1. Cultivation flask for continuous culture of mycobacteria. 1, Pipette for regulating the influx of the medium; 2, neck of the cultivation flask for introducing the inoculum; 3, constant-level device; 4, dropping device to regulate the discharge of the medium into the collecting flask. For the cultivation of Mycobacterium lepraemurium the seeds are placed at the bottom of the flask. original suspension received from London contained approximately 1.6 x 1010 - 1.8 x 1011 viable units per ml. This suspension was diluted 1 10 and was inoculated intravenously into " Zverez" strain mice in amounts of 0.2 ml. The mice were killed with ether 6 months after inoculation. Small pieces of liver from 20 mice were frozen for 2 hours and were then crushed in a porcelain dish. Unwanted tissue fragments were removed by slow centrifugation and washing with saline. The local suspension thus obtained contained approximately 5 x 108-5 x 109 viable units per ml. The culture media were inoculated with 1 ml of the local suspension and were incubated at 37°C for 1-8 months. As there were no visible colonies even after 8 months' incubation all the cultures were centrifuged. After centrifugation the deposit was resuspended in 1 ml of saline and this was used for continuous cultures. For electron microscopy the material obtained after 15 min centrifugation at 4000 rev/min was fixed with 1% OSO4 in acetate-barbital buffer. Further centrifugation for 15 min at 4 000 rev/min was followed by 3 washes with saline and centrifuga- tion at 4 000 rev/min for 15 min after each wash. RESULTS Altogether 3 continuous cultures ofMyco. leprae- murium incubated at 37°C for 1-8 months were 210 Fig. 2. Suspension of the Douglas strain of Mycobacterium lepraemurium prepared from mouse liver injected with material supplied by Dr Rees. Small longitudinally oriented clumps of short acid-fast bacilli, mostly of homogeneous appearance. Fig. 3. Distinct elongation of acid-fast rods in a 1 -month- old static culture in Hart's medium (x 750). Fig. 4. Single acid-fast rod of Mycobacterium lepraemurium cultured for 1 month in the placenta medium (x 22 500). . tr' %JX , ,. 'I t^ * a. -1i '.:.. ft. 1'44, s S e P w itn~~~~~~~~~~~~~~~i 'ltThr '. i' ' W: >>Y','"CjX' 1,,$ tRA ,"s '\ e'; >> ¢s. ls''' ;* .v-rki$$t; w >* 4t, AVt i ... .. ..... I Fig. 5. Incomplete fission and characteristic curving of the Douglas strain of Mycobacterium lepraemurium cultured in placenta medium (x 31 200). -p A' dit 4{' A l~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~A 'i'i'IS-0 ' '.. , j , 0-:: : 72 iR|w.,,fT A..T4| | E .............................. w s .... ,;7. / r i; : ff.ff: .. ,'h AW F050~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Douglas strain of Mycobacterium lepraemurium cultured in placenta mediumFig. 6. Complete fission of the( x 34 500). CULTIVATION OF MYCOBACTERIUM LEPRAEMURIUM IN CONTINUOUS CULTURE performed. For a 1-month culture the medium was changed every week; for cultures of 2 or more months the medium was changed once a month. The culture remained transparent during the whole period of incubation; some of the culture vessels, after a period of incubation of 3-8 months, showed a fine powder deposit. No colonies, in the form of either small granules or a surface pellicle, were visible macroscopically as with the typical mycobacteria, Myco. tuberculosis and Myco. bovis, even when the incubation was extended to 8 months. Cultures incubated for 1 month were observed with the light microscope and the electron micro- scope and the results are illustrated in Fig. 2-6. For comparison, a photomicrograph showing the results of culture for I month in Hart's elongation medium is included. The suspension prepared from mouse liver is char- acterized by short acid-fast rods that are not dis- tinctly granular; some of them are very thin, faintly acid-fast, with homogeneous cytoplasm (Fig. 2). Most of the rods are arranged in small longitudinal clumps. After one month's incubation in Hart's medium (Fig. 3) only a few small clumps of acid-fast rods and isolated granules are found with- out distinct signs of multiplication. After continuous culture, for 1 month some of the rods are distinctly elongated and this is clearly demonstrated in Fig. 4. Active multiplication of Myco. lepraemurium in placenta medium is shown in Fig. 5 and 6. In Fig. 5 the fission of the cells is not yet completed and indivi- dual rods in the process of fission are still connected with thin cytoplasic bridges. In Fig. 6 division of the cells is complete. Discussion In contrast with the mycobacteria that cause tuber- culosis in humans and in domestic and wild animals and birds, which are easily cultivated on solid and liquid media, Myco. kepraemurium and Myco. leprae are not yet routinely cultivable. Limited multiplica- tion in tissue culture has been observed by many authors (Rees & Wong, 1958; Murohashi & Yoshida, 1970) using tissue culture obtained from spleen explants of infected mice enriched with chicken embryo extract and 20% horse serum. Multiplica- tion of the Myco. lepraemurium was limited to the first 14 days when the cells remained viable, and the medium was changed twice weekly. The rate of multiplication was assayed by counting the number of bacilli in three cultures. No photographic evidence of the multiplication of Myco. lepraemurium was given. In our experiments the bacilli were not counted because the multiple changes of medium caused some loss of mycobacteria from the cultivation flask. Thus the only possible way to demonstrate the multi- plication of the Myco. kepraemurium was by electron microscopy. The technique of continuous culture has not previ- ously been employed for cultivating Myco. lepraemu- rium. B6nicke (1970) developed a special device in the form of U-shaped tubes with a sintered-glass filter that enabled the medium to be changed regu- larly. In this device a limited amount of growth of Myco. keprae was observed after 9-12 months' incu- bation, with a generation time between 20.8 and 41.3 days in a certain number of specimens. No cul- tivation trials with Myco. lepraemurium were under- taken. Thus ours are the first studies using continuous culture techniques for growing Myco. lepraemurium in liquid medium. The first trials made with gula's liquid medium containing 10% bovine serum, which stimulates the growth of Myco. tuberculosis and Myco. bovis, were unsuccessful, perhaps because of the inhibiting effects of serum on endogenous meta- bolism previously observed by Hanks & Gray (1954). After experimenting with Hart's liquid medium applied in static and continuous culture conditions, which led only to the elongation of the rods but not to their active fission, trials were started with the acid digest of human placenta considered by Weiszfeiler (1969) to be a suitable medium for growing mycobacteria in a surface pellicle. This medium is very simple to prepare in large quantities and chromatographic analysis showed 8 different amino acids: leucine, valine, alanine, serine, glycine, cysteine, arginine, and lysine; of these alanine, in particular, proved efficient for growing mycobacteria in deep liquid culture. The addition of other amino acids or their com- pound derivatives, such as asparagine and histidine, not contained in placenta digest led to no improvement in the growth ofMyco. lepraemurium in static cultures. Glycerol was added to the medium as an energy and carbon source, as Tepper (1970) demonstrated that this compound is metabolized by Myco. lepraemu- rium and that carbon from it is incorporated into cells. 211 212 L. §ULA & J. DUBINA ACKNOWLEDGEMENTS Thanks are due to Dr Rees, National Institute for Medical Research, London, England, for providing us with the Douglas strain of Mycobacterium lepraemurium and to Mrs L. Benesovi and Mr Winter for photographic work. R1SUMA CULTURE CONTINUE DE LA SOUCHE DOUGLAS DE MYCOBACTERIUM LEPRAEMURIUM On sait que le m6tabolisme de Mycobacterium leprae- murium est tr6s lent et que le bacille exige en moyenne 14 a 21 jours pour se reproduire. La technique de culture continue mise au point par les auteurs permet de renou- veler r6guli6rement le milieu et de lui conserver ses qualites nutritives pendant plusieurs mois. La culture est r6alise dans un flacon de 800 ml environ, pourvu d'un dispositif pour l'admission de milieu frais et l'evacuation du milieu us6. On a choisi un milieu liquide a base de placenta humain auquel on a incorpore l'inoculum, soit un millilitre d'une suspension de Myco. lepraemurium renfermant de 5 x 108 a 5 x 109 organismes viables. Trois cultures continues ont ete effectu6es, la periode d'incubation variant de 1 a 8 mois, avec renouvelle- ment du milieu chaque semaine ou chaque mois selon le cas. On n'a obtenu aucune colonie visible macroscopique- ment. Cependant, l'examen au microscope electronique du depot, apres centrifugation du mat6riel de culture, a montr6 des images de division cellulaire, t6moins d'une multiplication active de la souche Douglas de Myco. lepraemurium. REFERENCES B6nicke, R. (1970) The present state of growth of Myco. leprae under in vitro conditions. In: Sum- maries of the papers presented at the International Leprosy Colloquium, Borstel, 26-27 August 1970, pp. 29-30 Hanks, J. H., Gray, C. T. (1954) Int. J. Leprosy, 22, 147 Hart, P. D'Arcy & Valentine, R. C. (1960) Nature (Lond.), 185, 58-60 Murohashi, T., Yoshida, K. (1970) Attempts to the cul- tivation of Myco. leprae in cell-free, semi-synthetic media. In: Summaries of the papers presented at the International Leprosy Colloquium, Borstel, 26-27 August 1970, pp. 25-26 Nakamura, M. (1968) Factors affecting elongation of Myco. lepraemurium in vitro. In: Abstracts ofpapers presented at the Ninth International Leprosy Congress, London, 1968, p. 21 Rees, R. J. W. & Wong, P. C. (1958) Nature (Lond.), 181, 359-360 Sula, L. (1963) Bull. Wld Hlth Org., 29, 589-606 Tepper, B. S. (1970) Problems in the cultivation of Myco. leprae. Related cultivation and biochemical studies with Myco. lepraemurium. In: Summaries of papers pre- sented at the International Leprosy Colloquium, Borstel, 26-27 August 1970, p. 28 Weiszfeiler, J. G. (1969) Die Biologie und Variabilitat des Tuberkelbakteriums und die atypischen Mykobakterien, Budapest, Akad6miai Kiad6.