185058a0Nature18547051960010258600028-0836196010.1038/185058a0ukNatureNatureNATUREnatureNature is a weekly international journal publishing the finest peer-reviewed research in all fields of science and technology on the basis of its originality, importance, interdisciplinary interest, timeliness, accessibility, elegance and surprising conclusions. Nature also provides rapid, authoritative, insightful and arresting news and interpretation of topical and coming trends affecting science, scientists and the wider public./nature/journal/v185/n4705issueJournal homeArchiveCurrent issueAdvance online publicationPrivacy policySubscribeNature Publishing GroupCurrent issue185058a0Elongation of a Leprosy Bacillus (Mycobacterium lepraemurium) in a Cell-free Medium
AU  - HART, P. D'ARCY
AU  - VALENTINE, R. C.National Institute for Medical Research, Mill Hill, London, N.W.7. July 19.THE bacillus of human leprosy (Mycobacterium leprae) was one of the first bacteria to be identified as the causative organism of a disease; but it remains to this day among the very few that have failed to grow in any type of culture medium. There is indeed doubt as to whether it has ever even been transmitted to an experimental animal. Much of the laboratory work on leprosy has therefore been concerned with the only other closely related organism, the rat leprosy bacillus (Mycobacterium lepraemurium), which infects rats and mice, causing disease having many of the characteristics of human leprosy; in common with M. leprae it is unusually slow-growing in the body, with at least 10 days elapsing between each generation.An important advance is the recent observation of limited multiplication of M. lepraemurium in tissue culture1"3, but the organism, like M. leprae, has remained uncultivated so far in any cell-free medium, despite a vast, painstaking effort by many workers, using the most varied media4-5. Moreover, studies on the respiratory metabolism of M. lepraemurium have shown an almost complete lack of response to the many substances tested; even albumin and yeast and liver extracts, which do stimulate respiration, are apparently not themselves oxidized, nor do they promote utilization of other substrates5.
For some time, workers in these laboratories have found it possible with the electron microscope to distinguish, under certain conditions, a completely degenerate form of M. lepraemurium that is no longer able to produce disease6. In this way we have found that in the conventional culture media the bacilli not only fail to multiply but also are degenerate after a few weeks of incubation at 37 C. In one of our experiments, however, involving a liquid nutrient medium of the type used for culturing tubercle bacilli, with 20 per cent sucrose added in the hope of achieving a beneficial stabilizing effect, electron microscopy at 2 months revealed that, although the bacilli had degenerated, a number looked unusually long. It seemed possible that before death some actual, if limited, growth might have occurred in this medium. We therefore followed up this observation by investigating the frequency distributions of the lengths of rat leprosy bacilli after varying times of incubation in different media.
Table 1. MEAN LENGTHS AND PERCENTAGE DEGENERATION OF Mycobacterium lepraemurium AFTER INCUBATION AT 37 C. IN VARIOUS MEDIA Mean length of bacilli after up to 24 hr. incubation (base line) = 1-64 [plusmn]0 -01//, with 5 per cent degenerate
Medium	Days incubated
		7		14	28	63
	Mean length (P)	Degenerate (per cent)	Mean length 0")	Degenerate (per cent)	Mean length (/[ast])	Degenerate (per cent)	Mean length	Degenerate (per cent)
A Water B Phosphate buffer C As B + 20 per cent sucrose D Nutrient E As D + sugar: El +10 per cent sucrose E2 + 20 per cent sucrose E3 +8 per cent glucose F As El + isoniazid	1 75 1-79	(70) (10)	1-58 1-551-91[ast]2-85[ast] 2-21[ast] 2-44[ast] l-70f	(50) (50)(W)(10) (10) (5) (20)	1-57 1-59 1-642-14[ast]3-12[ast] 2-42[ast] 2-70[ast] l-64f	(50) (80) (90) (80)(20) (30) (10) (40)	2^24[ast]3-44[ast] 3-12[ast]2-88[ast]	(80)(95) (95) (80)
The table gives the summated results derived from all experiments performed.
[ast] The difference between these figures and the base line length (1-64^), as well as between the figures for D and all E, far exceed any possible chance fluctuation (P <[pound] 0-0001).
f These figures are not significantly above the base line length of 1 -64ju (P > 0-3); they are lower than the lengths of El by an amount far exceeding any possible chance fluctuation (P<| 0-0001).
Bacilli of M. lepraemurium, partly freed from tissue components, were provided by our colleague Dr. !R. J. W. Rees from the homogenized livers of intravenously infected mice. The bacilli were added to the following liquid media, contained in 5-ml. amounts in 25-mm. diameter test-tubes, to make final concentrations of about 5 x 107 bacilli/ml.: (A) distilled water; (B) phosphate buffer 0 -01 M, pH 7 -0; (C) phosphate buffer plus sucrose 20 per cent (w/v); (D) a basal nutrient medium composed of Difco 'Casamino' acids, 2-5 gm.; asparagine, 0-3 gm.; anhydrous disodium hydrogen phosphate, 2-5 gm.; potassium dihydrogen phosphate, 1-0 gm.; sodium citrate, 1-5 gm.; crystalline magnesium sulphate, 0-6 gm.; glycerol, 25 ml.; distilled water to 1,000 ml. To this basal medium, sterilized by autoclaving, bovine plasma albumin (fraction V), sterilized by filtration, was added to make 0 -25 per cent; (E) as (D), plus sucrose or glucose (sterilized by autoclaving in high concentrations) to give a range from 1 to 20 per cent (w/v), or 0-5-8 per cent (w/v), respectively; (F) as (E) (with 10 per cent sucrose), plus isoniazid 5 jjigm./ml.
The bacillary suspensions in these media were incubated at 37 C. Some were fixed within 24 hr. (base line) by adding formaldehyde to make 2 per cent, while others were incubated for various periods and then similarly fixed. Samples were examined in the electron microscope, set at a magnification of 10,000 by means of standard 0-26-pi diameter spheres of polystyrene latex. A series of concentric circles, the circumferences of which were spaced by 0 -5 cm., drawn on the fluorescent screen on which the final image was seen, allowed the lengths of the bacilli to be grouped in intervals of 0-5pi. The lengths of 100 or more bacilli from each sample were measured in this way, and the frequency distributions of lengths and their means were calculated. At the same time the proportion of these bacilli appearing completely degenerate was estimated, giving a rough minimum value for a non-viable count. Of 1,700 bacilli from six samples fixed within 24 hr., the lengths of 74 per cent lay between 1-0 and 2-0(ji, and 92 per cent between 1-0 and 2-5pi; the mean was 1-64 [plusmn] 0-Olpi a figure very similar to that already reported from the more difficult measurements made with the light microscope7. The average proportion degenerate in these base-line samples was 5 per cent.
The mean lengths after incubation (Table 1) indicate no elongation in the three non-nutrient media (A, B and C), compared with the base line at 24 hr.; and degeneration was rapid. On the other hand, a small amount of lengthening could occur even in the ordinary nutrient culture medium (D), though again degeneration was fairly rapid. However, in the same medium with 10 per cent sucrose or 8 per cent glucose (E), the mean length nearly doubled, and the proportion of bacilli longer than 2-5{ji rose from 6 to 67 per cent (sucrose) and to 51 per cent (glucose) in a month (the greater part of the increase being in the first 2 weeks). Moreover, the appearances of degeneration were distinctly delayed, though occurring ultimately and presumably terminating the lengthening process. The contrast in increased length and slowed degeneration between the sugar-containing medium and the same medium alone was evident through the whole range of concentrations of sucrose from 1 to 20 per cent (optimal at 10 per cent), and with 4 and 8 per cent glucose.
The effect of the anti-leprosy drug isoniazid (medium F) in preventing the lengthening excludes the possibility that we are witnessing only a passive stretching. Moreover, the electron density (and therefore the weight density) of the bacilli was unchanged in the long bacilli with, if anything, a slight increase in width. This again points to a real increase in bacterial protoplasm in the cultures. We now, therefore, regard the failure to obtain multiplication of the rat leprosy bacillus in culture medium as being due to a failure of the bacilli to divide, and not to their complete inability to metabolize and grow. If means could be found to encourage division, the culture of leprosy bacilli in cell-free media might at last become possible.
We are indebted to Dr. R. J. W. Bees for providing the M. lepraemurium and for his interest and advice ; and to Miss B. Cooling for undertaking many of the length determinations.Rees, , R. J. W., and Wong, , P. C., Nature, 181, 359 (1958).PubMedISIChemPortGarbutt, , E. W., Rees, , R. J. W., and Barr, , Y. M., Lancet, 2, 127 (1958).PubMedISIChemPortWallace, , J. H., Elek, , S. D., and Hanks, , J. H., Proc. Soc. Exp. Biol., 97, 101 (1958).ISIChemPortEddy, , B. E., Internat. J. Leprosy, 5, 31 (1937).Gray, , C. T., J. Bact., 64, 305 (1952).PubMedISIChemPortMcFadzean, , J. A., and Valentine, , R. C., Trans. Roy. Soc. Trop. Med. Hyg., 53, 414 (1959).ArticleChemPortHilson, , G. R. F., and Elek, , S. D., Internat. J. Leprosy, 25, 380 (1957).PubMedChemPort