Tubercle, Lond., (I959) , 4% 121

N O T E

The Effect of Surface Moisture on Colonial

Morphology of BCG

Mycobacterium luberculosis on solid media shows variations in colonial morphology; ' rough ' and 'smooth' colony types occur. The ability of virulent strains to grow in serpentine colonies was first illustrated by Koch in x884. More recently Middlebrook, Dubos and Pierce (1947) related the microscopic arrangement of cells in these serpentine cords to virulence. In virulent strains tile cells were found to be orientated in parallel along the cords, whereas in the avirulent strains a less orientated arrangement occurred. The formation of such growth was found by Yegian and Kurung (I952) to be favoured by the addition of normal blood serum or plasma to liquid culture medium. These workers also related the 'tightness' of the cord to the invasiveness of the culture hi v ivo-wi th the exception only of avirulent streptomycin-resistant H37Rv, which showed good cord formation.

Experiments in this laboratory with various substrains of BCG have shown that the appearance of serpentine colonies on blood agar medium can be influenced by the physical environment. Cultures normally growing as discrete colonies will form serpentine colonies on media with excess surface moisture. Nutrient agar with IO per cent human blood (Ungar and others, i956 ) allows the growth of BCG as discrete colonies, and two forms, ' rough' and 'smooth' , can be seen. These terms are defined by the appearance of the colonies: ' r o u g h ' - s p r e a d i n g colonies with umbonate surface and crenated edges; ' smoo th ' - co lon ie s that either form as

~Fm. I. - BCG 'Copenhagen' substraln. Mixed 'rough' and 'smooth' colony forms on 'dry' b!ood agar medium. • 15.

I 2 2 T U B E R O L E

FIG. 2. - - Serpentine colonies on 'wet' blood agar. X I2.

Fio. 3. - Part of smooth serpentine colony on 'wet' blood agar showing 'loose' cord formation • 1,2oo.

a s ingle d o m e - s h a p e d d rop or show a h e a p e d - u p a p p e a r a n c e (Fig. I ) . These two forms have b e e n isolated, a n d it was fo u n d tha t bo th types gave rise, o n 'we t ' aga r , to s e r p e n t i n e colonies, wh i c h were maeroscop ica l ly s imi la r (Fig. 2). S u b c u l t u r e

T U E E F F E C T OF S U R F A C E 31OISTURE ON C O L O N I A L M O R P t I O L O G Y OF BCG I2 3

l:lo. 4- - Rough serpentine colon)" on 'wet' blood agar showing 'tight' cord. • 325 .

of these serpentine colonies on to medium with a ' d ry ' surface produced discrete colonies as in the original inoculum.

The cultures were prepared on medium filled into vertical 6 ml. bottles closed by a rubber pad held in position by a screw cap (Yegian and Kurung , 1952 ). The free moisture present on the medium surface was controlled by part ly or completely screwing down this cap dur ing the first days of incubation.

Under microscopic examinat ion it was seen that the cords formed by the ' rough ' variants consisted of cells more tightly held together than the ' smooth ' variants (Figs. 3 and 4). Ttfis correlated with results from experiments in guinea-pigs (Jensen tests) in which the former caused larger local lesions than the latter. The Moreau substrain was found to form only loose cords, similar to those on the slide cultures illustrated by Kurylowicz (I957) , a l though both 's inooth ' and ' rough ' colony forms were observed in this substrain. This observation correlates with the reported lower invasiveness of this substrain in m a n (Assis, 1956 ).

Glaxo Laboratories Ltd., PAULINE FAR.',IER. Greelford, Middlesex.

Grateful acknowledgment is made to Mr J. Dudley for technical assistance and Mr D. F. Boxall, who took the photograp.~s

R e f e r e n c e s Assis, A. de (z956) l'r. reed., 64, 44 I. Koch, R. (1884) Mitt. k. GesundhAmt., 2, z.

Kurylowicz, W. (I957) Acta tuberc, stand., 33, 354. �9 Middlebrook, G., Dubos, R.J., and Pierce, (2. (z947) 07. exp. Med., 86, I75.

O'tlea, A.J. (z955) 07. Path. Bact., 69, I69. Ungar, J., Farmer, P., and Muggleton, P. W. (z956) Brit. reed. aT, ii, 568. Yeglan, D., and Kurung, J. 0952) Amer. Rev. Tuberc., 65, 18I.