Clostridium species are widely distributed in nature and are also associated with humans, either as non-pathogens at a variety of anatomic locations or at infected sites. Diseases caused by members of the genus Clostridium generally fall into one of the three categories:
a. non-invasive disease in which toxin(s) is responsible for all the symptoms
b. invasive (histotoxic) disease in which a progressive infections process and tissue destruction occur and
c. purulent disease in which a closed-space mixed infection involving multiple organisms is present (1).
Histotoxic clostridia can be isolated on egg yolk containing medium, as demonstrated by McClung and Toabe (2). This medium was further supplemented with additional milk and lactose to differentiate clostridia on the basis of lecithinase production, casein hydrolysis and lactose fermentation (3). Selectivity was obtained by the incorporation of neomycin sulphate. Subsequently, eggs were replaced by purified lecithin, to obtain an egg-free medium (4). This egg-free medium was further modified with reduced concentration of neomycin and additional sodium azide, which enhanced the selective properties of the medium (5). This refined medium was designated as Lactose Lecithin Agar, which is used for isolation and differentiation of histotoxic clostridia from clinical specimens.
Casein enzymic hydrolysate, peptic digest of animal tissue and pancreatic digest of heart muscles provide carbonaceous and nitrogenous compounds essential for the growth of bacteria. Lactose is the fermentable carbohydrate with bromocresol purple being the pH indicator. L-cysteine helps to create anaerobic conditions. Yeast extracts supplies vitamin B-complex nutrients. Corn starch neutralizes toxic fatty acids if any, present in the medium. Neomycin and sodium azide inhibit accompanying gram-negative and gram-positive organisms.
Before inoculation, prepared media plates should be pre-reduced by placing under anaerobic conditions for 18-24 hours. Specimens should be inoculated on these pre-reduced plates. A non-selective media should be inoculated simultaneously (1, 6). An opalescent zone surrounding the colonies indicates lecithinase production. Yellow colour around colonies indicates lactose fermentation.
Storage and Shelf-life:
Store dehydrated powder and prepared medium at 2-8°C in tightly closed container. Use before expiry date on the label.
References:
1. Murray P. R., Baron J. H., Pfaller M. A., Jorgensen J. H. and Yolken R. H., (Eds.), 2003, Manual of Clinical Microbiology, 8th Ed., American Society for Microbiology, Washington, D.C.
2. McClung L. S. and Toabe R., 1947, J. Bacteriol., 53:139.
3. Willis A. T. and Hobbs G., 1959, J. Pathol. Bacteriol., 77:511.
4. Willis A. T., 1960, J. Pathol. Bacteriol., 80:379.
5. Ellner P. D. and O. Donnell D., 1971, Am. J. Clin. Pathol., 56:197.
6. MacFaddin J. F., 1985, Media for Isolation-Cultivation-Identification-Maintenance of Medical Bacteria., Vol. 1, Williams and Wilkins, Baltimore.