HiCombi™ XLD - MacConkey Agar Plate

Principle And Interpretation

XLD Agar

XLD Agar has been recommended for the identification of Enterobacteriaceae (1) and for the microbiological testing. XLD Agar was formulated by Taylor (2-6) for the isolation and differentiation of enteric pathogens including Salmonella Typhi from other Salmonella species of foods, water and dairy products (7,8,9,10). XLD Agar exhibits increased selectivity and sensitivity as compared to other plating media e.g. SS Agar (M108), EMB Agar (M022) and Bismuth Sulphite Agar (M027) (10,8,11,3,5,12,13). The media formulation does not allow the overgrowth of other organisms over Salmonella and Shigella (14). Samples suspected of containing enteric pathogens, along with other mixed flora, are initially enriched in Modified Semisolid RV Medium Base (M1482) (15).

The medium contains yeast extract, which provides nitrogen and vitamins required for growth. Though the sugars xylose, lactose and sucrose provide sources of fermentable carbohydrates, xylose is mainly incorporated into the medium since it is not fermented by Shigellae but practically by all enterics. This helps in the differentiation of Shigella species. Sodium chloride maintains the osmotic balance of the medium. Lysine is included to differentiate the Salmonella group from the nonpathogens. Salmonellae rapidly ferment xylose and exhaust the supply. Subsequently lysine is decarboxylate by the enzyme lysine decarboxylase to form amines with reversion to an alkaline pH that mimics the Shigella reaction. However, to prevent this reaction by lysine-positive coliforms, lactose and sucrose are added to produce acid in excess. Degradation of xylose, lactose and sucrose to acid causes phenol red indicator to change its colour to yellow.

Bacteria that decarboxylate lysine to cadaverine can be recognized by the appearance of a red colouration around the colonies due to an increase in pH. These reactions can proceed simultaneously or successively, and this may cause the pH indicator to exhibit various shades of colour or it may change its colour from yellow to red on prolonged incubation. To add to the differentiating ability of the formulation, an H2S indicator system, consisting of sodium thiosulphate and ferric ammonium citrate, is included for the visualization of hydrogen sulphide produced, resulting in the formation of colonies with black centers. The non-pathogenic H2S producers do not decarboxylase lysine; therefore, the acid reaction produced by them prevents the blackening of the colonies (9).

XLD Agar is both selective and differential medium. It utilizes sodium deoxycholate as the selective agent and therefore it is inhibitory to gram-positive microorganisms. Some Proteus strains may give red to yellow colouration with most colonies developing black centers, giving rise to false positive reactions. Non-enterics like Pseudomonas and Providencia may exhibit red colonies. S. Paratyphi A, S. Choleraesuis, S. Pullorum and S. Gallinarum may form red colonies without H2S, thus resembling Shigella species (16).

MacConkey Agar

MacConkey agars are slightly selective and differential plating media mainly used for the detection and isolation of gram-negative organisms from clinical (17), dairy (18), food (19,20), water (7), pharmaceutical (21,22) and industrial sources (23). It is also recommended for the selection and recovery of the Enterobacteriaceae and related enteric gram-negative bacilli. USP recommends this medium for use in the performance of Microbial Limit Tests (22).

These agar media are selective since the concentration of bile salts, which inhibit gram-positive microorganisms, is low in comparison with other enteric plating media. The medium M081, which corresponds with, that recommended by APHA can be used for the direct plating of water samples for coliform bacilli, for the examination of food samples for food poisoning organisms (20) and for the isolation of Salmonella and Shigella species in cheese (24). Other than that this medium is also used for count of coli-aerogenes bacteria in cattle and sheep faeces (25), the count of coli-aerogenes and nonlactose fermenters in poultry carcasses (25), bacterial counts on irradiated canned minced chicken and the recognition of coli-aerogenes bacteria during investigations on the genus Aeromonas (26).

MacConkey Agar is the earliest selective and differential medium for cultivation of enteric microorganisms from a variety of clinical specimens (24,27). The original medium contains protein, bile salts, sodium chloride and two dyes. The selective action of this medium is attributed to crystal violet and bile salts, which are inhibitory to most species of gram-positive bacteria. Gram-negative bacteria usually grow well on the medium and are differentiated by their ability to ferment lactose. Lactose-fermenting strains grow as red or pink colonies and may be surrounded by a zone of acid precipitated bile. The red colour is due to production of acid from lactose, absorption of neutral red and a subsequent colour change of the dye when the pH of medium falls below 6.8. Lactose non-fermenting strains, such as Shigella and Salmonella are colourless, transparent and typically do not alter appearance of the medium.

Peptone, Tryptone and gelatin peptone are sources of nitrogen, carbon, long chain amino acids and other nutrients. Lactose is a fermentable carbohydrate, Sodium chloride maintains the osmotic equilibrium. Bile salts and crystal violet are selective agents that inhibit growth of gram-positive organisms. Neutral red is the pH indicator dye.