HiCombi™ CLED - MacConkey Agar Plate

Principle And Interpretation

C.L.E.D. Agar w/Bromo Thymol Blue

On a solid medium, Sandys reported that swarming of Proteus species can be controlled by restricting the electrolytes (1). Formerly swarming of Proteus was controlled by adding alcohol, surface-active agent, sodium azide, boric acid etc. to the medium (1). Later on Sandy's medium was modified by Mackey and Sandys (2), by replacing mannitol by lactose and sucrose and elevating concentration of agar and bromothymol blue. This formulation was further modified by the same authors, called C.L.E.D. (Cystine-Lactose-Electrolyte-Deficient) by deleting the sucrose and by including Lcystine for promoting the growth of cystine dependent dwarf colony coliforms (3). This medium is recommended for use in urinary bacteriology, promoting the growth of all urinary pathogens. C.L.E.D. Medium is also recommended for dip stick procedures and as dip inoculum transport medium for urine specimens (2,3,4).

Peptone, Tryptone and HM Peptone B provides nitrogenous and carbonaceous compounds, long chain amino acids, vitamins and other essential growth nutrients. Lactose is the fermentable sugar. L-cystine supports the growth of dwarf coliform colony. Bromo thymol blue is the pH indicator which turns yellow at acidic pH.

Bacteriuria may be quantitated by inoculating the surface of an agar medium by proper dilution. Inoculate the medium immediately after urine collection. It can also be inoculated by calibrated loop or duplicate dilution pour plate methods (5,6).

MacConkey Agar

MacConkey agars are slightly selective and differential plating media mainly used for the detection and isolation of gram-negative organisms from clinical (7), dairy (8), food (9,10), water (11), pharmaceutical (12,13) and industrial sources (14). 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 (13). 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 (17) and for the isolation of Salmonella and Shigella species in cheese (18). Other than that this medium is also used for count of coli-aerogenes bacteria in cattle and sheep faeces (13), the count of coli-aerogenes and non-lactose fermenters in poultry carcasses (13), bacterial counts on irradiated canned minced chicken (16) and the recognition of coli-aerogenes bacteria during investigations on the genus Aeromonas (19). MacConkey Agar is the earliest selective and differential medium for cultivation of enteric microorganisms from a variety of clinical specimens (11,13). 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.