HiCombi™ Cetrimide - MacConkey Agar Plate

Principle And Interpretation

Cetrimide Agar Base

Pseudomonas aeruginosa grows well on all normal laboratory media but specific isolation of the organism, from environmental sites or from human, animal or plant sources, is best carried out on a medium, which contains a selective agent and also constituents to enhance pigment production. Most selective media depend upon the intrinsic resistance of the species to various antibacterial agents. Cetrimide inhibits the growth of many microorganisms whilst allowing Pseudomonas aeruginosa to develop typical colonies.

Cetrimide is a quaternary ammonium salt, which acts as a cationic detergent that reduces surface tension in the point of contact and has precipitant, complexing and denaturing effects on bacterial membrane proteins. It exhibits inhibitory actions on a wide variety of microorganisms including Pseudomonas species other than Pseudomonas aeruginosa. King et al developed Medium A for the enhancement of pyocyanin production by Pseudomonas (1). Cetrimide Agar developed by Lowburry (1) is a modification of Tech Agar (Medium A) with addition of 0.1% cetrimide for selective isolation of P.aeruginosa. Later, due to the availability of the highly purified cetrimide, its concentration in the medium was decreased (2,3). The incubation was carried out at 37°C for a period of 18-24 hours (4).

P.aeruginosa can be identified due to their characteristic production of pyocyanin, a blue, water-soluble, non-fluorescent phenazine pigment coupled with their colonial morphology and the characteristic grape-like odor of aminoacetophenone (5). P.aeruginosa is the only species of Pseudomonas or gram-negative rod known to excrete pyocyanin. These media are therefore, important in the identification of P.aeruginosa. These media are used for the examination of cosmetics (6) and clinical specimens (5,7) for the presence of P.aeruginosa, as well as for evaluating the efficacy of disinfectants against this organism (8). Gelatin peptone provide necessary nutrients for P.aeruginosa. Sodium chloride maintains osmotic equilibrium in the medium. Magnesium chloride and potassium sulfate stimulates pyocyanin production (9).

For the isolation of P.aeruginosa, plates of Cetrimide Agar should be inoculated from non-selective medium such as Brain Heart Infusion Broth (M210) or Soyabean Casein Digest Medium (M011). If the count is high, the test sample can be directly inoculated onto Cetrimide Agar. P.aeruginosa colonies may appear pigmented blue, blue-green or non-pigmented. Colonies exhibiting fluorescence at 250nm and a blue green pigmentation are considered as presumptive positive. P.aeruginosa may lose its fluorescence under UV if the cultures are left at room temperature for a short time. Fluorescence reappears after the plates are re-incubated (4). Type of peptone used in the base may also affect pigment production (4,10). Certain strains of P.aeruginosa may not produce pyocyanin. Other species of Pseudomonas do not produce pyocyanin but fluoresce under UV light. Most non-Pseudomonas species are inhibited on Cetrimide Agar, and some species of Pseudomonas may also be inhibited. Some non-fermenters and some aerobic spore formers may exhibit a water-soluble tan to brown pigmentation on this medium. Serratia may exhibit pink pigmentation (3). Biochemical tests and serological procedures should be performed to confirm the findings.

MacConkey Agar

MacConkey agars are slightly selective and differential plating media mainly used for the detection and isolation of gram-negative organisms from clinical (12), dairy (13), food (14,15), water (16), pharmaceutical (17,18) and industrial sources (19). 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 (18). 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 (21). Other than that this medium is also used for count of coli-aerogenes bacteria in cattle and sheep faeces (18), the count of coli-aerogenes and non-lactose fermenters in poultry carcasses (18), bacterial counts on irradiated canned minced chicken (22) and the recognition of coli-aerogenes bacteria during investigations on the genus Aeromonas (23). MacConkey Agar is the earliest selective and differential medium for cultivation of enteric microorganisms from a variety of clinical specimens (16,18). 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.