MacConkey No. 3 Agar EP/USP/ISO

Description

Advancements in the understanding of bacterial physiology have meant that the medium has now been adapted to facilitate the detection of coliforms. The two most significant modifications to the original formulation are as follows:

• The substitution of ox bile by purified bile salts that improves the selectivity and avoids the inherent turbidity, which is due to the fat composition of bile. The efficiency of the inhibition due to bile salts is variable and depends on the relative concentration of cholate and taurocholate.
• The inclusion of supplementary inhibitors such as crystal violet and/or brilliant green. A popular formulation in America, but not in Europe where lower selectivity is preferred.
• Lactose-positive bacteria grown on this medium form red colonies due to acid production resulting from lactose fermentation and thus Escherichia coli colonies can be easily distinguished as they also form a small precipitation zone of bile salts around them.

Some enterococci may also grow, but they are easy to distinguish from coliforms, as they form smaller colonies and have no precipitation zone.

Technique

For plate inoculation, follow the laboratory's standard methods or the applicable norms (spiral plating method, ecometric methods, streak plating, dilution banks, spread plating with Drigalski rod, etc.).

For enumeration, after an incubation of 24 hours at 35 °C, select plates with 30-150 colonies. The characteristic colonies must be confirmed as coliforms by gas production from lactose in a broth culture.

Physical/chemical control

Microbiological control

Growth Promotion Test 50-100 CFU according to harmonized Pharmacopoeia monographs (EP) and test methods & ISO 11133:2014/Amd 1:2018.

Inoculate: practical range 100 ± 20 CFU, min. 50 CFU (productivity) / 10⁴-10⁶ (selectivity) and < 100 CFU (specificity, Ph. Eur.) and ≥ 10³ CFU (specificity, ISO).

Analytical methodology according to ISO 11133:2014/Amd 1:2018; A2:2020.

Aerobiosis. Incubation at 30-35 °C. Reading at 18-72 h.

S. sonnei incubation at 37 °C for 20-24 h.

Sterility control

Incubation 48 h at 30-35 °C and 48 h at 20-25 °C: NO GROWTH.

Check at 7 days after incubation in same conditions.

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