BS EN 15788:2021 pdf free download – Animal feeding stuffs: Methods of sampling and analysis – Detection and enumeration of Enterococcus (E. faecium) spp. used as feed additive.
9.4.3 Pour plate method
Transfer 0,1 ml to 1 ml of each suitable dilution step into at least two plates, so that not less than 10 colonies and not more than 200 colonies per plate are to be expected.
Pour about 15 ml of the medium (5.2.3.1 or 5.2.3.2) according to 9.2 previously maintained at 44 °C to 47 °C in a water bath or incubator. Carefully mix the inoculum with the medium and allow the mixture to solidify, by leaving the plates to stand on a cool horizontal surface.
Incubate the inverted plates aerobically at 37 °C ± 1 °C for 24 h (bile aesculin azide agar) or 48 h (Slanetz and Bartley agar). Up to four plates can be stacked for incubation.
9.5 Enumeration of colonies
After incubation under the conditions specified above, all plates showing more than 10 and no more than
200 presumptive enterococci (E.faecium)-positive colonies according to the phenotypic characterization
(9.6) are used for the enumeration of CFU.
For the calculation of the number of enterococci per unit of sample (CFU/g or CFU/ml) a lower limit of 10 colonies per plate corresponds to the recommendations of EN ISO 7218 [6]. Fora higher precision and increased confidence level a lower limit of 20 colonies per plate is recommended.
If possible, count at least four plates (of two successive dilutions) or three plates (of one dilution). If no suitable incubated plates with colony counts between> 10 and  200 are available, the analysis shall be repeated with appropriate dilutions.
9.6 Confirmation
Presumptive enterococci (E. faecium) show the following colony morphology after applying the spread plate method:
a) on the surface of bile aesculin azide agar: colony size between 1 mm and 2 mm in diameter, circular, convex to dome-shaped, entire, white, glistening surface, opaque. The medium surrounding the colonies shows a dark brown to black coloration, due to the hydrolysis of aesculin;
b) on the surface of Slanetz and Hartley agar: colony size between 1 mm and 2 mm in diameter, circular, convex to dome-shaped, entire, red, maroon or pink with a narrow whitish border, glistening surface.
Selected colonies are checked microscopically by suspension in a drop of 085 % sterile saline with a cover slip and a suitable magnification (e.g. oil immersion) for morphology. Enterococci colonies are cocci in pairs and in short chains.
A test for catalase is done with a drop of 3 % hydrogen peroxide (11202) on a colony. The formation of bubbles is a positive reaction. Only bacteria that are cocci in pairs or short chains and catalase negative are considered to be enterococci.
Since E.faecium belongs to the intestinal enterococci, the bacteria are able to hydrolyse aesculin at 44 °C. Selected colonies from Slanetz and Bartley agar can he streaked on the surface of bile aesculin acide agar plates and incubated at 44°C ± 0,5 °C for 2 h under aerobic conditions. E.faecium-positive colonies show dark brown to black coloration of the medium surrounding the colonies.
In case of doubt, the presence of enterococci is confirmed by biochemical characterization (for example commercial available test kits), polymerase chain reaction (PCR) analysis or via matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry of exemplary colonies.