The Animal By-Products (Wales) Regulations 2006

PART III METHOD FOR THE ISOLATION OF ENTEROBACTERIACEAE

1.  Tests must be begun on receipt of the sample or on the first working day which allows this method to be completed. If the test is not begun on the day of receipt the sample must be stored in a refrigerator until required at between 2°C and 8°C. If the sample has been refrigerated it must be removed from the refrigerator and stored at room temperature for at least one hour before the test is started.

Samples

2.  Tests must be carried out using five 10 gram portions of each sample submitted for testing. Each 10 gram sample must be placed aseptically in a sterile container containing 90 ml Buffered Peptone Water and mixed thoroughly until the sample is evenly suspended.

Inoculations

3.  For each portion of the sample 1 ml of solution must be transferred to a sterile 90 mm petri dish (in duplicate). The plates must be labelled to identify the portion of sample they were taken from. 15 ml of Violet Red Bile Glucose Agar (VRBGA)(1) at a temperature of 47°C±2°C must be added to each petri dish and immediately gently mixed by swirling the dish with five clockwise and five anticlockwise circular movements.

4.  Once the agar has set, each agar plate must be overlaid with a further 10 ml VRBGA at a temperature of 47°C±2°C. Once the overlay has set, the plates must be inverted and incubated aerobically at 37°C±1°C for 20 hours±2 hours.

Samples with colonies of Enterobacteriaceae

5.  After incubation each set of duplicate plates must be examined for colonies characteristic of Enterobacteriaceae (purple colonies 1 – 2 mm in diameter). All characteristic colonies on each plate must be counted and the arithmetic mean of the duplicate plates taken.

The sample provisionally fails if either–

(a) any arithmetic mean is above 30(2); or

(b) three or more arithmetic means are above 10;

in which case the following procedure must be followed to establish whether or not the colonies are Enterobacteriaceae.

6.  After counting the colonies, characteristic colonies must be taken at random from the agar plates, the number being at least the square root of the colonies counted. The colonies must be subcultured onto a blood agar plate and incubated aerobically at 37°C±1°C for 20 hours±2 hours.

Examination of subcultures

7.  An oxidase test and a glucose fermentation test must be performed on each of the five subcultured colonies. Colonies which are oxidase-negative and glucose fermentation-positive must be considered to be Enterobacteriaceae.

8.  If not all of the colonies prove to be Enterobacteriaceae, the total count in paragraph 5 must be reduced in proportion prior to establishing whether or not the sample should fail.

Controls

9.  Control tests must be carried out each day that a test is initiated using–

(a) Escherichia coli NCTC 10418 no more than seven days old at time of use; and

(b) processed animal protein or compost or digestion residue which is free of Enterobacteriaceae.

10.  A 10 gram portion of the rendered animal protein must be placed aseptically in a sterile container containing 90 ml BPW and mixed thoroughly until the sample is evenly suspended.

11.  One colony of Escherichia coli must be placed in 10 ml BPW and mixed to form an even suspension. 0.1 ml of the suspension must be added to the suspension in the preceding paragraph.

12.  This is then treated and examined in the same way as test samples. If no typical colonies are formed then that day’s testing must be invalid and must be repeated.

(1)

Violet Red Bile Glucose Agar – See Mossell D A A, Eelderink I, Koopmans M, van Rossem F (1978) Laboratory Practice 27 No. 12 1049-1050; Emap Maclaren, PO Box 109, Maclaren House, 19 Scarbrook Road, Croydon CR9 1QH.

(2)

An arithmetic mean of 30 is equivalent to 3x102 colony forming units per gram of original sample.