For each portion of the sample 1 ml of solution must be transferred to a sterile 90 mm petri dish (in duplicate), to which 15 ml of Shahidi – Ferguson agar (SF agar)34 at a temperature of 47°C±1°C must be added and immediately gently mixed by swirling the dish with 5 clockwise and 5 anticlockwise circular movements.

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

After incubation each set of duplicate plates must be examined for colonies characteristic of Clostridium perfringens (black). The sample provisionally fails if any colonies characteristic of Clostridium perfringens are present, in which case the following procedure must be followed to establish whether or not the colonies are Clostridium perfringens.

In the case of each plate, 10 characteristic colonies of Clostridium perfringens must be subcultured on to a further SF agar plate. If there are less than 10 colonies on the plate, all characteristic colonies must be subcultured on to the further plate. The plates must be incubated anaerobically at 37°C±1°C for 20 hours±2 hours.

If the surface area of the plates is overgrown and it is not possible to select well isolated characteristic colonies, 10 suspect colonies must be subcultured on to duplicate SF agar plates and incubated anaerobically at 37°C±1°C for 20 hours±2 hours.

One characteristic colony from each plate must be subcultured on to SF agar and incubated anaerobically at 37°C±1°C for 20 hours±2 hours.

The lactose gelatin medium must be examined for the presence of small gas bubbles in the medium.

The lactose gelatin medium must be examined for colour. A yellow colour indicates fermentation of lactose.

The lactose gelatin medium must be chilled for one hour at 2 – 8°C and then checked to see if the gelatin has liquefied. If the medium has solidified it must be re-incubated anaerobically for a further 18 – 24 hours, the medium chilled for a further one hour at 2 – 8°C and again checked to see if the gelatin has liquefied.

The presence of Clostridium perfringens must be determined on the basis of the results from paragraphs 10 to 14. Bacteria which produce black colonies on SF agar, are non-motile, reduce nitrate to nitrite, produce gas and acid from lactose and liquefy gelatin within 48 hours must be considered to be Clostridium perfringens.

10 gram portions of the rendered animal protein must be placed aseptically in each of two sterile containers containing 90 ml Buffered Peptone Water (BPW)39 and mixed thoroughly until the samples are evenly suspended.

One colony of Clostridium perfringens 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. This must be repeated for Escherichia coli.

These are 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.

On day one, each 25 gram sample must be placed aseptically in a container containing 225 ml Buffered Peptone Water (BPW) and incubated at 37°C±1°C for 18 hours±2 hours.

On day two, 0.1 ml from the container of incubated BPW must be inoculated into 10 ml Rappaports Vassiliadis broth (RV broth)40 and incubated at 41.5°C±0.5°C for 24 hours ± 3 hours.

The reincubated RV broth must be plated out as described in paragraph 4.

Tests must be begun on receipt of the sample or on the first working day which allows the following 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. If the sample has been refrigerated it must be stored at room temperature for at least four hours before the test is started.

On day one tests must be carried out in duplicate using two 25 gram portions of each sample submitted for testing. Each 25 gram sample must be placed aseptically in a sterile container containing 225 ml Buffered Peptone Water/Lysine/Glucose (BPW/L/G)44 and incubated at 37°C for 18 hours.

On day two the incubated BPW/L/G must be added to Selenite Cystine Trimethylamine-N-Oxide Dulcitol (SC/T/D)45 and Lysine Decarboxylase Glucose (LD/G)46 media in electrical conductance cells or wells. For cells or wells containing more than 5 ml medium 0.2 ml of the BPW/L/G must be added and for cells or wells containing 5 ml or less medium 0.1 ml of the BPW/L/G must be added. Cells or wells must be connected to appropriate electrical conductance measuring equipment set to monitor and record changes in electrical conductance at 6 minute intervals over a 24 hour period. The temperature of cells and wells must be kept at 37°C.

On day three, at the end of the 24 hour period, the information recorded by the conductance measuring equipment must be analysed and interpreted using criteria defined by the manufacturers of the equipment. Where a well or cell is provisionally identified as being positive for Salmonella, the result must be confirmed by subculturing the contents of the well or cell on to two 90 millimetre plates of BGA or on to one 90 millimetre plate of BGA and one 90 millimetre plate of Xylose Lysine Deoxycholate Agar (XLD) using a 2.5 mm diameter loop. The plates must be inoculated with a droplet taken from the edge of the surface of the fluid by drawing the loop over the whole of one plate in a zig zag pattern and continuing to the second plate without recharging the loop. The space between the loop streaks must be 0.5 cm – 1.0 cm. The plates must be incubated at 37°C overnight.

On day five the incubated composite media or equivalent must be examined and the findings recorded, discarding cultures which are obviously not Salmonella. Slide serological tests must be performed using Salmonella polyvalent “O”and polyvalent “H” (phase 1 and 2) agglutinating sera on selected suspect colonies collected from the blood agar or MacConkey plates. If reactions occur with one or both sera, the colonies must be typed by slide serology. If requested in writing by the National Assembly , the operator of the laboratory must send a subculture to a Regional Veterinary Laboratory of the Veterinary Laboratories Agency of the Department for Environment, Food and Rural Affairs for further typing.

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)47 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.

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.

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.

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.

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.

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.

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.

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.