The Processed Animal Protein Order 1989

b. Electrical conductance method for the detection and isolation of salmonella from animal protein

Samples of processed animal protein submitted for testing shall be examined on the first working day which allows the following method to be completed. Samples not examined on the day of receipt shall be stored in a refrigerator until required. Examination shall be carried out in duplicate using two 25 gram portions of each sample submitted for testing.

Day 1

The sample shall be received or removed from refrigeration and left at room temperature for at least 4 hours. Thereafter 25 grams shall be added aseptically to a jar containing 225 ml BPW/L/G(a) and incubated at 37°C for 18 hours.

Day 2

Volumes of the incubated BPW/L/G inoculated with the samples under test shall be transferred to SC/T/D(b) and LD/G(c) media in electrical conductance cells or wells. For cells or wells containing >5 ml medium 0.2 ml shall be transferred and for cells or wells containing <5 ml medium 0.1 ml shall be transferred.

Cells or wells shall 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 shall be controlled at 37°C.

Day 3

At the end of the 24 hour period, the information recorded by the conductance measuring equipment shall be analysed and interpreted using criteria defined by the manufacturers of the equipment.

Where a well or cell is identified as being positive for salmonella, the result shall be confirmed by subculturing the contents of the well or cell on to two plates BGA(d) using a 2.5 mm diameter loop. The BGA plates shall 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 shall be 0.5 cm–1.0 cm. The plates shall be incubated at 37°C overnight.

Day 4

The plates of BGA shall be examined and a minimum of 3 colonies from the plates showing suspicion of salmonella growth shall be subcultured on to a blood agar plate and a MacConkey agar plate and into biochemical composite media or equivalent. These media shall be incubated at 37°C overnight.

Day 5

The incubated composite media or equivalent shall be examined and the findings recorded, discarding cultures which are obviously not salmonella. Slide seriological tests shall 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 shall be typed by slide serology and a subculture sent (in Scotland) to the Lasswade Veterinary Laboratory situated at Penicuik, Midlothian, and (in England and Wales) to a Veterinary Investigation Centre of the Ministry for further typing.

(a) Buffered Peptone Water/Lysine/Glucose (BPW/L/G)—Ogden (1988)

(b) Selenite Cystine TMAO Dulcitol (SC/T/D)—Eastern and Gibson (1985)

(c) Lysine Decarboxylase Glucose (LD/G)—Ogden (1988)

(d) Brilliant Green Agar (Modified) (BGA)—Edel and Kampelmacher (1969)

References:

Ogden I. D. (1988) International Journal of Food Microbiology 7 287-297.

Easter M. C. and Gibson D. M. (1985) Journal of Hygiene 94 245-262.

Edel W. & Kampelmacher E. H. (1969) Bulletin of the World Health Organisation 41 297-306.

Edel W. & Kampelmacher E. H. (1973) Bulletin of the World Health Organisation 48 167-174.