The Poultry Breeding Flocks and Hatcheries Order 1993

1.  Bacteriological method (Rappaports) for the detection of salmonella in chick box liners, composite faeces samples, meconium samples and carcases.

Samples submitted for testing for the presence of salmonella shall be examined in the following prescribed manner on consecutive days and, where a laboratory at which samples have been received for testing on any day is unable to commence such an examination on that day, the samples shall be stored in a refrigerator at between 1 degree C and 4 degrees C until required for examination.

• Day 1

  (a)

  Chick box liners: a one gram portion shall be taken from a soiled area on each liner and the portions from separate liners shall be bulked together and placed in Buffered Peptone Water (BPW)(a), at the rate of 1 gram of liner in 10 ml of BPW up to a maximum of 10 grams in 100 ml of BPW.

  (b)

  Composite faeces and meconium samples: the samples shall be thoroughly mixed and a sub-sample weighing not more than 10 grams shall be placed in BPW at the rate of 1 gm sample to 10 ml BPW to a maximum of 10 grams in 100 ml BPW.

  (c)

  Carcases of chicks: there shall be removed samples of the yolk sac, liver and terminal intestines to include portions of small intestines, large intestine and caecal tonsil.The samples of organs taken from the carcases of chicks submitted shall then be bulked together and placed in BPW at the rate of 1 gram of bulked tissue in 10 ml BPW up to a maximum of 10 grams of tissue in 100 ml BPW.

  The inoculated BPW shall then be incubated at 37°C for 18-24 hours.

• Day 2

  0.1 ml from the incubated BPW shall be inoculated into 10 ml of Rappaports Vassiliadis (RV) broth or Rappaports Vassiliadis Soya Peptone (RVS)(b) broth and incubated at 41.5 degrees C ± 0.5° C for 18-24 hours.

• Day 3

  The RV or RVS broth shall be plated out on to two plates of Brilliant Green Agar (BGA)(c) using a 10 microlitres loop. The BGA plates shall be inoculated with a droplet taken from the edge of the surface of the fluid and drawing the loop over the whole of one plate in a zigzag pattern and continuing to the second plate without recharging the loop. The space between the loop streaks shall be 0.5-1.0 cm. The plates shall be incubated at 37 degrees C for 18-24 hours, and the RV or RVS broth reincubated at 41.5 degrees C ± 0.5° C for a further 18-24 hours.

• Day 4

  (i)

  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 degrees C for 18-24 hours.

  (ii)

  The reincubated RV or RVS broth shall be plated out, and the plates incubated, as described in Day 3.

• Day 5

  (i)

  The incubated plates and composite media or equivalent shall be examined and the findings recorded, discarding cultures which are obviously not salmonella. Slide serological tests shall be performed using salmonella polyvalent “O”(Groups A-S) 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 to Group level by slide serology.

  (ii)

  The plates of BGA prepared at Day 4(ii) shall be examined and further action taken as described in Day 4(i) and Day 5(i).