Search Legislation

Animal By-Products Order (Northern Ireland) 2002

What Version

 Help about what version
  • Latest available (Revised)
  • Original (As made)

Status:

This is the original version (as it was originally made). This item of legislation is currently only available in its original format.

Articles 2, 9 and 10

SCHEDULE 3Sampling and Testing Methods

Part IManner of Sampling

Method 1

1.  In accordance with the following table, samples of approximately equal size shall be extracted evenly from the whole of the rendered material. These samples shall then be divided into groups of approximately equal numbers, the number of groups being the number of aggregate samples specified in the table. The samples in each group shall then be mixed together to form aggregate samples.

Total quantity of rendered material consigned from the premisesNumber of samples extractedNumber of aggregate samples obtained by mixing the relevant number of samples
Loose animal protein
up to 1 tonne71
1–2.5 tonnes72
2.5–10 tonnes√20 × weight of sampled portion in tonnes2
10–40 tonnes√20 × weight of sampled portion in tonnes3
over 40 tonnes√20 × weight of sampled portion in tonnes4
(maximum – 40 incremental samples)
Bagged animal protein
1–16 bags41
17–200 bagsNo. of bags of sampled portion2
201–800 bags√No. of bags of sampled portion3
over 800 bags√No. of bags of sampled portion4
(maximum – 40 incremental samples)

2.  Each aggregate sample shall be placed into a separate sterile receptacle and each shall be thoroughly mixed by stirring or shaking.

3.  Approximately equal amounts shall be taken from each aggregate sample and mixed so as to provide a single final sample of approximately 500 grammes. This final sample shall be transferred into a suitable sterile screw top container which shall then be sealed and marked to indicate its identity.

Method 2

1.  In accordance with the following table, samples of approximately equal size shall be extracted evenly from the whole of the rendered material. These samples shall then be divided into groups of approximately equal numbers, the number of groups being the number of aggregate samples specified in the table. The samples in each group shall then be mixed together to form aggregate samples.

Total consignments consigned from the premisesNumber of samples extractedNumber of aggregate samples obtained by mixing the relevant number of samples
Loose or bagged animal protein
1–5 consignments1 per consignment1
6–10 consignments1 per consignment2
11–15 consignments1 per consignment3
Over 15 consignments1 per consignment4

For the purpose of this method “consignment” means the total quantity of rendered material loaded onto a single vehicle or trailer.

2.  Each aggregate sample shall be placed into a separate sterile receptacle and each shall be thoroughly mixed by stirring or shaking.

3.  Approximately equal amounts shall be taken from each aggregate sample and mixed so as to provide a single final sample of approximately 500 grammes. This final sample shall be transferred into a suitable sterile screw top container which shall then be sealed and marked to indicate its identity.

Part IIMethod for the Isolation of clostridium perfringens

Time of testing

1.  Tests shall 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 shall be stored in a refrigerator at between 2°C and 8°C until required. If the sample has been refrigerated it shall be removed from the refrigerator and stored at room temperature for at least one hour before the test is started.

Samples

2.  Tests shall be carried out using two 10 gramme portions of each sample submitted for testing. Each 10 gramme sample shall be placed aseptically in a jar containing 90 ml Clostridium perfringens diluent consisting of 0.1% peptone and 0.8% sodium chloride at a pH of 7 and mixed thoroughly until the sample is evenly suspended.

Inoculations

3.  For each portion of the sample 1 ml of suspension/solution shall be transferred to a sterile 90 mm petri dish (in duplicate), to which 15 ml of egg yolk-free tryptose sulphite cycloserine agar (EY-free TSC agar)(1) at a temperature of 46°C ± 1°C shall be added and immediately gently mixed by swirling the dish with 5 clockwise and 5 anticlockwise circular movements.

4.  Once the agar has set, each agar plate shall be overlaid with a further 10 ml EY-free TSC agar at a temperature of 46°C ± 1°C. Once the overlay has set and with the plate lids uppermost the plates shall be incubated anaerobically at 37°C ± 1°C for 20 hours ± 2 hours.

Samples with colonies of Clostridium perfringens

5.  After incubation each set of duplicate plates shall 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 shall be followed to establish whether or not the colonies are Clostridium perfringens.

6.  In the case of each plate 10 characteristic colonies of Clostridium perfringens shall each be subcultured onto a further EY-free TSC agar plate. If there are less than 10 colonies on the plate, all characteristic colonies shall be subcultured onto further plates. The plates shall be incubated anaerobically at 37°C ± 1°C for 20 hours ± 2 hours.

7.  If the surface area of the plates is overgrown and it is not possible to select well isolated characteristic colonies, 10 suspect colonies shall each be subcultured onto duplicate EY-free TSC agar plates and incubated anaerobically at 37°C ± 1°C for 20 hours ± 2 hours.

8.  One characteristic colony from each plate shall be subcultured onto EY-free TSC agar and incubated anaerobically at 37°C ± 1°C for 20 hours ± 2 hours.

Subcultured colonies

9.  After incubation each plate shall be examined for colonies characteristic of Clostridium perfringens. All colonies characteristic of Clostridium perfringens shall be—

(a)stab inoculated into motility nitrate medium(2); and

(b)inoculated into either lactose gelatine medium(3) or charcoal gelatine discs(4) and incubated anaerobically at 37°C ± 1°C for 20 hours ± 2 hours.

Examination of subcultures

Motility

10.  The motility nitrate medium shall be examined for the type of growth along the stab line. If there is evidence of diffuse growth out into the medium away from the stab line, the bacteria shall be considered to be motile.

Reduction of nitrate to nitrite

11.  After examination of the motility nitrate medium 0.2ml to 0.5ml of nitrite detection reagent shall be added to it. The formation of a red colour confirms that the bacteria have reduced nitrate to nitrite. Cultures that show a faint reaction (i.e. a pink colour) should be discounted. If no red colour is formed within 15 minutes, a small amount of zinc dust shall be added and the plate allowed to stand for 15 minutes. If a red colour is formed after the addition of zinc dust no reduction of nitrate to nitrite has taken place.

Production of gas and acid from lactose and liquefaction of gelatine

12.  The lactose gelatine medium shall be examined for the presence of small gas bubbles in the medium.

13.  The lactose gelatine medium shall be examined for colour. A yellow colour indicates fermentation of lactose.

14.  The lactose gelatine medium shall be chilled for one hour at 2–8°C and then checked to see if the gelatine has liquefied. If the medium has solidified it shall 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 gelatine has liquefied.

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

Control Tests

16.  Control tests shall be carried out each day that a test is initiated using

(a)Clostridium perfringens NCTC 10662(5) no more than seven days old at the time of use;

(b)Escherichia coli NCTC 10418 or equivalent not more than seven days old at the time of use; and

(c)rendered animal protein which is free of Clostridium perfringens.

17.  10 gramme portions of the rendered animal protein shall be placed aseptically in each of two jars containing 90 ml Buffered Peptone Water (BPW)(6) and mixed thoroughly until the samples are evenly suspended.

18.  One colony of Clostridium perfringens shall be placed in 10 ml BPW and mixed to form an even suspension. 0.1 ml of the suspension shall be added to the suspension in the preceding paragraph. This shall be repeated for Escherichia coli.

19.  These are then treated and examined in the same way as test samples. If no typical colonies are formed then that day’s testing shall be invalid and shall be repeated.

Part IIIMethods for the Isolation of Salmonella

A. Bacteriological Method

1.  Tests shall 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 shall be stored in a refrigerator until required. If the sample has been refrigerated it shall be removed from the refrigerator and stored at room temperature for at least four hours before the test is started.

Day 1

2.  Tests shall be carried out in duplicate using two 25 gramme portions of each sample submitted for testing. Each 25 gramme sample shall be placed aseptically in a jar containing 225 ml Buffered Peptone Water (BPW) and incubated at 37°C ± 1°C for 18 hours.

Day 2

3.  0.1 ml from the jar of incubated BPW shall be inoculated into 10 ml Rappaport Vassiliadis (RV) broth(7) and incubated at 41.5°C ± 0.5°C for 24 hours.

Day 3

4.  The RV broth shall be plated out onto two 90 mm plates of Brilliant Green Agar(BGA)(8) or onto one 90mm plate of BGA and one 90 mm plate of Xylose Lysine Deoxycholate Agar (XLD)(9) using a 2.5 mm diameter loop. The 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 ± 1°C overnight.

5.  The residual RV broth shall be reincubated at 41.5°C ± 0.5°C for a further 24 hours.

Day 4

6.  The plates shall be examined and a minimum of 3 colonies from each plate showing suspicion of Salmonella growth shall be subcultured—

(a)onto a nutrient agar plate;

(b)onto a MacConkey agar plate(10); and

(c)into biochemical media suitable for the identification of Salmonella.

These media shall be incubated at 37°C ± 1°C overnight.

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

Day 5

8.  The incubated 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” and polyvalent “H” (phase 1 and 2) agglutinating sera on selected suspect colonies collected from the nutrient agar or MacConkey agar plates. If reactions occur with one or both sera, the colonies shall be typed by slide serology and a subculture sent to one of the Department’s laboratories at either Food Science Division, Newforge Lane, Belfast, BT9 5PX or, Veterinary Science Division, Stoney Road, Belfast, BT4 3SD, for further typing.

9.  The plates referred to in paragraph 7 shall be examined and further action taken as in paragraphs 6 and 8.

B. Electrical conductance method

1.  Tests shall be begun on receipt of the samples of processed animal protein 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 shall be stored in a refrigerator until required. If the sample has been refrigerated it shall be removed from the refrigerator and stored at room temperature for at least four hours before the test is started.

Day 1

2.  Tests shall be carried out in duplicate using two 25 gramme portions of each sample submitted for testing. Each 25 gramme sample shall be placed aseptically in a jar containing 225 ml Buffered Peptone Water (BPW) and incubated at 37°C ± 1°C for 18 hours.

Day 2

3.  The incubated BPW shall be added to Rappaport Vassiliadis Broth in electrical conductance cells or tubes to be inserted into electrical conductance cells. Detection of growth will utilise indirect impediometry as in the method of Donaghy and Madden (1993)(11). For cells or tubes containing more than 5 ml medium 0.2 ml of the BPW shall be added and for cells or tubes containing 5ml or less medium 0.1 ml of the BPW shall be added. Cells or tubes 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 tubes shall be kept at 42°C.

Day 3

4.  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 tube or cell is provisionally identified as being positive for Salmonella, the result shall be confirmed by subculturing the contents of the tube or cell onto two 90 mm plates of BGA or onto one 90 mm plate of BGA and one 90mm plate of Xylose Lysine Deoxycholate Agar (XLD) using a 2.5 mm diameter loop. The 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 ± 1°C overnight.

Day 4

5.  The plates shall be examined and a minimum of 3 colonies from each plate showing suspicion of Salmonella growth shall be subcultured—

(a)onto a nutrient agar plate;

(b)onto a MacConkey agar plate; and

(c)into biochemical media suitable for the identification of Salmonella.

These media shall be incubated at 37°C ± 1°C overnight.

Day 5

6.  The incubated 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” and polyvalent “H” (phase 1 and 2) agglutinating sera on selected suspect colonies collected from the nutrient agar or MacConkey plates. If reactions occur with one or both sera, the colonies shall be typed by slide serology and a subculture shall be sent to one of the Department’s laboratories at either Food Science Division, Newforge Lane, Belfast, BT9 5PX or Veterinary Science Division, Stoney Road, Belfast, BT4 3SD, for further typing.

Part IVMethod for the isolation of enterobacteriaceae

1.  Tests shall 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 shall be stored in a refrigerator until required at between 2°C and 8°C. If the sample has been refrigerated it shall be removed from the refrigerator and stored at room temperature for at least one hour before the test is started.

Samples

2.  Tests shall be carried out using five 10 gramme portions of each sample submitted for testing. Each 10 gramme sample shall be placed aseptically in a jar containing 90 ml Buffered Peptone Water (BPW) and mixed thoroughly until the sample is evenly suspended.

Inoculations

3.  For each portion of the sample 1 ml of solution shall be transferred to a sterile 90 mm petri dish (in duplicate). The plates shall be labelled to identify the portion of sample they were taken from. 15 ml of Violet Red Bile Glucose Agar (VRBGA)(12) at a temperature of 46°C ± 1°C shall 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 shall be overlaid with a further 10 ml VRBGA at a temperature of 46°C ± 1°C. Once the overlay has set, the plates shall 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 shall be examined for colonies characteristic of Enterobacteriaceae (purple colonies 1–2mm in diameter). All characteristic colonies on each plate shall be counted and the arithmetic mean of the duplicate plates taken.

The sample provisionally fails if either—

(a)any arithmetic mean is above 30(13); or

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

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

6.  After counting the colonies, characteristic colonies shall be taken at random from the agar plates, the number being at least the square root of the colonies counted. The colonies shall be subcultured onto a nutrient 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 shall be performed on each of the five subcultured colonies. Colonies which are oxidase-negative and glucose fermentation-positive shall be considered to be Enterobacteriaceae.

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

Controls

9.  Control tests shall 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)rendered animal protein which is free of Enterobacteriaceae.

10.  A 10 gramme portion of the rendered animal protein shall be placed aseptically in a jar containing 90 ml BPW and mixed thoroughly until the sample is evenly suspended.

11.  One colony of Escherichia coli shall be placed in 10 ml BPW and mixed to form an even suspension. 0.1 ml of the suspension shall 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 shall be invalid and shall be repeated.

(1)

EY- free TSC agar. See Hauschild, A.H.W. and Hilsheimer, R. (1974) Applied Microbiology 27:78–82. American Society for Microbiology, 1913 1 St N.W., Washington DC 20006, USA Back [1]

(2)

Motility nitrate medium. See Hauschild AHW, Gilbert RJ, Harmon SM, O'Keefe MF, Vahlefeld R, (1997) ICMSF Methods Study VIII, Canadian Journal of Microbiology 23, 884–892. National Research Council of Canada, Ottawa ON K1A OR6, Canada

(3)

Lactose gelatine medium. See Hauschild AHW, Gilbert R J, Harmon S M, O'Keefe M F, Vahlefeld R, (1997) ICMSF Methods Study VIII, Canadian Journal of Microbiology 23, 884–892

(4)

Charcoal gelatine discs. See Mackie and McCartney, (1996) Practical Medical Microbiology 14,509. Churchill Livingstone, Robert Stevenson House, 1–3 Baxter’s Place, Leith Walk, Edinburgh EH1 3AF

(5)

The National Collection of Type Cultures, Central Public Health Laboratory, 61 Colindale Avenue, London NW9 5HT.

(6)

Buffered Peptone Water. See Edel, W. and Kampelmacher, E.H. (1973) Bulletin of World Health Organisation, 48: 167–174, World Health Organisation Distribution and Sales, CH-1211, Geneva 27, Switzerland (ISSN 0042-9686)

(7)

Rappaport Vassiliadis Broth. See Vassiliadis, P., Pateraki, E., Papaiconomou, N., Papadakis, J. A,. and Trichopoulos, D. (1976) Annales de Microbiologie (Institut Pasteur) 127B; 195–200. Elsevier, 23rue Linois, 75724 Paris, Cedex 15, France

(8)

Brilliant Green Agar. See Edel, W and Kampelmacher, E.H. (1969) Bulletin of World Health Organisation, 41:297–306, World Health Organisation Distribution and Sales, CH-1211, Geneva 27, Switzerland (ISSN 0042-9686)

(9)

Xylose Lysine Deoxycholate Agar. See Taylor, W.I. (1965) American Journal of Clinical Pathology, 44:471–475, Lippincott and Raven, 227 E. Washington Street Philadelphia PA19106, USA

(10)

MacConkey agar. See (1963) International Standards for Drinking Water. World Health Organisation Distribution and Sales, CH-1211, Geneva 27, Switzerland

(11)

Donaghy and Madden. See Donaghy, J.A. and Madden R.H. (1993) International Journal of Food Microbiology. 17;281–288

(12)

Violet Red Bile Glucose Agar-See Mossel, 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.

(13)

An arithmetic mean of 30 is equivalent to 3×102 colony forming units per gramme of original sample

Back to top

Options/Help

Print Options

Close

Legislation is available in different versions:

Latest Available (revised):The latest available updated version of the legislation incorporating changes made by subsequent legislation and applied by our editorial team. Changes we have not yet applied to the text, can be found in the ‘Changes to Legislation’ area.

Original (As Enacted or Made):The original version of the legislation as it stood when it was enacted or made. No changes have been applied to the text.

Close

Opening Options

Different options to open legislation in order to view more content on screen at once

Close

More Resources

Access essential accompanying documents and information for this legislation item from this tab. Dependent on the legislation item being viewed this may include:

  • the original print PDF of the as enacted version that was used for the print copy
  • lists of changes made by and/or affecting this legislation item
  • confers power and blanket amendment details
  • all formats of all associated documents
  • correction slips
  • links to related legislation and further information resources
Close

More Resources

Use this menu to access essential accompanying documents and information for this legislation item. Dependent on the legislation item being viewed this may include:

  • the original print PDF of the as made version that was used for the print copy
  • correction slips

Click 'View More' or select 'More Resources' tab for additional information including:

  • lists of changes made by and/or affecting this legislation item
  • confers power and blanket amendment details
  • all formats of all associated documents
  • links to related legislation and further information resources