1、 Collection of SANS standards in electronic format (PDF) 1. Copyright This standard is available to staff members of companies that have subscribed to the complete collection of SANS standards in accordance with a formal copyright agreement. This document may reside on a CENTRAL FILE SERVER or INTRA
2、NET SYSTEM only. Unless specific permission has been granted, this document MAY NOT be sent or given to staff members from other companies or organizations. Doing so would constitute a VIOLATION of SABS copyright rules. 2. Indemnity The South African Bureau of Standards accepts no liability for any
3、damage whatsoever than may result from the use of this material or the information contain therein, irrespective of the cause and quantum thereof. ISBN 978-0-626-20606-2 SANS 5762:2008Edition 2SOUTH AFRICAN NATIONAL STANDARD Examination for the presence of viable Bacillus anthracis in foods Publishe
4、d by Standards South Africa 1 dr lategan road groenkloof private bag x191 pretoria 0001 tel: 012 428 7911 fax: 012 344 1568 international code + 27 12 www.stansa.co.za Standards South Africa SANS 5762:2008 Edition 2 Table of changes Change No. Date Scope Foreword This South African standard was appr
5、oved by National Committee StanSA TC 5140.26, Microbiological evaluation of foods, feeds and beverages, in accordance with procedures of Standards South Africa, in compliance with annex 3 of the WTO/TBT agreement. This document was published in February 2008. This document supersedes SABS SM 762:197
6、5 (first edition). SANS 5762:2008 Edition 2 1 Contents Page Foreword 1 Scope . 3 2 Normative references 3 3 Apparatus 3 4 Media and reagents 3 5 Storage and dispersion of the sample 5 6 Procedure . 5 SANS 5762:2008 Edition 2 2 This page is intentionally left blank SABS 5762:2008 Edition 2 3 Examinat
7、ion for the presence of viable Bacillus anthracis in foods 1 Scope This standard specifies a method for the examination for the presence of viable Bacillus anthracis in foods. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated
8、references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. Information on currently valid national and international standards can be obtained from Standards South Africa. SANS 171, Glassware and equipment for
9、 microbiological tests. SANS 5553 (SABS SM 553), Media and reagents for microbiological tests. 3 Apparatus The apparatus and equipment shall comply with the requirements of SANS 171. 4 Media and reagents 4.1 General The general requirements for the ingredients and for the preparation of media and re
10、agents shall comply with those given in SANS 5553. 4.2 Nutrient medium 4.2.1 Ingredients Peptone .10 g Beef extract . 5 g Sodium chloride. 5 g NOTE Commercially available nutrient broth of the same recipe may be used. SANS 5762:2008 Edition 2 4 4.2.2 Preparation Dissolve the ingredients in water and
11、 make up to 1 L. Adjust the pH value to 7,2. Dispense 10 mL volumes into bottles and sterilize by autoclaving at a temperature of (03120+) C for 15 min. 4.3 Nutrient agar To the nutrient medium (4.2) add 1,5 % of agar. Heat to dissolve the agar. Dispense 15 mL volumes into bottles and sterilize by a
12、utoclaving at a temperature of (03120+) C for 15 min. NOTE Commercially available nutrient agar of the same recipe may be used. 4.4 Charcoal-gelatin pellets1)4.5 Sterile filter paper strips Cut coarse, low ash filter paper into strips of size 10 mm 100 mm and sterilize these by autoclaving them (in
13、a suitable container) at a temperature of (03120+) C for 20 min. Take a representative sample 2 d after autoclaving and test it for sterility by immersing it aseptically in nutrient medium (see 4.2). Incubate at 37 C 2 C for 72 h. At the end of this time there shall be no evidence of bacterial growt
14、h. 4.6 Sodium chloride solution Dissolve 9 g of sodium chloride in 1 L of water. Dispense 10 mL volumes into bottles and sterilize by autoclaving at a temperature of (03120+) C for 15 min. 4.7 Blood agar 4.7.1 Basal medium ingredients Agar .15 g Peptone .10 g Beef extract .10 g Sodium chloride. 5 g
15、4.7.2 Preparation Dissolve the ingredients in water by boiling, and make up to 1 L. Adjust the pH value to 7,5. Dispense 15 mL volumes into bottles and sterilize by autoclaving at a temperature of (03120+) C for 15 min. Immediately before use, melt the basal medium, cool to 45 C to 50 C, and add to
16、each bottle 1 mL of fresh sterile blood (horse blood gives excellent results). Mix the blood and the basal medium well and aseptically dispense 15 mL volumes into sterile Petri dishes. The blood agar plates, when poured and set, should show no haemolysis. NOTE If commercially available blood agar ba
17、se is used, add 5 % sterile, defibrinated mammalian blood before pouring 15 mL quantities into Petri dishes for blood agar plates. 1) These are available from commercial sources. SABS 5762:2008 Edition 2 5 4.8 Diluent Dissolve 1 g of peptone and 8,5 g of sodium chloride in 1 L of water. Adjust the p
18、H so that after sterilization its value will be 7,0 0,1. Dispense 9 mL and 200 mL volumes into bottles and sterilize by autoclaving at a temperature of (03120+) C for 15 min. 5 Storage and dispersion of the sample 5.1 Storage Store the sample (of mass at least 200 g) for the minimum practicable peri
19、od under such conditions that changes in composition are prevented or kept to a minimum. 5.2 Dispersion If frozen, allow the sample to thaw in a refrigerator at 5 C to 10 C for not longer than 18 h. Using a pair of sterile forceps, transfer approximately 20 g of sample to a previously tared and ster
20、ile sample bottle. Add enough diluent (see 4.8) to give a 1 in 10 mass per volume dispersion, and macerate the mixture for sufficient time to allow 15 000 to 20 000 revolutions of the macerator blades, but in no case for longer than 2,5 min. Use the 1 in 10 dispersion of the sample so obtained for t
21、he procedure in clause 6. 6 Procedure 6.1 Pasteurization So heat 50 mL of the 1 in 10 dispersion (see 5.2) in a water bath at 82 C to 85 C, that the temperature of the contents is maintained at 80 C for 60 s. Quickly cool the heated dispersion to below 45 C in running tap water. 6.2 Sporulation Dip
22、five filter paper strips (see 4.5) into the pasteurized dispersion and then place each strip in a large sterile test tube loosely plugged with cotton wool. Keep these tubes at 25 C 5 C for 10 d to 14 d under aerobic conditions. 6.3 Cultivation Transfer 1 mL of the pasteurized dispersion to a bottle
23、of nutrient medium (see 4.2) and incubate at 37 C 2 C for 18 h to 24 h. Streak a loopful of this culture on to a plate of nutrient agar (see 4.3). Incubate under adequately aerobic conditions at 37 C 2 C for 20 h to 24 h. Examine the plate for large (3 mm to 5 mm), flat, white to grayish-white circu
24、lar colonies that appear like ground glass. Remove material from suspected colonies for subculture and for microscopical examination. 6.4 Microscopical examination Prepare Gram-stained smears from suspected colonies (see 6.3) and examine these under the microscope at a magnification of 800 to 1 000.
25、 Straight, rectangular shaped, relatively large rods (of size 4 m to 8 m by 1 m to 1,5 m), which are strongly Gram-positive and are usually arranged in chains end to end, but which can occur singly in pairs, are suspect. SANS 5762:2008 Edition 2 6 6.5 Gelatinase production Transfer a portion of the
26、material from suspected colonies (see 6.3) to a bottle of nutrient medium (see 4.2) to which a charcoal-gelatin pellet (see 4.4) has been added and incubate this culture at 37 C 2 C for 18 h to 24 h. At the end of this time examine for gelatinase production. 6.6 Motility Suspend a portion of the mat
27、erial from suspected colonies (see 6.3) in sodium chloride solution (see 4.6) as to produce a dilute suspension. Examine the organisms in the suspension for motility by the “hanging drop“ method. 6.7 Haemolysis Streak a portion of the material from suspected colonies (see 6.3) on to blood agar plate
28、s (see 4.7) and incubate these at 37 C 2 C for 18 h to 24 h. Examine the resulting growth for haemolytic action. 6.8 Conclusion Consider organisms to be presumptive Bacillus anthracis that a) show the microscopical characteristics described in 6.4, b) produce gelatinase (6.5), c) are non-motile (6.6
29、), and d) are usually non-haemolytic (6.7). 6.9 Confirmation Forward pure cultures of the organisms considered as Bacillus anthracis to a recognized pathological laboratory for confirmation by animal inoculation and biochemical test work. WARNING Destroy all material contaminated with suspected Baci
30、llus anthracis by incineration or sterilize by autoclaving at a temperature of (03120+) C for 30 min to 40 min to prevent cross-infection by these organisms. Handle all suspect cultures and material with the utmost care and get prompt medical attention if any of the personnel develop carbuncle-like
31、skin lesions. Bring the possibility of anthrax infection to the notice of the physician as rapid diagnosis is essential for successful treatment. 6.10 Examination for spores (indirect procedure) NOTE In material that is moderately or heavily contaminated with other bacteria, growth of these other or
32、ganisms makes the detection of B. anthracis by the procedure described in clause 6 difficult or impossible. In this case proceed as in 6.10. Transfer the five strips of filter paper inoculated in 6.2 to a bottle of nutrient medium (see 4.2), pasteurize it and its contents as described in 6.1, and subsequently incubate and examine this culture as described in 6.3 to 6.9. Standards South Africa
