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 978-0-626-21009-0 SANS 5761:2008Edition 2SOUTH AFRICAN NATIONAL STANDARD Examination for the presence of viable spores of mesophilic Clostridium org
4、anisms in foods Published 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 5761:2008 Edition 2 Table of changes Change No. Date Scope Foreword This South A
5、frican standard was approved 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 su
6、persedes SABS SM 761:1975 (first edition). SANS 5761:2008 Edition 2 1 Contents Page Foreword 1 Scope . 3 2 Normative references 3 3 Media and reagents. 3 4 Storage and dispersion of the sample. 5 5 Procedure 5 SANS 5761:2008 Edition 2 2 This page is intentionally left blank SANS 5761:2008 Edition 2
7、3 Examination for the presence of viable spores of mesophilic Clostridium organisms in foods 1 Scope This standard specifies a method for the determination of the presence of viable spores of mesophilic Clostridium organisms in foods. 2 Normative references The following referenced documents are ind
8、ispensable for the application of this document. For dated 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 St
9、andards South Africa. SANS 5553 (SABS SM 553), Media and reagents for microbiological tests. 3 Media and reagents 3.1 General The general requirements for the ingredients and for the preparation of media and reagents shall comply with those given in SANS 5553. 3.2 Sodium sulfite solution Dissolve 1
10、g of sodium sulfite in 25 mL of water, sterilize by filtration using a filter with a diameter of 0,22 m, and store at 3 C to 5 C in a stoppered bottle. Use within 14 d. 3.3 Ferric citrate solution Dissolve 1,75 g of ferric citrate scales in 25 mL of water, heating for about 5 min to aid solution. Co
11、ol, sterilize by filtration using a filter with a diameter of 0,22 m, and store at 3 C to 5 C in a stoppered bottle. Use within 14 d. 3.4 Differential reinforced Clostridium medium (double strength) 3.4.1 Basal medium ingredients Peptone .10 g Meat extract.10 g Sodium acetate 3 g Yeast extract1,5 g
12、Soluble starch 1 g SANS 5761:2008 Edition 2 4 Glucose . 1 g L-cysteine 0,5 g Agar .0,5 g 3.4.2 Preparation Add the peptone, meat extract, sodium acetate, and yeast extract to 400 mL of water. Prepare a starch solution in a further 100 mL of water by making a cold slurry in a small volume of the wate
13、r, boiling the rest, and then stirring it into the paste. Add this starch solution to the other mixture, steam for 30 min to dissolve all the ingredients, and then add the glucose and cysteine (which dissolve readily). Adjust the pH value to 7,1 and filter while hot through paper pulp. Dispense 13 m
14、L volumes of this basal medium into bottles and sterilize by autoclaving at a temperature of (03120+) C for 15 min. On the day that the medium is to be used, steam it for approximately 10 min, allow to cool to 50 C, and to each bottle of the medium add 0,25 mL of the sodium sulfite solution (see 3.2
15、) and 0,25 mL of the ferric citrate solution (see 3.3). NOTE Commercially available reinforced Clostridium medium may also be used but made up to double strength with 500 mL of water and to which 0,25 mL sodium sulfite (see 3.2) and 0,25 mL ferric citrate (see 3.3) solutions are added on the day of
16、use. 3.5 Reinforced Clostridium agar 3.5.1 Ingredients Agar .15 g Peptone .10 g Meat extract.10 g Sodium acetate 3 g Sodium chloride. 5 g Yeast extract 3 g Soluble starch 1 g Dextrose 5 g L-cysteine 0,5 g 3.5.2 Preparation Prepare the agar medium as for the basal medium in 3.4.2, but use 1 L of wate
17、r. Filter through paper pulp. Adjust the pH value to 7,4 units, and sterilize by autoclaving at a temperature of (03120+) C for 15 min. Aseptically dispense 15 mL volumes into sterile Petri dishes and allow to solidify. 3.6 Hydrogen peroxide solution, 3 % by mass SANS 5761:2008 Edition 2 5 3.7 Dilue
18、nt Dissolve 1 g of peptone and 8,5 g of sodium chloride in 1 L of water. Adjust the pH 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. 4 Storage and dispersion of the sample 4
19、.1 Storage Store the sample, of mass at least 200 g, for the minimum practicable period under such conditions that changes in composition are prevented or kept to a minimum. 4.2 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
20、sterile forceps, transfer approximately 20 g of the sample to a previously tared and sterile sample bottle. Add enough diluent (see 3.7) 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
21、 case for longer than 2,5 min. Use the 1 in 10 dispersion of the sample so obtained for the procedure in clause 5. 5 Procedure 5.1 Pasteurization So heat 50 mL of the 1 in 10 dispersion (see 4.2) in a water bath at 82 C to 85 C, that the temperature of the contents is maintained at 80 C for 60 s. Qu
22、ickly cool the heated dispersion to below 45 C in running tap water. 5.2 Differential cultivation (incubation at 30 C) Transfer 13 mL of the pasteurized dispersion of the sample to a bottle of differential reinforced Clostridium medium (see 3.4) and incubate at 30 C for 120 h. Examine the contents a
23、t intervals for possible black coloration. Confirm the presence of anaerobes in cultures showing black coloration by subculturing on to plates of reinforced Clostridium agar (see 3.5), incubating these both aerobically and anaerobically at 30 C for 48 h, and examining for any colonies which develop.
24、 Consider the test positive if growth of colonies occurs on the anaerobic plate, and the aerobic plate shows scanty or no growth. If any clostridia are present, test them for catalase production (see 5.5). SANS 5761:2008 Edition 2 6 5.3 Differential cultivation (incubation at 35 C) Repeat the proced
25、ure in 5.2 but incubate at 35 C to 37 C instead of 30 C to ensure detection of C. perfringens. 5.4 Confirmation Where growth occurs both aerobically and anaerobically, suspect colonies from the anaerobic plate shall be resubcultured on to duplicate plates as in 5.2 and 5.3 and this process shall be
26、repeated until the presence or absence of Clostridium organisms is confirmed. 5.5 Catalase production In the case where growth occurs anaerobically but not aerobically (see 5.2 and 5.3), flood the surface of the anaerobically incubated plate with hydrogen peroxide solution (see 3.6). Consider the te
27、st for catalase production negative when no visible gas formation takes place within 10 min. 5.6 Microscopical examination Prepare a Gram-stained slide of the culture and examine it microscopically. 5.7 Conclusion Consider organisms to be members of the genus Clostridium that: a) grow anaerobically, but grow weakly or not at all aerobically; b) appear as Gram-positive rods under the microscope; and c) give a negative result in the catalase production test. Standards South Africa
