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SN/T 1071-2014: Method for the determination of anaerobic sulfite-reducing clostridia in food for export
SN/T 1071-2014
SN
ENTRY-EXIT INSPECTION and QUARANTINE STANDARD
OF THE PEOPLE’S REPUBLIC OF CHINA
Replacing SN/T 1071-2002
Method for the determination of anaerobic
sulfite-reducing clostridia in food for export
ISSUED ON. JANUARY 13, 2014
IMPLEMENTED ON. AUGUST 01, 2014
Issued by. General Administration of Quality Supervision, Inspection and
Quarantine of PRC
Table of Contents
Foreword ... 3 
1 Scope ... 4 
2 Terms and definitions ... 4 
3 Equipment and materials ... 4 
4 Media and reagents ... 5 
5 Storage and transportation of samples ... 5 
6 Sample preparation ... 5 
7 Testing steps and calculations ... 6 
8 Confirmation test ... 7 
Appendix A (Normative) Culture medium and reagents ... 8 
Appendix B (Normative) Most proximate number (MPN) of 1 g (mL) anaerobic
sulfite-reducing clostridia ... 11 
Foreword
This standard was drafted in accordance with the rules given in GB/T 1.1-2009.
This standard replaces SN/T 1071-2002 “Method for the determination of
anaerobic sulfite-reducing clostridia in food for import and export”. As compared
with SN/T 1071-2002, the main technical changes of this standard are as
follows.
- CHANGE the original standard name “Method for the determination of
anaerobic sulfite-reducing clostridia in food for import and export” into
“Method for the determination of anaerobic sulfite-reducing clostridia in food
for export”;
- ADD normative references;
- MODIFY and STANDARDIZE “equipment and materials”;
- ADD the methods for the collection, storage, transportation and sample
preparation of “non-frozen and corrosive samples”;
- ADD that “respectively pipette 1 mL of blank dilution into two sterile dishes
for blank control” in the plate counting method;
- ADD examples of calculation of results in the plate counting method;
- MODIFY the numerical unit “/g (mL)” in the plate counting method to “CFU/g
(mL)”;
- MODIFY the numerical unit “/g(mL)” in the most approximate number (MPN)
method to “MPN/g (mL)”;
- In Appendix A, ADD the ingredients and preparation method of 2% sterile
agar.
This standard was proposed by and shall be under the jurisdiction of the
National Certification and Accreditation Administration.
Drafting organizations of this standard. Liaoning Entry-Exit Inspection and
Quarantine Bureau of the People's Republic of China.
The main drafters of this standard. Cao Jijuan, Wang Jinling, Zhang Ying, Xu
Junyi, Li Junhuan.
This Standard replaces the standard previously issued as follows.
- SN/T 1071-2002.
Method for the determination of anaerobic
sulfite-reducing clostridia in food for export
1 Scope
This standard specifies the detection method of anaerobic sulfite-reducing
clostridia in food for export.
This standard applies to the detection of anaerobic sulfite-reducing clostridia in
food for export.
2 Terms and definitions
The following terms and definitions apply to this document.
2.1
Anaerobic sulfite-reducing clostridia
A group of anaerobic, catalase-negative, Gram-positive Clostridia that can
reduce sulfite to sulfide. It is one of the evaluation indicator bacteria for food,
water, food processing equipment, food production environment and other
hygiene conditions.
3 Equipment and materials
3.1 Refrigerator. 2 °C ~ 5 °C.
3.2 Constant temperature water bath. 50 °C ± 1 °C.
3.3 Constant temperature incubator. 36 °C ± 1 °C.
3.4 Balance. Sensitivity 0.1 g.
3.5 Homogenizer and homogenizing cup or homogenized bag.
3.6 Anaerobic conditions and related equipment. SELECT an appropriate size
anaerobic tank, PLACE it in a sealed box with good sealing effect, or SELECT
other equipment that maintains anaerobic conditions and PLACE it in an
anaerobic thermostatic workstation for culturing.
3.7 Sterile straws. 1 mL (with 0.01 scale), 10 mL (with 0.1 scale) or
micropipettes and tips.
sample contamination. If the spores of the anaerobic sulfite-reducing clostridia
are tested, it may heat the 1.10 sample homogenate at 75 °C for 20 min or BOIL
it and MAINTAIN 10 min heating, then USE running water to quickly cool it to
room temperature, for further serial 10-fold incremental dilution.
7 Testing steps and calculations
7.1 Plate counting method
7.1.1 It is suitable for testing the unprocessed fresh foods and the food with
anaerobic sulfite-reducing clostridia > 10 CFU/g (mL).
7.1.2 SELECT the appropriate three consecutive dilutions, respectively
PIPETTE 1 mL of diluted sample solution from each dilution in the sterile culture
dish. MAKE two plates for each dilution. At the same time, respectively
PIPETTE 1 mL of blank dilution into each of the two sterile dishes for blank
control.
7.1.3 In a timely manner, POUR about 15 mL of ferrous sulfite agar medium
(which can be placed in a constant temperature water bath at 50 °C ± 1 °C)
cooled at 50 °C into a plate, TURN the plate to mix it evenly. From the end of
the preparation of the initial dilution to the pouring of the medium in the last
plate, the time consumed shall not exceed 15 min.
7.1.4 After the mixture is solidified, POUR 10 mL of 2% sterile agar onto the
solidified medium as a barrier layer.
7.1.5 After the barrier layer is solidified, PLACE the prepared plate at 36 °C ±
1 °C for anaerobic culture for 24 h ~ 48 h. If there are special requirements for
the culture temperature (e.g. 46 °C), it can be cultured as required.
7.1.6 Anaerobic sulfite-ring clostridia is black colonies on iron sulfite agar.
7.1.7 Randomly TAKE 10 typical colonies from the counted plates (with 20 ~
200 colonies), respectively PERFORM take cultures for confirmation test.
COUNT the positive results.
7.1.8 The number of typical colonies on the plate is multiplied by the proportion
of colonies in the of the confirmed anaerobic sulfite-reducing clostridia colonies,
and multiplied by the dilution factor of the sample, to obtain the count of
anaerobic sulfite-reducing clostridia in the sample, it is reported as the
anaerobic sulfite-reducing clostridia CFU g (mL). For example, there are 85 and
80 colonies in each of the two plates of the 10-1 sample solution, whilst it is
confirmed that 8 and 4 of the 10 colonies are respectively anaerobic sulfite-
reducing clostridia, then the number of anaerobic sulfite-reducing clostridia per
gram or milliliter of food is (85 × 8/10 + 80 × 4/10)/2 × 10 = 500.
Appendix A
(Normative)
Culture medium and reagents
A.1 Sodium chloride tryptone dilution
A.1.1 Ingredients
Tryptone. 1.0 g
Sodium chloride (NaCl). 8.5 g
Distilled water. 1000.0 mL
A.1.2 Preparation methods
MIX the above ingredients, HEAT to dissolve it, ADJUST the pH to 7.2 ± 0.1,
AUTOCLAVE it at 121 °C for 15 min.
A.2 Ferric sulfite agar
A.2.1 Ingredients
Tryptone. 15.0 g
Soy protein. 5.0 g
Yeast extract. 5.0 g
Sodium metabisulfite (Na2S2O5). 1.0 g
Ammonium ferric citrate [(NH4)3FeCl2H10O4]. 1.0 g
Agar. 20.0 g
Distilled water. 1000.0 mL
A.2.2 Preparation method
MIX the above ingredients, HEAT to dissolve it, ADJUST the pH to 7.6 ± 0.1,
AUTOCLAVE it at 121 °C for 15 min.
A.3 Froth medium
A.3.1 Ingredients