GB/T 13091-2018 English PDF (GBT13091-2018)

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GB/T 13091-2018: Determination of Salmonella in feeds
GB/T 13091-2018
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
ICS 65.120
B 46
Replacing GB/T 13091-2002
Determination of Salmonella in feeds
ISSUED ON: SEPTEMBER 17, 2018
IMPLEMENTED ON: APRIL 01, 2019
Issued by: State Administration for Market Regulation;
Standardization Administration of PRC.
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Normative references ... 4
3 Medium or material... 4
4 Instruments and equipment ... 5
5 Samples ... 5
6 Test method (see Appendix A for the test procedure diagram) ... 6
7 Expression of results ... 10
Appendix A (Normative) Test procedure diagram ... 11
Appendix B (Normative) Preparation and test methods of medium and reagents ... 12
Determination of Salmonella in feeds
1 Scope
This standard specifies the inspection methods for Salmonella in feed.
This standard applies to the inspection of Salmonella in feed and feed additives.
2 Normative references
The following documents are essential to the application of this document. For the dated
documents, only the versions with the dates indicated are applicable to this document;
for the undated documents, only the latest version (including all the amendments) is
applicable to this standard.
GB/T 6682 Water for analytical laboratory use - Specification and test methods
3 Medium or material
Unless otherwise stated, only reagents confirmed to be of analytical grade are used in
the analysis.
3.1 Water: It shall meet the requirements of grade-3 water in GB/T 6682.
3.2 Buffered peptone water (BPW): See B.1 in Appendix B.
3.3 Magnesium chloride malachite green (RV) enrichment solution: See B.2 in
Appendix B.
3.4 Cystine selenite (SC) enrichment solution: See B.3 in Appendix B.
3.5 Bismuth sulfite (BS) agar: See B.4 in Appendix B.
3.6 Deoxycholate-Hydrogen-Sulfide-Lactose (DHL) agar: See B.5 in Appendix B.
3.7 Salmonella chromogenic medium.
3.8 Nutrient agar (NA): See B.6 in Appendix B.
3.9 Trisaccharide iron agar (TSI): See B.7 in Appendix B.
3.10 Peptone water and indigo-based reagents: See B.8 in Appendix B.
3.11 Urea agar (pH7.2): See B.9 in Appendix B.
3.12 Potassium cyanide (KCN) medium: See B.10 in Appendix B.
3.13 Lysine decarboxylase test medium: See B.11 in Appendix B.
3.14 Sugar fermentation tube: See B.12 in Appendix B.
3.15 Ortho-nitrophenol β-D-galactopyranoside (ONPG) medium: See B.13 in
Appendix B.
3.16 Semi-solid agar: See B.14 in Appendix B.
3.17 Sodium malonate medium: See B.15 in Appendix B.
3.18 Salmonella factor O and H polyvalent diagnostic serum, Vi factor diagnostic serum.
4 Instruments and equipment
4.1 Refrigerator: 2 °C ~ 5 °C.
4.2 Constant temperature incubator: 36 °C ± 1 °C, 42 °C ± 1 °C.
4.3 Homogenizer.
4.4 Oscillators.
4.5 Electronic balance: Sensitivity 0.1 g.
4.6 Sterile conical flasks: Capacity 500 mL, 250 mL, or sterile homogeneous bag.
4.7 Sterile pipettes: 1 mL (with 0.01 mL scale), 10 mL (with 0.1 mL scale), or
micropipette and tip.
4.8 Sterile petri dishes: Diameter 60 mm, 90 mm.
4.9 Sterile test tubes: 3 mm × 50 mm, 10 mm × 75 mm.
4.10 pH meter or pH colorimetric tube or precision pH test paper.
4.11 Automatic microbial biochemical identification system.
5 Samples
5.1 Sampling principles
The collection of samples shall follow the principles of randomness and
representativeness. The sampling process shall follow aseptic procedures, to prevent all
possible external contamination.
5.2 Sampling method
5.2.1 Samples shall be collected from the same batch of products. The sampling volume
of each sample shall meet the requirements for microbiological index inspection, which
is generally not less than 500 g (mL).
5.2.2 For solid products not larger than 500 g or liquid products not larger than 500 mL
in independent packaging, take the complete package.
5.2.3 For individually packaged liquid products larger than 500 mL, it shall be shaken
or stirred with a sterile stick before sampling, to make them uniform, then collect an
appropriate amount of samples and put them into a sterile sampling container, as a
sample.
5.2.4 For solid products with individual packages greater than 500 g, use a sterile
sampler to take appropriate samples from different parts of the same package; put them
into the same sterile sampling container, as one sample.
5.3 Storage and transport of collected samples
5.3.1 The samples shall be sent to the laboratory for testing, as soon as possible.
5.3.2 The samples shall be kept intact during transportation.
5.3.3 The samples shall be stored under conditions close to the original storage
temperature, OR necessary measures shall be taken to prevent changes in the number
of microorganisms in the samples.
6 Test method (see Appendix A for the test procedure
diagram)
6.1 Pre-enrichment
Weigh 25g (mL) of sample under sterile conditions. Add it into a 500 mL sterile conical
flask, which was filled with 225 mL of sterile BPW. Place it in an oscillator. Oscillate
at 8000 r/min ~ 10000 r/min for 2 min ~ 3 min. If the sample is in liquid form, shake
and mix well. Incubate it at 36 °C ± 1 °C for 18 h ± 2 h.
Note: A sterile homogenizing bag can be used instead of the conical flask. Then beating with a
homogenizer for 2 min ~ 3 min, but the homogenizing bag cannot be used for hard or angular
samples, only the conical flask can be used, to prevent the homogeneous bag from being
ruptured and leaked, in the homogenization and enrichment process, which may cause pollution.
b) Reaction No. A2: Supplementarily conduct mannitol and sorbitol tests; the results
of the two tests for the indigase-positive variant of Salmonella are all positive,
but it needs to be judged in combination with the results of serological
identification.
c) Reaction No. A3: Supplementarily conduct ONPG test. If ONPG is negative, then
it is Salmonella, while lysine decarboxylase is positive; if lysine decarboxylase is
negative, then it is Salmonella paratyphi A.
d) If necessary, carry out identification of Salmonella biochemical groups according
to Table 6.
6.4.3 If a biochemical identification kit or an automatic microbial biochemical
identification system is selected, suspicious colonies can be picked from the nutrient
agar plate, according to the preliminary judgment results in 6.4.1. A bacterial
suspension with appropriate turbidity can be prepared with physiological saline. Use a
biochemical identification kit or an automatic microbial biochemical identification
system for identification.
6.5 Serological identification
6.5.1 Checking the culture for autoagglutination
Use 1.2% ~ 1.5% agar culture as the antigen, for slide agglutination test. Firstly, to rule
out self-agglutination reaction, add a drop of normal saline to a clean glass slide; mix
the culture to be tested in the normal saline, to make a homogeneous turbid suspension;
shake the slide gently for 30 s ~ 60 s; observe the reaction in dark (observe with a
magnifying glass if necessary). If there is visible bacterial agglutination, it is considered
to have self-agglutination; otherwise, there is no self-agglutination. For the non-self-
agglutinating culture, refer to the following method for serological identification.
6.5.2 Identification of the O antigen
Draw 2 areas of about 1 cm × 2 cm on the slide. Pick 1 loop of bacteria to be tested. Put
1/2 loop on the upper part of each area on the slide. Add 1 drop of multivalent bacteria
O serum, to the lower pa...