SN/T 1353-2004 English PDF (SNT1353-2004)
SN/T 1353-2004 English PDF (SNT1353-2004)
SN/T 1353-2004: Protocol of inspection and quarantine for import fishmeal
ENTRY-EXIT INSPECTION AND QUARANTINE INDUSTRY
STANDARDS OF THE PEOPLE REPUBLIC OF CHINA
Protocol of inspection and
quarantine for import fishmeal
ISSUED ON. JUNE 1, 2004
IMPLEMENTED ON. DECEMBER 1, 2004
Issued by. General Administration of Quality Supervision, Inspection and Quarantine of the PEOPLE Republic of China
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Normative references ... 4
3 On-site inspection and quarantine ... 5
4 Sampling ... 5
5 Sample preparation ... 6
6 Laboratory inspection and quarantine ... 7
7 Assessment and disposal of inspection and quarantine results ... 8
Annex A (Normative) Salmonella test method ... 9
Annex B (Normative) Test method for total bacterial count ... 13
Annex C (Normative) Mold count test method ... 16
Annex D (Normative) Test method for pathogenic vibrio ... 18
Protocol of inspection and
quarantine for import fishmeal
This Standard specifies the methods for on-site inspection and quarantine for imported fishmeal, methods for extraction and preparation of inspection and quarantine samples, laboratory inspection and quarantine, and assessment of inspection and quarantine results.
This Standard applies to the inspection and quarantine for import fishmeal. 2 Normative references
The provisions in the following documents become the provisions of this Standard through reference in this Standard. For dated references, the
subsequent amendments (excluding corrections) or revisions do not apply to this Standard. However, parties who reach an agreement based on this
Standard are encouraged to study if the latest editions of these documents are applicable. For undated references, the latest editions apply to this Standard. GB/T 5009.45 Method for analysis of hygienic standard of fish and other aquatic products
GB/T 6432 Determination of crude protein in feeds
GB/T 6433 Determination of crude fat in feeds
GB/T 6435 Method for the determination of moisture in feedstuffs
GB/T 6438 Method for the determination of crude ash in feedstuffs
GB/T 6439 Determination of water-soluble chlorides in feeds
GB/T 9825 Inspection of grain and oils - Determination of insoluble dietary fiber in cereals
GB/T 18088 Sampling for entry and exit animal quarantine
SN/T 0800.1 Inspection of cereals oils and feedstuffs for import and export - Methods of sampling and preparation of samples
bag per 50kg, and 50 bags below 100t; in case of above 100t, TAKE 1% of the total number of bags in batches, SAMPLE 15g per bag, and TAKE one original sample per 500t (calculated as 500t in case of less than 500t).
4.4.2 Quarantine samples of bagged fishmeal shall be taken as one bag per 50kg, and 40 bags below 100t; in case of above 100t, TAKE 1% of the total number of bags in batches. Each bag is sampled at 15g, and the collected samples from 20 bags are integrated into one sample, with a sample weight of about 300g (calculated as 500t in case of less than 500t).
4.4.3 Inspection and quarantine samples of bulk fishmeal shall be taken according to 4.4.1 and 4.4.2 after the fishmeal is filled into the warehouse. 4.5 Sampling method
4.5.1 The number of samples is calculated according to 4.4. Samples are randomly taken around each stack, as well as at the upper, middle and lower parts in a curved pattern.
4.5.2 The sampling port is faced down, and inserted into the bag from the diagonal direction of the angle of fishmeal bag, then rotate it upwards to pull it out, and put it into the sampling bag.
5 Sample preparation
USE a cleaned, dry, and sterilized aluminum or a stainless steel sample shovel (two sheets of thin iron plate or stainless steel plate).
5.2.1 Quartering. EXECUTE according to SN/T 0800.1.
5.2.2 The quarantine sample is made into a laboratory sample every 100t; the test sample is made into a laboratory sample every 3000t.
The sample label shall indicate the inspection number, product name, quantity (weight), inspection items, sample delivery date, sample delivery organization, etc.
5.4 Sample retention
The samples shall be sealed and stored in a cool and dry place.
Salmonella test method
A.1 Pre-enrichment culture
TAKE 25g of quarantine sample and add it to a 500mL jar containing 225mL of buffered peptone water (the block is granulated with a homogenizer and
crushed for 1min at 8000 r/min~10000 r/min). INCUBATE at 36??C ?? 1??C for 16h- 20h.
A.2 Selective enrichment culture
10mL of pre-enrichment culture is taken and inoculated into a culture flask containing 100mL of TTB enrichment broth. Another 10mL of pre-enrichment culture is inoculated into a culture flask containing 100mL of SC enrichment broth. The TTB culture flask is incubated at 43??C for 24h. The SC culture flask is incubated at 36??C ?? 1??C for 24h.
A.3 Quick screening
SELECT commercially available ?€?Salmonella rapid detection kit" or related microbiological testing instrument supporting reagents or similar commercial reagents for rapid screening of Salmonella, according to the relevant
commercial instructions, TREAT and TEST TTB and SC selective enrichment cultures and DETERMINE the positive or negative reaction results. For
negative results, "Salmonella not detected" can be reported; positive results must be isolated, cultured and identified.
A.4 Isolated culture
A.4.1 TAKE the TTB or SC selective enrichment culture, which is positive in Chapter A.3, the inoculating loop, and INSCRIBE the phenol red brilliant green (BS) agar plates and DHL agar plates, respectively, and INOCULATE two plates each. The bottom of the plate is incubated at 36??C ?? 1??C upward. If necessary, REPEAT the isolated culture of the selective enrichment culture in Chapter A.2. A.4.2 After isolated culture for 20h-24h, CHECK for the typical colonies of Salmonella in the plate, the typical colonies of Salmonella grown on BS agar, TURN the color of the medium change from pink to red, and the colonies are red and transparent. A typical colony of Salmonella grown on DHL medium is a yellow-brown transparent, black in center, or a small yellow-brown colony. becomes purple after growth, indicating a positive reaction. About 95% of the Salmonella reactions are positive.
A.6 Antigen reaction
A.6.1 The presence of the antigen is examined by pure culture of colonies using Salmonella factor serum O, Vi or H type by plate agglutination. On a carefully cleaned glass plate, a drop of saline is placed to partially disperse the colonies in the saline. After mixing evenly, gently SHAKE for 30s-60s and OBSERVE the black shadow. If the bacteria have condensed into more or less clear units, this strain is considered to be self-coagulated and shall not be provided for antigen identification.
A.6.2 O antigen inspection. Using pure colonies considered to have no self- coagulation, REPLACE the saline with 1 drop of O-type serum according to the method of A.6.1. If agglutination occurs, it is judged as positive.
A.6.3 Vi antigen inspection. Using pure colonies considered to have no self- coagulation, REPLACE the saline with 1 drop of Vi-type serum according to the method of A.6.1. If agglutination occurs, it is judged as positive.
A.6.4 H antigen inspection. Pure colonies which are considered to have no self-coagulation are inoculated in semi-solid nutrient agar and cultured at 36??C ?? 1??C for 18h-20h, and this culture is used as an H antigen for inspection. In accordance with the method of A.6.1, 1 drop of H serum is used instead of saline. If agglutination occurs, it is judged as positive.
A.7 Determination of biochemical and serum reaction results
Only the above biochemical reactions (including instrument identification) are typical, and the O, Vi or H antigens are seropositive, and the "Salmonella is detected" can be determined.
A.8 Inspection report
In combination with the above biochemical tests and serum identification results, REPORT whether the inspection samples contain Salmonella. The hygienic
standard is ?€?not detectable/25g?€?.
A.9 Inspection procedure
The Salmonella inspection procedure is shown in Figure A.1.
Test method for total bacterial count
B.1 Operating procedures
B.1.1 Sample dilution and culture
B.1.1.1 10.0g of the sample is aseptically weighed and placed in a sterile Erlenmeyer flask containing 90mL of diluent (the appropriate amount of glass beads are pre-filled in the bottle). After shaking well, make a 1.10 uniform diluent. It is best to treat the oscillator at a speed of 8000r/min-10000r/min for 2min-3min.
B.1.1.2 PIPETTE 1mL of 1.10 diluent with a 1mL sterile pipette and slowly INJECT into the tube containing 9mL of diluent along the tube wall (note that the tip of the pipette does not touch the diluent in the tube). SHAKE the tube and MIX well to make a 1.100 diluent.
B.1.1.3 TAKE another 1mL sterile pipette and make 10 times incremental
dilution according to the above operation sequence. In this way, each time the dilution is incremented, a pipette is replaced.
B.1.1.4 According to the requirements of fishmeal hygiene standards or the estimation of the degree of contamination of the sample, SELECT 2 to 3
suitable dilutions, respectively, while making 10 times of incremental dilution, i.e., PIPETTE 1mL of diluent into the sterilized plate using a pipette that draws the dilution. Two plates are made for each dilution.
B.1.1.5 After the diluent is pipetted to the plate, 15mL of plate counting medium (can be placed in a 46??C ?? 1??C water bath) cooled to 46??C ?? 1??C shall be injected into the plate. Carefully ROTATE the plate to mix the sample well with the medium. The time between dilution of the sample and pouring of the medium shall not exceed 15min. If it is estimated that the microorganisms contained in the sample may grow on the surface of the agar plate, after the agar is completely solidified, 4mL of water agar medium cooled to 46??C ?? 1??C can be poured onto the medium surface.
B.1.1.6 After the agar is solidified, the inverted plate is taken out and then cultured in a 30??C ?? 1??C incubator for 72h ?? 3h. COUNT the number of colonies in the plate. Multiplying the number of colonies by the dilution factor gives the total number of bacteria per gram of sample.
Mold count test method
C.1 Operating procedures
C.1.1 25g (or 25mL) of sample is weighed in aseptically and placed in a conical flask with a glass stopper containing 225mL of sterile diluent. PLACE on the shaker and SHAKE for 30 min, which is a 1.10 diluent.
C.1.2 PIPETTE 10mL of 1.10 dilution with a sterile pipette. INJECT it into a test tube with glass beads. MIX it on a micro-mixer for 3min, or INJECT into a test tube. In addition, a 1mL sterile pipette with a rubberized nipple is repeatedly used for 50 times to spread the mold spores.
C.1.3 TAKE 1mL of 1.10 dilution. INJECT into a test tube containing 9mL of sterile diluent. Another pipette is used to pipette five times. This solution is a 1.100 diluent.
C.1.4 A 10-fold incremental diluent is prepared in the order described above. For each dilution, replace with a 1mL sterile pipette. According to the requirements of fishmeal hygiene standards or the estimation of the degree of contamination of the sample, SELECT 3 suitable dilutions. While making a 10- fold dilution, PIPETTE 1mL of diluent into a sterile plate. PREPARE two plates for each dilution. Then, the Salt Czapek Dox medium cooled to about 45??C is poured into the dish and mixed well. After the agar solidified, it is placed in a (25??C - 28??C) ?? 1??C incubator. The observation is started after 3 days of culture. It shall be observed for 5d.
C.2 Calculation method
In general, SELECT the plates with the number of colonies between 30 and 100 to count. The average number of colonies of two plates with the same dilution is multiplied by the dilution factor, which is the number of molds contained per gram (or per milliliter) of the sample.
The number of molds per gram of fishmeal is expressed in molds/g. The
sanitary standard is ?€?< 20 000 molds/g?€?.
C.4 Mold inspection procedure
The mold inspection procedure is shown in Figure C.1.
Test method for pathogenic vibrio
D.1 Inspection steps
WEIGH 25g of sample. ADD to a jar containing 225mL of alkaline peptone water (APW). Solid samples shall be broken with a homogenizer at 8000 r/min or fully shredded with scissors. After incubating at 37??C for 8h-16h, 10mL of culture solution is added to 10mL of double-basic alkaline peptone water (APW), and cultured at 37??C for 6h-8h.
The surface growth of the above-mentioned enrichment broth is inoculated with 3mm to 5mm. Two TCBS agar plates are inoculated by a circular streak, and cultured at 37??C for 20h to 24h. On TCBS agar, vibrio colonies are yellow or green colonies, smooth and slightly flat.
D.1.3 Determination of vibrio
D.1.3.1 Several suspected colonies on the TCBS agar plates are picked with an inoculating loop for Gram staining and microscopic examination. Vibrio shall be G-negative or Campylobacter and have power.
D.1.3.2 Several suspected colonies are picked with an inoculating loop, and oxidase reaction is carried out by adding an oxidase reagent to a sterile white filter paper or a sterile glass slide. Vibrio shall be positive for oxidase. D.1.3.3 The culture in which the oxidase reaction is positive is inoculated in the D-mannose solution. Vibrio shall be positive for the D-mannose
D.1.3.4 The culture in which the D-mannose reaction is positive is inoculated, the O/129 test medium is inoculated, and 150??g of O/129 paper is applied thereto. INCUBATE at 37??C for 18h-24h. Vibrio is sensitive to the O/129 bacteriostatic test.
D.1.4 Identification of pathogenic vibrio
D.1.4.1 The above-mentioned vibrio qualitative biochemical reaction-positive culture is picked with an inoculating loop, and inoculated with TSI agar, KIA agar,