GB/T 4789.5-2003 Microbiological examination of food hygiene - Shigella test

This standard specifies the test method for Shigella in food. This standard is applicable to the test for Shigella in various types of food and food poisoning samples. GB/T 4789.5-2003 Food Hygiene Microbiological Test Shigella Test GB/T4789.5-2003 Standard Download Decompression Password: www.bzxz.net

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National Standard of the People's Republic of China
GB/T4789.5-—2003
Replaces GB/T4789.5—1994
Microbiological examination of food hygiene—Examination of Shigella
Promulgated on August 11, 2003
Ministry of Health of the People's Republic of China
Standardization Administration of the People's Republic of China
Implementation on January 1, 2004
GB/T4789.5—2003
This standard revise GB/T4789.5—1994 "Microbiological examination of food hygiene—Examination of Shigella". Compared with GB/T4789.5-1994, this standard has the following major revisions: The format and text of the standard text are revised in accordance with GB/T1.1-2000. The "equipment and materials" in the original standard are revised and standardized. From the date of implementation of this standard, GB/T4789.5-1994 will be abolished at the same time. This standard is proposed and managed by the Ministry of Health of the People's Republic of China. The drafting units of this standard are: Jiangxi Provincial Health and Epidemic Prevention Station, Nutrition and Food Safety Institute of China Center for Disease Control and Prevention. The main drafters of this standard are: He Xiaoqing, Ran Lu, Fu Ping, Yang Baolan, Yao Jinghui. This standard was first issued in 1984, revised for the first time in 1994, and this is the second revision. 34
1 Scope
Food Hygiene Microbiological Examination
Shigella Examination
This standard specifies the test method for Shigella in food. This standard is applicable to the test of Shigella in various types of food and food poisoning samples. 2 Normative references
GB/T4789.5—2003
The clauses in the following documents become the clauses of this standard through reference in this standard. For all dated references, all subsequent amendments (excluding errata) or revisions are not applicable to this standard. However, parties to agreements based on this standard are encouraged to study whether the latest versions of these documents can be used. For all undated references, the latest versions are applicable to this standard. GB/T4789.28--2003 Food hygiene microbiological examination staining methods, culture media and reagents 3 Equipment and materials
3.1 Refrigerator: 0℃~4℃.
3.2 Constant temperature incubator: 36℃±1℃, 42℃. 3.3 Microscope: 10×~100×.
3.4 ​​Homogenizer or sterile mortar.
3.5 Plate-mounted pharmaceutical balance: 0g~500g, accurate to 0.5g. 3.6 Sterilized wide-mouth bottle: 500mL.
3.7 Sterilized conical flask: 500mL, 250mL. 3.8 Sterilized culture blood: 90mm in diameter.
3.9 Nitrocellulose filter: 150mm×50mm, 0.45μm. Cut into two pieces before use, each 70mm×50mm, and mark with a pencil, each grid 6mm×6mm. Each row has 10 grids, divided into 6 rows. Sterilize for use. 4 Culture medium and reagents
4.1 GN enrichment solution: in accordance with 4.17 of GB/T4789.28-2003. 4.2 HE agar: in accordance with 4.21 of GB/T4789.28-2003. 4.3 SS agar: in accordance with 4.22 of GB/T4789.28-2003. 4.4 MacConkey agar: in accordance with 4.24 of GB/T4789.28-2003. 4.5
Eosin-methylene blue agar (EMB): in accordance with 4.25 of GB/T4789.28-2003. 4.6 Trisaccharide iron agar (TSI): in accordance with 4.26 and 4.27 of GB/T4789.28-2003. 4.7 Glucose semisolid tube: in accordance with 4.31 of GB/T4789.28-2003. 4.8 Semisolid tube: in accordance with 4.30 of GB/T4789.28-2003. 4.9 Glucose ammonium agar: in accordance with 3.8 of GB/T4789.28-2003. 4.10 Urea agar (pH 7.2): in accordance with 3.15 of GB/T4789.28-2003. 4.11 Simon's citrate agar: in accordance with 3.5 of GB/T4789.28-2003. 4.12 Potassium cyanide (KCN) medium: in accordance with 3.16 of GB/T4789.28--2003. 4.13 Amino acid decarboxylase test medium: lysine, ornithine and control medium, in accordance with 3.1235 of GB/T4789.28-2003
GB/T4789.5--2003
4.14 Sugar fermentation tube: raffinose, mannitol, glycerol, esculin and salicin, in accordance with 3.2 of GB/T4789.28-2003. 4.155% lactose fermentation tube: in accordance with 4.32 of GB/T4789.28-2003. 4.16 Protein stale water, indigo matrix reagent: in accordance with 3.13 of GB/T4789.28-2003. 4.17 Shigella diagnostic serum.
5 Test procedure
See Figure 1 for the Shigella test procedure.
25g+GN225mL
36℃±1c.6h~8h
HE agar or SS
TSI bottom layer produces acid, slant surface produces chloramine, H, S-
No gas, no power
Serotyping
Biochemical grouping test
Shigella
Grouping and typing results
36C±1C,18h～24h
Pick out suspicious seedlings
TSI, glucose semisolid
Further biochemical tests
Glucose ammonium test, western bacterium citric acid salt, lysine, urea, potassium cyanide (KCN), salicin and esculin glucose Ammonium +
or any other positive result
non-Shigella
McConkey or EMB
non-as described
various reaction results
non-Shigella
6 Operation steps
6.1 Enrichment
GB/T4789.5--2003
Take 25g (mL) of the test sample by aseptic operation and add it into a wide-mouth bottle containing 225mL GN enrichment solution. Use a homogenizer at 8000r/min~10000r/min to break up solid food for 1min, or use a mortar with sterilized sand to grind it. Use a metal spoon or glass rod to grind powdered food to emulsify it, and culture it at 36℃ for 6h~8h. The culture time depends on the growth of bacteria. When the culture solution becomes slightly turbid, the culture should be stopped. 6.2 Isolation and preliminary biochemical test
6.2.1 Take 1 loop of the enrichment solution and streak it on a HE agar plate or SS agar plate, and take another loop and streak it on a MacConkey agar plate or eosin-methylene blue agar plate, and culture it at 36℃ for 18h~24h. Shigella will appear as colorless, transparent colonies that do not ferment lactose on these culture media.
6.2.2 Pick suspicious colonies on the plate and inoculate one tube each of triple sugar iron agar and glucose semisolid. Generally, you should pick a few more colonies to prevent omissions, and culture them at 36℃ for 18h~24h, and observe the results respectively. 6.2.3 The following cultures may be discarded:
Cultures that grow in a spreading manner on the three-sugar iron agar slant; a)
Cultures that ferment lactose and sucrose within 18h~24h; c) Cultures that do not decompose glucose and only grow on semi-solid surfaces; d)
Cultures that produce gas;
Motile cultures;
Cultures that produce hydrogen sulfide.
6.2.4 All strains that do not ferment lactose and sucrose, produce acid but not gas from glucose (Shigella flexneri type 6 can produce a small amount of gas), and have no motility can be subjected to serological typing and further biochemical tests. 6.3 Serological typing and further biochemical tests 6.3.1 Serological typing
Pick the culture on the three-sugar iron agar and do the slide agglutination test. First use the four Shigella polyvalent sera to check. If agglutination does not occur due to the presence of K antigen, the bacterial solution should be boiled before checking: if agglutination is present, use A1, A2, B group polyvalent and D group sera to test respectively. If it is group B Shigella flexneri, use group and type factor sera to check respectively. The type and group antigens of each type and subtype of Shigella flexneri are shown in Table 1. You can first use the group factor sera to check, and then use the type factor sera to check in turn according to the result of agglutination of the group factor sera. Strains that do not agglutinate with the four Shigella polyvalent sera can be checked with Boyer polyvalent 1, 2, and 3 respectively, and further checked with 1 to 15 type factor sera. If Boyer polyvalent sera do not agglutinate, Shigella dysenteriae 312 type polyvalent sera and various type factor sera can be used for inspection. Table 1 Type antigens and group antigens of various types and subtypes of Shigella flexneri Type and subtype
X variant
Y variant
Type antigen
Note: Ten agglutinations and one non-agglutination: () yes or no. Group antigen
1,2,4,5.9....
1,2,4,5,9....
1.7,8.9..
1.6,7.8,9....
1.(3.4)..
1.3,4,6..
1,5,7,9....
1,2,(4)......
1. 7.8,9..
Agglutination in group factor blood digestion
GB/T4789.5—2003
6.3.2 Further biochemical tests
Further biochemical tests should be performed at the same time as serological typing, namely: ammonium glucose, Simon's citrate, lysine and ornithine decarboxylase, pH7.2 urea, potassium cyanide (KCN) growth, and salicin and esculin decomposition. Except for sonnei and boydii type 13, which are ornithine positive, the cultures of Shigella are all negative. If necessary, Gram staining and oxidase tests should also be performed, which should be oxidase-negative Gram-negative rods. Strains that do not meet the biochemical reaction should not be judged as Shigella cultures even if they can agglutinate with a certain Shigella typing serum. The culture identified as Shigella should be further tested for 5% lactose fermentation, fermentation of mannitol, raffinose and glycerol, and indigo test. The culture of the four biochemical groups of Shigella should conform to the biochemical characteristics of the group. However, the biochemical characteristics of flexneri type 6 are similar to those of group A or group C, see Table 2.
Group A: Shigella flexneri
Group B: Shigella flexneri
Group C, Shigella boydii
Group D: Shigella sonnei
5% lactose
Biochemical characteristics of the four groups of Shigella
Mannitol
Raffinose
Note: + positive; - negative, ~/10 mostly negative, a few positive; (10) delayed positive; d has different biochemical types. 6.4 Result Report
Combined biochemical and serological test results to determine the bacterial type and make a report. 38
Indigo matrix
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