GB/T 4789.31-2003 Microbiological examination of food hygiene - Method for the examination of Enterobacteriaceae phages for Salmonella, Shigella and diarrhea-causing Escherichia coli

This standard specifies the basic requirements, operating procedures and result judgment for the application of Enterobacteriaceae phage diagnostic method to test for Salmonella, Shigella and diarrhea-causing Escherichia coli in food. This standard is applicable to the testing of various foods and food poisoning, and also to the testing of intestinal Salmonella and Shigella in food practitioners. GB/T 4789.31-2003 Food Hygiene Microbiology Test Method for Enterobacteriaceae Phage Test for Salmonella, Shigella and Diarrhea-causing Escherichia coli GB/T4789.31-2003 Standard Download Decompression Password: www.bzxz.net

ICS07.100.30
National Standard of the People's Republic of China
GB/T4789.31—2003
Replaces GB/T4789.31—1994
Microbiological examination of food hygieneExamination of Salmonellae,Shigellae,and diarrhoea causativeEscherichia coli by means of the diagnostic typing phage setfor enterobacteriaceae
2003-08-11 Issued
Ministry of Health of the People's Republic of China
Standardization Administration of the People's Republic of China
2004-01-01 Implementation
GB/T4789.31--2003
This standard amends GB/T4789.31--1994 "Test Methods for Salmonella, Shigella and Diarrhea-causing Escherichia coli Enterobacteriaceae Phages in Food Hygiene Microbiology". Compared with GB/T4789.31-1994, this standard has the following major revisions: The format and text of the standard text are revised in accordance with GB/T1.1-2000. Standardize "equipment and materials" in the original standard. From the date of implementation of this standard, GB/T4789.31-1994 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. This standard was drafted by: Jiangxi Provincial Health and Epidemic Prevention Station, Institute of Nutrition and Food Safety, Chinese Center for Disease Control and Prevention. The main drafters of this standard are: He Xiaoqing, Fu Ping, Ji Rong. This standard was first issued in 1994, and this is the first revision. 250
1 Scope
Food Hygiene Microbiological Examination
GB/T4789.31—2003
Test Method for Enterobacteriaceae Phage for Salmonella, Shigella and Diarrhea Escherichia coli
This standard specifies the basic requirements, operating procedures and result judgment for the application of Enterobacteriaceae pyrimidine diagnostic method for testing Salmonella, Shigella and diarrhea Escherichia coli in food. This standard is applicable to the inspection of various foods and food poisoning, and also to the inspection of intestinal Salmonella and Shigella for food practitioners.
2 Normative References
The clauses in the following documents become the clauses of this standard through reference in this standard. For any dated referenced document, all subsequent amendments (excluding errata) or revisions are not applicable to this standard. However, parties to an agreement based on this standard are encouraged to study whether the latest versions of these documents can be used. For any undated referenced document, the latest version shall apply to this standard. GB/T4789.4 Food Hygiene Microbiology Test Salmonella Test GB/T4789.5-2003 Food Hygiene Microbiology Test Shigella Test GB/T4789.6 Food Hygiene Microbiology Test Diarrhea Escherichia Coli Test GB/T4789.28-2003 Food Hygiene Microbiology Test Staining Method, Culture Medium and Reagents 3 Equipment and Materials
3.1 Constant Temperature Incubator: 36℃±1℃.
3.2 Level.
3.3 Sterile Culture III: 90mm in diameter.
3.4 ​​Quantitative Dropper: Each drop is about 10μL. Sterile glass pipette for infusion with No. 4 needle.
3.5 Sterile cotton swabs.
4 Culture media and reagents
4.1 Nutrient agar, nutrient broth: according to 3.7 and 3.8 of GB/T4789.28-2003. 4.2 Protein water, indigo matrix reagent: according to 2.13 of GB/T4789.28-2003. 4.3 Trisaccharide iron agar: according to 3.24 and 3.25 of GB/T4789.28-2003. 4.4 Phages for diagnosis of Enterobacteriaceae, 7 and 3 species. After opening the anus, use a sterile capillary pipette to suck out and transfer into a sterile small test tube, plug it with a sterile rubber stopper, and put it in a 4℃ refrigerator for use. 5 Test procedures
5.1 Diagnosis of Enterobacteriaceae The procedure for testing Salmonella with bacteriophage is shown in Figure 1. 251
GB/T4789.31-2003
O-1 double solution
Others do not lyse
Salmonella
Serological test
Which bacterial test
Not as described on the left
Various lysis results
Pre-enrichment, enrichment and separation, according to GB/T4789.4, carry condensable blue drop
TSI (slant, bottom, gas production, H2S) protein stale water (indigo matrix)
Urea (pH7.2), KCN, whipping
Various magnetic bacteria
Do not lyse
H, S+, acid matrix-|| tt||Urea-KCN-，
Lysine+
Non-Salmonella
Salmonella
Serological test
H2S+, indigo matrix+，
Urea—，KCN—，
Lysine+
Mannitol, sorbitol
Salmonella
Serological test
H2S-, knock matrix
Urea—,KCN—，
Carboxylic acid+/-
Non-Salmonella
Figure 1 The procedure for phage testing of Salmonella for diagnosis of Enterobacteriaceae is not as shown on the left
Various reaction results
Non-Salmonella
5.2 The procedure for phage testing of Shigella for diagnosis of Enterobacteriaceae is shown in Figure 2. Sample inspection
Bacteria enrichment and separation, pick suspicious colonies according to GB/T4789.5
TSI microbial semi-structure
TSI bottom layer produces acid, slant surface produces alkali, H2S-no gas production, no power
Phage test
Sh lysis, and various lysis patterns are expected
Serological test is consistent with the lysis pattern
and is decomposed by 1RTDSh
Biochemical grouping test
Shimao bacteria are decomposed
Grouping and typing results
Serological test is inconsistent with the lysis pattern or is not decomposed by 1RTDSh
Glucosamine test Test
If necessary, add sodium acetate test, Klebsiella citrate test, mucic acid, esculin, salicin test
Ammonium glucose +
Or any other positive result
Non-Shigella
GB/T4789.31-2003
Non-As described on the left
Various reaction results
Sh does not lyse
Non-Shigella
Figure 2 Procedure for phage testing of Shigella for diagnosis of Enterobacteriaceae Non-Shigella
GB/T4789.31-2003
5.3 The procedure for phage testing of diarrhea-causing Escherichia coli for diagnosis of Enterobacteriaceae is shown in Figure 3. Sample
Bacterial enrichment and separation, according to GB/T4789.6
Lactoethanol or non-fermented colonies 3 to 5 phage tests
E and or Sh, E-4
Phage does not lyse,
Other phages lyse
Enterotoxin +
Non-coli
Escherichia
Enterotoxin-producing
Enterotoxin-producing Escherichia
E and or Sh, E-4
Only bacteriophage lysis
Bacteria isolated from different sources Socks
Its lysis pattern
has the same
TSI bottom layer+
HS-,CN
serological test
EPEC+ or
EHEC(O157)
pathogenic or
enterohemorrhagic
Escherichia coli
various bacteria are not lysed
oxidase-,G-bacillus
TSI. Indigo matrix,
PH7.2 Urea, KCN,
Lysine, power
Not as described on the left
Various reaction results
Not as described on the left
Various reaction results
Lysine waist one, knockout matrix ten,
Power (0124 power +/-)
Broken channel invasiveness
Escherichia coli
Non-diarrheagenic
Escherichia coli
Figure 3 Procedure for phage detection of enterobacteriaceae for diarrheagenic Escherichia coli Non-diarrhea
Escherichia
6 Test steps
6.1 Pre-enrichment, enrichment and separation
6.1.1 Pre-enrichment, enrichment and separation of Salmonella shall be carried out in accordance with GB/T4789.4. 6.1.2 The enrichment and separation of Shigella shall be carried out in accordance with GB/T4789.5. 6.1.3 The enrichment and separation of diarrhea-causing Escherichia coli shall be carried out in accordance with GB/T4789.6. 6.2 Bacteriophage test
6.2.1 Preparation of culture medium
GB/T4789.31--2003
Nutrient agar plate (containing 1% to 1.5% agar, the amount of agar depends on its coagulation, about three-quarters of the general amount), about 25mL of each 90mm culture Ⅲ, placed on a horizontal surface and wait for it to solidify, turn the plate over, and invert it at 36℃ half-open Ⅲ for about 1h to dry the surface moisture of the culture medium and make the agar have significant water absorption. 6.2.2 Preparation of test bacterial solution
Pick colonies from the selective identification plate. Generally, you should pick more colonies to prevent omission. When testing Salmonella, not only lactose-negative colonies that produce HS and no H2S should be picked, but also lactose-positive colonies that produce H and S. When testing Escherichia coli, 3 to 5 lactose-positive or -negative colonies should be picked. When testing Shigella, suspicious colonies should be picked and inoculated into three-sugar iron agar and glucose semisolid tubes, cultured at 36°C overnight, and the next day, the culture that produces acid on the bottom of the three-sugar iron agar and alkali on the slant, does not produce H2S, does not produce gas and has no power should be selected. The following two methods are available for selection.
6.2.2.1 Method: Inoculate the colony to be tested into a nutrient broth tube and culture it at 36°C overnight. Pick a full loop of this broth culture and dilute it in a test tube containing 1mL~2mL of protein stale water to make a (1:200)~(1:400) diluted bacterial solution with a bacterial count of about 1×10/mL. 6.2.2.2 Method 2: Use an inoculation needle to gently dip the suspected colony on the identification plate. The amount of bacteria picked should not be too much. Dilute it in a test tube containing 1mL to 2mL of protein water so that the bacterial content is similar to that of method 1. 6.2.3 Smearing test solution
6.2.3.1 Spot smearing method: Divide the surface of the nutrient agar plate into three equal parts from the center of the circle. Each equal part can be used to smear a bacterial culture. Pick a full circle of test solution and smear a plaque with a diameter of about 1cm. Smear seven plaques for each culture, four on the outer circle and three on the inner circle.
6.2.3.2 Cotton swab smearing method: Use a sterile cotton swab to take the test solution and slightly squeeze out the excess liquid. Smear it on one-third of the surface of the agar plate as above. Keep an appropriate distance between the three smearing areas. Wait for a few minutes for the solution to dry. 6.2.4 Adding bacteriophages
Use a quantitative nipple dropper to drop a drop of bacteriophage on a bacterial plaque, in the order of OI, C.Sh, E, CE, E-4 and Ent. Install a No. 4 needle on the dropper, about 100 drops per milliliter. Each dropper only drops one type of bacteriophage, and the needle tip must not touch the surface of the plate to strictly prevent cross contamination. Each dropper can be used to drop all the plaques where the bacteriophages should be added. When adding bacteriophages, the agar plate must be placed on a horizontal surface. After the 7 types of bacteriophages are added, wait for a few minutes for the bacteriophage liquid to dry. Turn the plate over, incubate at 36℃ for 5h~6h, and observe the results overnight. If only one or two cultures are used for phage testing, phages can be picked up with an inoculation loop with a diameter of 3mm and dripped on the bacterial plaques in turn. The hot inoculation loop must be completely cooled before picking up phages. 6.2.5 Determination of test results
Determine the results of the phage test according to Table 1. If necessary, take a small amount of the remaining protein stale water culture for indigo test. Escherichia coli culture is generally indigo positive. 255
GB/T4789.31-2003
Table 1 Diagnostic table of Enterobacteriaceae phages
Phage lysis pattern
Note: CL fusion lysis, - no lysis; · lysis or no lysis (-) Very few strains lyse. a Combined with bacterial type prediction, see Table 3.
6.2.6 The simplified phage diagnostic method for rapid detection of Salmonella uses the following three phages:
Salmonella CI Xi bacteriophage;
b) E polyvalent phage, including E and Sh;
c) C polyvalent phage, including C, CE and Ent. Preparation of culture medium and test bacterial solution is the same as the previous method. Ent
Judgment result
Salmonella fluoride
Salmonella (indole-1)
Escherichia coli (indole+)
Citrobacter freundii
Shigella coli
Escherichia coli
Escherichia coli
Citrobacter freundii, coli
Escherichia coli
Escherichia coli
Enterobacter cloacae
Phage-negative strains (identified by biochemical tests)
Smear test bacterial solution: The spot smear method divides the surface of the agar plate into four equal parts, and each equal part can be used to smear a bacterial culture. Three plaques are applied to each culture, two in the outer circle and one in the inner circle. The cotton swab smearing method can smear the test bacterial solution on the agar plate into a strip shape, and each plate can be smeared with about five bacterial strips. Wait for a few minutes until the plaque is dry. Add the above three phages in turn, culture at 36℃ for 5h~6h and overnight, and observe the results once each time. Determine the results of the simplified diagnosis method according to Table 2.
Table 2 Simplified diagnosis table for three-drop method phage test o1
E multivalent
Note: CL fusion lysis, - non-lysis · lysis or non-lysis.
aIncludes Enterobacter cloacae and a few Escherichia coli. 6.3 Supplementary biochemical test of culture of non-lysed bacteria C multivalent
Determination result
Salmonella
Escherichia coli
Citrobacter freundii
Phage-negative culture
There are a few Salmonella cultures that are not lysed by OI phage, and a few Escherichia coli cultures that are not lysed by the corresponding phage 256
GB/T4789.31-2003
. Therefore, the cultures that are not lysed by various phages should be inoculated with three-sugar iron agar. The following shall be carried out according to GB/T4789.4 and GB/T4789.6 respectively.
6.4 Serological typing identification
6.4.1 Salmonella
Perform according to GB/T4789.4.
6.4.2 Diarrhea Escherichia coli
Perform according to GB/T4789.6. Www.bzxZ.net
6.4.3 Shigella
6.4.3.1 Record the results of the phage test and determine the direction of serological typing identification according to Table 3. Cultures that are not lysed by Sh phage do not belong to Shigella. It is known that all Shigella cultures can be lysed by Sh phage, and when diluted to 1RTD, 99% of Shigella cultures should still be lysed. Table 3 Results of phage test and direction of serological typing of Shigella No.
Phage lysis pattern
Note, CL fusion lysis, I no lysis, () indicates rare results, CE
Shigella serological typing
Direction of identification
Flexneri 1~5 (Boweri 11)
Flexneri 1, 4, Xiangji 2 Boweri
5,7,11,16,17, Sonnei
Shigella 1)
Sonnei I, Xiangji 2, Celsius
4, Boweri 5, 16,
Sonnei!, Flexneri 3. …
Flexerella 6, Bowerella 1~4, even
type 618 (except 16),
epilepsy 3~12
Bowerella 9, 15
residual disease 1 (Sonne II)
Bowerella 13
Pick the culture on triple sugar iron agar, select the corresponding Shigella typing factor serum according to the phage lysis pattern, and do a slide agglutination test. If agglutination does not occur due to the presence of K antigen, the bacterial solution should be boiled and then checked. 6.4.3.2 If the phage lysis pattern indicates that it is Shigella flexneri type 1~5, the agglutination test can be performed first with flexerella polyvalent serum. If agglutination is present, each type and group factor serum should be used for examination to determine the serotype and subtype (see GB/T4789.5-2003 Table 2).
6.4.3.3 If the phage lysis pattern indicates that it is flexneri type 6, Boyer type or dysentery type 3-12, the flexneri type 6 serum can be used for agglutination test first. If flexneri type 6 serum does not agglutinate, Boyer polyvalent serum or dysentery type 3-12 polyvalent serum can be used for agglutination test. If agglutination is present, then use each type of factor serum to check separately. 6.4.3.4 If the phage lysis pattern indicates that it is Sonne II phase bacteria but it is not agglutinated by Sonne serum, it can be directly judged according to the phage lysis pattern and rough colony characteristics.
6.4.3.5 For those who do not agglutinate in the direction suggested by the phage lysis pattern, or those who are found to be agglutinated by a certain typing factor serum but do not match the phage lysis pattern, or those who match the phage lysis pattern but are not lysed by the 1RTD Sh phage, the ammonium glucose test should be performed to distinguish them from Escherichia coli. Those who are positive in the ammonium glucose test are Escherichia coli. If necessary, the following biochemical tests can also be performed, namely: sodium acetate, Klebsiella citrate and mucolytic acid utilization tests, lysine and ornithine decarboxylase, and esculin 257
GB/T4789.31—2003
decomposition tests. Except for Sonnelia and Boyer 13 types, which are ornithine positive, a few strains of Sonnelia can utilize mucolytic acid, and a few strains of Flexner 4a type can utilize sodium acetate, the cultures of Shigella are all negative. Any positive result in these tests indicates Escherichia coli.
6.4.3.6 The detected Shigella culture should conform to the biochemical characteristics of the group (see Table 3 of GB/T4789.5-2003), but flexneri type 6 is similar to group A or group C. The indigo-positive Shigella serotypes are dysentery type 2, 7 and 8, and boydii type 5, 7, 9, 11, 13, 15, 16 and 17, but the indigo reaction of flexneri type 1 and type 5 is weak: the indigo-negative Shigella serotypes are dysentery type 1, type 3 to type 6, type 9 to type 12, flexneri type 6, boydii type 1 to type 4, type 6, type 8, type 10, type 12, type 14 and type 18 and sonnei. 6.5 Escherichia coli enterotoxin test
Perform according to GB/T4789.6.
6.6 Result Report
6.6.1 Report of Result of Salmonella Test
6.6.1.1 If OI phage is lysed and others are not, report after Salmonella serotyping: or if the above lysis results are met but serotyping test has not been done, it can be reported as Salmonella untyped. 6.6.1.2 If all phages are not lysed but biochemical test confirms that it is Salmonella, report after Salmonella serotyping, or if the above biochemical test results are met but serotyping test has not been done, it can be reported as Salmonella untyped. 6.6.1.3 If the lysis results are not the above, or the biochemical test denies Salmonella, it can be reported as non-Salmonella. 6.6.2 Report of Shigella Test Results
6.6.2.1Sh phage lysis shows various lysis patterns. If the serological test is consistent with the lysis pattern, the Shigella typing results can be reported. Rare serotypes are required to be lysed by 1RTDSh phage and meet the results of Shigella biochemical grouping. 6.6.2.2 Test results other than the above are reported as non-Shigella. 6.6.3 Report of Diarrhea Escherichia coli Test Results6.6.3.1E and/or Sh, E-4 phage lysis, the lysis pattern of strains from different sources is identical. Those confirmed by serological typing can be reported as enterotoxigenic Escherichia coli (enterotoxin test results are required), invasive Escherichia coli (lysine negative and motility negative are required, but 0124 can also be motility positive), enterohemorrhagic Escherichia coli (limited to 0157) or enteropathogenic Escherichia coli.
6.6.3.2 All phages are not lysed, and the biochemical test is consistent with Escherichia coli, and the serological typing is confirmed, and various types of diarrhea-causing Escherichia coli can also be reported separately. The requirements are the same as 6.6.3.1. 6.6.3.3 Non-Escherichia coli or non-diarrhea-causing Escherichia coli results are reported as non-Escherichia coli or non-diarrhea-causing Escherichia coli. 258
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