This standard specifies the test method for hemolytic streptococci in food. This standard is applicable to the test for hemolytic streptococci in various types of food and food poisoning samples. GB/T 4789.11-2003 Food hygiene microbiological test Hemolytic streptococcus test GB/T4789.11-2003 Standard download decompression password: www.bzxz.net

ICS07.100.30
National Standard of the People's Republic of China
GB/T4789.11-2003
Replaces GB/T4789.11-1994
Microbiological examination of food hygiene—Examination of Streptococcus hemolyticus
Microbiological examination of food hygiene—Examination of Streptococcus hemolyticusPromulgated 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/T4789.11—2003
This standard revise GB/T4789.11—1994 “Microbiological examination of food hygiene—Examination of Streptococcus hemolyticus”. Compared with GB/T4789.11-1994, 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. GB/T4789.11-1994 will be abolished as of the date of implementation of this standard. 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: Beijing Municipal Health and Anti-epidemic Station, Institute of Nutrition and Food Safety, Chinese Center for Disease Control and Prevention. The main drafters of this standard are: Liu Yixian, Ran Lu, Fu Ping, Yao Jinghui. This standard was first issued in 1984, revised for the first time in 1994, and this is the second revision. 78
1 Scope
Food Hygiene Microbiological Examination
Hemolytic Streptococcus Examination
This standard specifies the test method for hemolytic streptococcus in food. This standard is applicable to the test of hemolytic streptococcus in various types of food and food poisoning samples. 2 Normative references
GB/T4789.11—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 materialswwW.bzxz.Net
3.1 Refrigerator: 0℃~4℃.
3.2 Constant temperature incubator: 36℃±1℃.
3.3 Constant temperature water bath: 36℃±1℃.
Microscope: 10×~100×.
3.5 Homogenizer or sterile mortar.
Centrifuge: 4000r/min.
Plate pharmaceutical balance: 0g~500g, accurate to 0.5g. 3.8 Sterile test tube: 10mmX100mm.16mmX160mm. 3.9 Sterile pipette: 1mL (with 0.01mL scale), 5mL, 10mL (with 0.1mL scale). Sterile conical flask: 100mL.
Sterile culture blood: 90mm in diameter.
Sterile cotton swabs, tweezers, etc.
4 Culture medium and reagents
4.1 Glucose meat extract broth: According to 4.1 of GB/T4789.28-2003. Add 1% glucose to the meat extract broth. 4.2 Meat extract broth: According to 4.1 of GB/T4789.28-2003. 4.3 Picolin broth: in accordance with 4.62 of GB/T4789.28-2003. 4.4 Blood agar plate: in accordance with 4.6 of GB/T4789.28-2003. 4.5 Human plasma.
0.25% calcium chloride.
4.70.85% sterile saline.
4.8 Bacitracin drug-sensitive paper (containing 0.04 units). 5
Test procedure
The test procedure is shown in Figure 1.
GB/T4789.112003
Glucose meat broth
or Pick's broth
Streptokinase test
6 Operating steps
6.1 Sample treatment
25g(mL)+225mL sterile saline
36℃±1℃
Bacillus sensitivity test
Observe bleeding
36℃±1℃
Blood plate
Blood plate (pure culture)
Gram staining
According to aseptic operation, weigh 25g(mL) of food sample, add 225mL sterile saline and grind into a homogenate to make a suspension. 6.2 Culture
Take 5mL of the above suspension and inoculate it into 50mL of glucose broth, or directly streak it into a blood plate. If the sample is seriously contaminated, you can inoculate it with the above amount of Pick's broth at the same time, culture it at 36℃±1℃ for 24h, inoculate it into a blood plate, and culture it at 36℃±1℃ for 24h. Pick up the small colonies with round protrusions of hemolysis, separate them on the blood plate, and then observe the hemolysis and Gram staining, and perform streptokinase test and bacitracin sensitivity test.
6.3 Morphology and staining
This bacterium is spherical or oval, with a diameter of 0.5m~1um, arranged in chains, and the length of the chain varies. The short one is composed of 4~8 cells, and the long one is 20~30 cells. The length of the chain is often related to the type of bacteria and the growth environment; it is easy to be a long chain in liquid culture; it is often a short chain in solid culture medium, does not form spores, has no flagella, and cannot move. 6.4 Culture characteristics
This bacterium has high nutritional requirements and grows poorly on ordinary culture media, but grows better in culture media with blood and serum. When hemolytic streptococci grow in serum broth, the bottom of the tube is floccose or granular. The colonies on the blood plate are grayish white, translucent or opaque, with a smooth surface and opalescence, about 0.5mm to 0.75mm in diameter, and are small colonies with round protrusions. There is a 2mm to 4mm clearly defined, colorless and transparent hemolytic zone around beta-hemolytic streptococci. 6.5 Streptokinase test
Pathogenic beta-hemolytic streptococci can produce streptokinase (i.e., fibrinolytic protease), which can activate plasma protease in normal human blood to form plasma protease, and then dissolve fibrin. Take 0.2ml of potassium oxalate plasma, add 0.8ml of sterile saline, mix well, then add 0.5ml of streptococcal culture cultured at 36℃±1℃ for 18h24h and 0.25ml of 0.25% calcium nitride (if calcium nitride has deliquesced, it can be increased to 0.3%~0.35%), shake well, place in a 36℃±1℃ water bath for 10min, the plasma mixture will coagulate by itself (coagulation degree is to the test tube inverted, the contents will not flow), then observe the time for the coagulation block to completely dissolve again, complete dissolution is positive, if it does not dissolve after 24h, it is negative. 80
GB/T4789.11—2003
Preparation of potassium oxalate human plasma: put 0.01g of potassium oxalate into a sterile small test tube, add 5mL of human blood, mix well, centrifuge and precipitate, and take the supernatant to be potassium oxalate human plasma.
6.6 Bacitracin sensitivity test
Pick the beta-hemolytic streptococcus liquid and spread it on the blood plate. Use sterilized tweezers to pick up each piece of bacitracin paper containing 0.04 units, place it on the above plate, and culture it at 36℃±1℃ for 18h~24h. If an inhibition zone appears, it is positive. At the same time, use a known positive strain as a control.
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