1 Scope
This standard specifies examination method of enterobacteriaceae in foods.
In this standard, Method I is applicable to the enumeration of enterobacteriaceae in foods with higher enterobacteriaceae content; Method II is applicable to the enumeration of enterobacteriaceae in foods with lower enterobacteriaceae content.
2 Terms and Definitions
2.1
Enterobacteriaceae
Oxidase-negative, aerobic or facultative anaerobic, Gram-negative non-spore bacillus that ferments and produces acid with glucose under given conditions.
2.2
Enumeration of enterobacteriaceae
Enumeration of the enterobacteriaceae in the sample per gram or milliliter according to the method specified in this standard.
2.3
Most probable number; MPN
An indirect enumeration method based on Poisson distribution.
3 Apparatus and Materials
In addition to the conventional sterilization and cultivation apparatuses in microbiological laboratory, other apparatuses and materials are as follows:
3.1 Constant temperature incubator: 36℃±1℃.
3.2 Refrigerator: 2℃~5℃.
3.3 Water bath: 46℃±1℃.
3.4 Balance: with sensibility of 0.1g.
3.5 Microscope: 10x~100x.
3.6 Homogenizer.
3.7 Oscillator.
3.8 Aseptic suction tube: 1mL (scale division of 0.01mL), 10mL (scale division of 0.1mL) or micropipettor and suction head.
3.9 Aseptic conical flask or equivalent container: with the volume of 150mL and 500mL.
3.10 Aseptic culture dish: 90mm in diameter.
3.11 Aseptic test tube: 18mm×l80mm, 15mm×150mm.
3.12 pH meter or pH colorimetric tube or precise pH paper .
4 Media and Reagents
4.1 Buffered peptone water (BPW): see B.1.
4.2 Buffered glucose brilliant green bile broth (EE Broth): see B.2.
4.3 Violet red bile glucose agar (VRBGA): see B.3.
4.4 Nutrient agar (NA): see B.4.
4.5 Glucose agar: see B.5.
4.6 Gram stain solution: see B.6.
4.7 Oxidase reagent: see B.7.
4.8 Aseptic 1mol/L NaOH: see B.8.
4.9 Aseptic 1mol/L HCL: see B.9.
Method I: Enumeration of Enterobacteriaceae by Plate Count
5 Examination Procedures
Examination procedures for the enumeration of enterobacteriaceae by plate count are shown in Figure 1.
Figure 1 Examination Procedures for the Enumeration of Enterobacteriaceae by Plate Count
6 Operation Steps
6.1 Dilution of sample
6.1.1 Solid and semi-solid sample: weigh 25g sample, put into an aseptic homogenizing cup with 225mL BPW, homogenize it for 1min~2min at 8000r/min~10000r/min, or put into an aseptic homogenizing bag with 225mL BPW, slap for 1min~2min with slap type homogenizer to prepare 1: 10 homogeneous sample solution.
6.1.2 Liquid sample: pipet 25mL sample with the aseptic suction tube and put into an aseptic conical flask with 225mL BPW (preset proper number of aseptic glass beads in the flask), shake the bottle well to prepare 1:10 homogeneous sample solution.
6.1.3 Pipet 1mL of 1: 10 homogeneous sample solution with 1mL aseptic suction tube or micropipettor and slowly pour it into the aseptic test tube with 9mL BPW along the tube wall (the suction tube or suction head tip shall not touch the diluent surface); shake the test tube, or use another 1mL aseptic suction tube to beat it repeatedly so that it is mixed uniformly and made into 1: 100 homogeneous sample solution.
6.1.4 According to the operation procedures stated in 6.1.3, make into 10-fold diluted homogeneous sample solution successively. Per increasing dilution, use another 1mL aseptic suction tube or suction head. From the preparation of the homogeneous sample solution to completion of the sample inoculation, it shall not exceed 15min in the whole process.
6.2 Pour plate and cultivation
6.2.1 According to the estimation on the sample contamination conditions and relevant limit requirements, select 2~3 portions of homogeneous sample solutions with appropriate continuous dilutions (stock solution may be selected as the liquid sample), inoculate in 2 aseptic plates per dilutions. Meanwhile, pipet 1mL BPW respectively and put into two aseptic plates as blank control.
6.2.2 Pour 10mL~15mL VRBGA cooled to 46℃ (it may be put in 46℃±1℃ thermostatic water bath for thermal insulation) into each plate. Rotate the plate carefully to mix the homogeneous sample solution and the medium sufficiently and uniformly.
6.2.3 After the agar is solidified, pour a thin layer of the same medium to cover the surface layer of the plate to avoid spreading and growth and make the characteristics of the colony more obvious.
6.2.4 After the upper layer of the agar on the VRBGA plate is solidified, turn the plate and cultivate for 18h~24h at 36℃±1℃.
6.3 Enumeration and confirmation of typical colony
6.3.1 Typical colony of the enterobacteriaceae is pink to red or purple colony with or without precipitation ring. Number of the selected typical colonies is between 15CFU~150CFU; for the plate without spreading colony growth, only the number of typical colonies is enumerated. The colony enumeration is represented in the colony forming unit (CFU).
6.3.2 Any plate thereof on which relatively large flake colony grows, the plate without flake colony shall be taken as the colony number of that dilutions ; if the flake colony is less than half of the plate and the colonies on the other half are distributed very uniformly, the colony number for the latter half may represent that for one plate if multiplied by 2.
6.3.3 Where the plate has such chain growth of colonies without obvious boundary, each single chain shall be taken as one colony enumeration.
6.3.4 Pick at least 5 typical colonies from each plate for confirmation (pick all of them if less than 5); if with different forms of typical colonies, at least 1 colony shall be picked for each form for confirmation.
6.3.5 Confirmation of typical colony:
a) For each of the selected colonies respectively, inoculate in the nutrient agar plate by streaking, cultivate for 18h~24h at 36℃±1℃, pick the colonies on the plate for Gram stain microscopy, oxidase test and glucose fermentation test.
b) Gram stain microscopy: enterobacteriaceae is non-spore Gram negative bacillus, with the size of (0.3~1.0)μm×(1.0~6.0)μm.
c) Oxidase test: with platinum/iridium inoculating loop or glass rod (nickel-chromium inoculating loop shall not be used), pick single colony and coat on the filter paper soaked with oxidase reagent; if the filter paper turns bluish violet within 10s, it will be judged as positive reaction.
d) Glucose fermentation test: pick a little of the same oxidase-negative colony with inoculating needle, puncture it into glucose agar, cultivate for 24h±2h at 36℃±1℃; if the content in the test tube turns yellow, it will be judged as positive reaction.
1 Scope
2 Terms and Definitions
3 Apparatus and Materials
4 Media and Reagents
5 Examination Procedures
6 Operation Steps
7 Report
8 Examination Procedures
9 Operation Steps
10 Report
Appendix A Examples for Calculation of Results
Appendix B Media and Reagents
Appendix C Most Probable Number (MPN) of Enterobacteriaceae
