This standard replaces GB 4789.15-2010 National Food Safety Standard Food Microbiological Examination: Enumeration of Moulds and Yeasts and SN/T 2552.3-2010 Microbiological Examination for Milk and Milk Products Hygiene - Part 3: Colony-count Method of Yeast and Moulds.
Compared with GB 4789.15-2010, the main changes in this standard are as follows:
- The Chapter "Apparatus and Materials" is modified;
- The Chapter "Culture Media and Reagents" is modified;
- The Chapters of “Examination Procedures” and “Operation Steps” are modified;
- The Chapter “Result and Report” is modified;
- Appendix A is modified;
- Appendix B is modified as Method II.
National food safety standard
Food microbiological examination: Enumeration of moulds and yeasts
1 Scope
This standard specifies the enumeration methods of moulds and yeasts in foods.
In this standard, Method I is applicable to the enumeration of molds and yeasts in various foods while Method II is applicable to that of moulds in canned tomato sauce and tomato juice.
2 Apparatus and Materials
In addition to the conventional sterilization and cultivation apparatus in microbiological laboratory, other apparatus and materials are as follows:
2.1 Incubator: 28±1℃.
2.2 Slap type homogenizer and homogenizing bag.
2.3 Electronic balance: with sensibility of 0.1g.
2.4 Aseptic conical flask: with volume of 500mL.
2.5 Aseptic suction tube: 1mL (with scale division of 0.01mL) and 10mL (with scale division of 0.1mL).
2.6 Aseptic test tube: 18×180 mm.
2.7 Vortex mixers.
2.8 Aseptic plate: with diameter of 90mm.
2.9 Thermostatic water bath: 46±1℃.
2.10 Microscope: 10×~100×.
2.11 Micropipettor and tip: 1.0mL.
2.12 Refractometer.
2.13 Howard measuring slide: special side with standard measuring chamber.
2.14 Cover glass.
2.15 Micrometer: slide with standard scale.
3 Culture Media and Reagents
3.1 Normal saline: see A.1.
3.2 Potato dextrose agar: see A.2.
3.3 Rose bengal agar: see A.3.
3.4 Phosphate buffer solution: see A.4.
Method I: Enumeration of Molds and Yeasts by Plate Count
4 Examination Procedure
Examination procedures for the enumeration of molds and yeasts by plate count are shown in Figure 1.
Figure 1 Examination Procedures for the Enumeration of Molds and Yeasts by Plate Count
5 Operation Steps
5.1 Dilution of sample
5.1.1 Solid and semi-solid sample: weigh 25g sample, add 225mL aseptic diluent (distilled water/normal saline/phosphate buffer solution) and shake well or slap for 1~2min with slap type homogenizer to prepare 1:10 homogeneous sample solution.
5.1.2 Liquid sample: pipet 25mL sample with aseptic suction tube and put into an appropriate container (proper number of aseptic glass beads may be preset in the flask) or aseptic homogenizing bag with 225mL aseptic diluent (distilled water/normal saline/phosphate buffer solution), shake the container well or slap for 1~2min with slap type homogenizer to prepare 1:10 homogeneous sample solution.
5.1.3 Pipet 1mL 1:10 homogeneous sample solution and inject into the test tube with 9mL aseptic diluent, use another 1mL aseptic suction tube to blow and suck repeatedly or mix it on vortex mixer so that it is mixed uniformly, thus such solution is the 1:100 homogeneous sample solution.
5.1.4 According to the operation procedures stated in 5.1.3, prepare 10-fold diluted homogeneous sample solution successively. Per increasing dilution, use another 1mL aseptic suction tube.
5.1.5 According to the estimation for the sample contamination status, select 2~3 portions of homogeneous sample solutions (stock solution may be selected as the liquid sample) with appropriate dilutions; during the 10-fold increasing dilution, pipet 1 mL homogeneous sample solution for each dilution respectively and then add them into two sterile plates. Meanwhile, take 1mL aseptic diluent and put into two sterile plates as blank control.
5.1.6 Pour 20~25mL potato dextrose agar or rose bengal agar cooled to 46℃ (it may be put in 46±1℃ thermostatic water bath for thermal insulation) into a plate timely, and rotate the plate to mix them uniformly. Place the plate on the horizontal stand until the culture medium is completely solidified.
5.2 Culture
After the agar is solidified, turn the plate, cultivate it in 28±1℃ incubator and observe and record the result on the 5th day.
5.3 Colony count
Carry out visual observation, if necessary, by magnifier or low power lens, and record the dilution times and the corresponding numbers of colonies in moulds and yeasts. The count is expressed in colony-forming units (CFU).
Select the plate with the colony number of 10~150 CFU is selected, and enumerate the moulds and yeasts respectively according to the colonial morphology. If the moulds spread and cover the whole plate, it may be recorded as colony spreading.
Foreword I
1 Scope
2 Apparatus and Materials
3 Culture Media and Reagents
4 Examination Procedure
5 Operation Steps
6 Result and Report
7 Operation Steps
Appendix A Culture Media and Reagents
