GB 4789.15-2016National food safety standard - Food Microbiological Examination: Enumeration of Moulds and Yeasts (English PDF)

2.1 Incubator. 28°C±1°C.

2.2 Beat-type homogenizer and homogeneous bag.

2.3 Electronic balance. sensitivity of 0.1g.

2.4 Sterile conical flask. capacity of 500mL.

2.5 Sterile pipette. 1mL (with 0.01mL scale), 10mL (with 0.1mL scale).

2.6 Sterile test tube. 18mm × 180mm.

2.7 Vortex mixer.

2.8 Sterile flat plate. diameter of 90mm.

2.9 Constant temperature water batch. 46°C±1°C.

2.10 Microscope. 10× ~ 100×.

2.11 Micro-pipettor and tip. 1.0mL.

2.12 Refractometer.

2.13 Hearst measuring slide. special side with standard measurement room.

2.14 Cover glass.

2.15 Micrometer. slide with standard scale.

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. see A.4.

5.1 Sample dilution

5.1.1 Solid and semi-solid samples. weigh 25g of sample; add 225mL of sterile

diluent (distilled water or normal saline or phosphate buffering solution), sufficiently

shake; or use beat-type homogenizer to beat for 1min ~ 2min; then 1.10 sample

homogenous solution is prepared.

5.1.2 Liquid sample. use the sterile pipette to absorb 25mL of sample into 225mL

appropriate container (can pre-set appropriate amount of sterile glass beads in the

Sample Examination

5.1.3 Take 1mL of 1.10 sample homogenous solution to inject into the test tube

containing 9mL of sterile diluent; change another 1mL sterile pipette to blow and

absorb repeatedly; or mix evenly on the vortex mixer; such solution is 1.100 sample

homogenous solution.

5.1.4 Operate as per 5.1.3, prepare the 10 times incremental serial sample

homogenous solution. Once incrementally dilute, replace 1 piece of 1mL sterile pipette.

5.1.5 According to the evaluation of the sample pollution, select the sample

homogenous solution (liquid sample can include the stock solution) with 2 ~ 3

appropriate dilution; when performing the 10 times incremental dilution, absorb 1mL of

sample homogenous solution for each dilution, and place into 2 sterile flat plates,

respectively.

5.1.6 Timely cool off the 20mL ~ 25mL of potato dextrose agar or rose Bengal agar

(can place into a 46°C±1°C constant temperature water bath for thermal insulation) to

46°C, then pour into the flat plate; rotate the flat plate to make it mix evenly.

5.2 Cultivation

After agar solidification, upright the flat plat, and place into 28°C±1°C incubator for

cultivation; observe and record the cultivation results to the first 5d.

5.3 Colony counting

Perform visual examination; if necessary, use magnifier or low power lens to record

dilution factor, and corresponding number of moulds and yeast colonies. It is expressed

by the Colony-Forming Unit (CFU).

6.1 Results

6.1.1 Calculate the average value of two flat plate colonies at the same dilution, then

multiply the average value by the corresponding dilution factor.

6.1.2 If the number of colonies on two dilution flat plate is in 10CFU ~ 150CFU,

calculate the corresponding values as per the provisions of GB 4789.2.

6.1.3 If the number of colonies on all flat plates are greater than 150CFU, count the

flat plate with the maximum dilution; other plates are recorded as unacceptable; the

result shall be calculated through the average number of colonies multiply by the

maximum dilution factor.

6.1.6 If the number of colonies on the flat plate with all dilution aren't in 10CFU ~

150CFU, thereof, one part is less than 10CFU or greater than 150CFU, then it shall be

calculated through the average number of colonies closest to 10CFU or 150CFU

multiply by the dilution factor.

6.2 Report

6.2.1 The number of colonies shall be rounded off as per the "tetrahedral

hybridization" principle. If the number of colonies are within 10, take one effective digit

to report; if the number of colonies are in 10 ~ 100, take two effective digits to report.

6.2.2 When the number of colonies are greater than or equal to 100, after the first

three digits are rounded off as per the "tetrahedral hybridization" principle, the result

shall be expressed by the first two digits plus "0" replacing the following digit capacities;

or expressed by the index number of 10;

6.2.3 If the colony appears on the blank control flat plate, then such test results are

invalid.

6.2.4 The weight sampling shall be reported in unit of CFU/g; while the volume

sampling shall be reported in unit of CFU/mL; report, or respectively report the number

of moulds and/or yeasts.

7.1 Preparation of the sample for examination. take appropriate amount of sample

for examination; add distilled water to dilute to the refractive index to be 1.3447 ~

1.3460 (i.e. concentration is 7.9% ~ 8.8%); then backup.

7.2 Microscope calibration of standard field of view. magnify the microscope for 90×

~ 125×, adjust the standard field of view; so that its diameter is 1.382mm.

7.3 Smear. 7.4 Observation. place the prepared slide under the standard field of view of

microscope to observe. Generally, one sample for examination shall be observed for

7.5 Results and calculation. under the standard field of view, if finding the mould

hypha length exceed 1/6 of standard field of view (1.382mm) or the total length of three

hyphae exceed 1/6 of standard field of view (i.e. 1 block of micrometer), then it shall

be recorded as positive (+), otherwise, it shall be recorded as negative (-).

7.6 Report. the whole number of positive field of views in every 100 field of views are

reported as the visual field percentage of moulds (visual field %).

Appendix A

Culture Medium and Reagents

A.1 Normal saline

A.1.1 Components

Sodium chloride 8.5g

Distilled water 1000mL

A.1.2 Preparation

Add sodium chloride into 1000mL of distilled water, mix till it is fully dissolved; after

packaging separately, sterilize for 15min at 121°C, then backup.

A.2 Potato dextrose agar

A.2.1 Components

A.2.2 Preparation

Peel the potato and cut it into blocks; add 1000mL of distilled water, and boil for 10min

~ 20min. Use gauze to filter, add distilled water to 1000mL. Add dextrose and agar,

heating for dissolution; after packaging separately, sterilize for 15min at 121°C, then

backup.

A.3 Rose Bengal agar

A.3.2 Preparation

Add the above components into the distilled water, heating for dissolution; make up

sufficient distilled water to 1000mL; after packaging separately, sterilize for 15min at

121°C; store in the dark for backup.

A.4 Phosphate buffering solution

A.4.1 Components

Potassium dihydrogen phosphate 34.0g

Distilled water 500mL

A.4.2 Preparation

Stock solution. take 34.0g of potassium dihydrogen phosphate and dissolve into

500mL of distilled water; adjust with 175mL of 1mol/L sodium hydroxide to pH value to

be 7.2±0.1; use distilled water to dilute to 1000mL then store in the refrigerator.

Diluent. take 1.25mL of stock solution; use distilled water to dilute to 1000mL;

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