GB 4789.2-2022National food safety standard - Microbiological examination of food: Aerobic plate count (English PDF)

2.1 Aerobic plate count

The total number of microbiological colonies formed in per g (mL) of test sample,

which is obtained after the food sample under test is processed and cultured under

certain conditions (such as culture medium, culture temperature, and incubation time,

etc.).

4.1 Plate count agar medium. See A.1.

4.2 Test piece of aerobic plate count. It shall meet the quality control requirements of

plate count agar medium in GB 4789.28.The main nutrients are consistent with the

formula of plate count agar medium.

4.3 Sterile phosphate buffer. See A.2.

6.1 Dilution of sample

6.1.1 Solid and semi-solid sample. Weigh 25 g of sample; place it in a sterile

homogeneous cup filled with 225 mL of sterile phosphate buffer or sterile normal saline.

Homogenize at 8000 r/min~10000 r/min for 1 min~2 min. Or put it into a sterile

homogeneous bag containing 225 mL of diluent. Use a slap-type homogenizer to beat

for 1 min~2 min, to make a 1.10 sample homogenate.

Test sample25 g (mL) of sample + 225 mL of diluent,homogenized10-fold serial dilution

Culture Add 15 mL~20 mL of plate count agar

medium to each dish and mix well Select 1~3 sample homogenates of

appropriate dilution; add 1 mL of each to asterile petri dish Count the number of colonies

on each plate Calculate the aerobic plate count Report Culture at 36 °C+/-1 °C for 48 h+/-2 h. Aquatic

products are cultured at 30 °C+/-1 °C for 72 h+/-3 h

6.1.2 Liquid sample. Use a sterile straw to pipette 25 mL of sample; place it in a sterile

conical flask containing 225 mL of sterile phosphate buffer or sterile normal saline (an

appropriate number of sterile glass beads can be preset in the bottle); and mix

thoroughly. Or put it into a sterile homogeneous bag containing 225 mL of diluent. Use

a slap-type homogenizer to beat for 1 min~2 min, to make a 1.10 sample homogenate.

When the result is required to be the aerobic plate count per g of sample, operate

according to 6.1.1.

6.1.3 Use a 1 mL sterile straw or micropipette to draw 1 mL of the 1.10 sample

homogenate; along the tube wall, slowly inject it into a sterile test tube containing 9 mL

of diluent (be careful not to touch the surface of the diluent with the pipette or the tip).

Oscillate and mix on an oscillator, to make a 1.100 sample homogenate.

6.1.4 According to the operation in 6.1.3, prepare a 10-fold serial dilution of the sample

homogenate. For each incremental dilution, use a new 1 mL sterile straw or tip.

6.1.5 According to the estimation of the contamination status of sample, select 1 to 3

sample homogenates with appropriate dilution (liquid samples can include stock

solution). Pipette 1 mL of the sample homogenate into a sterile petri dish; make two

petri dishes for each degree of dilution. At the same time, respectively pipette 1 mL of

blank diluent; add it to two sterile petri dishes as blank control.

6.1.6 In a timely manner, pour 15 mL~20 mL of plate count agar medium cooled to

6.2 Culture

6.2.1 Place horizontally until the agar solidifies; turn the plate over; incubate at

6.2.2 If the test piece of aerobic plate count is used, it shall be operated in accordance

with the relevant technical regulations provided for the test piece.

6.3 Colony count

6.3.3 When one of the plates has larger flaky colonies, it is not suitable to use it. The

plate without larger flaky colonies shall be used as the colony count of the degree of

dilution. If the flaky colonies are less than half of the plate, and the colonies in the

remaining half are evenly distributed, it is possible to calculate the number of the half

plate and multiply it by 2, to represent the number of colonies on one plate.

6.3.4 When there is a chain growth with no clear boundary between the colonies on the

plate, count each single chain as a colony.

7.1 Calculation method of aerobic plate count

7.1.1 If the number of colonies on only one dilution plate is within the appropriate count

range, calculate the average of the number of colonies on the two plates; then multiply

the average by the corresponding dilution factor as the aerobic plate count per g (mL)

of the sample. Example is given in B.1.

7.1.2 If the number of colonies on the plate with two serial dilutions is within the

appropriate count range, calculate according to formula (1). For an example, see B.2.

Where.

7.1.3 If the number of colonies on all dilution plates is greater than 300 CFU, the plate

with the highest dilution shall be counted. The other plates can be recorded as

uncountable. The result shall be calculated by multiplying the average number of

colonies by the highest dilution ratio. For an example, see B.3.

7.1.4 If the plate colony count of all dilutions is less than 30 CFU, it shall be calculated

by multiplying the average colony count with the lowest dilution by the dilution ratio.

For an example, see B.4.

7.1.5 If plates at all degrees of dilution (including liquid sample stock solution) have no

colony growth, it shall be calculated by multiplying less than 1 by the minimum dilution

ratio. For an example, see B.5.

7.1.6 If the number of colonies on the plates at all degrees of dilution is not between 30

CFU and 300 CFU, and some of them are less than 30 CFU or greater than 300 CFU,

it shall be calculated by multiplying the average colony number closest to 30 CFU or

7.2 Report of aerobic plate count

Appendix A

Medium and reagents

A.1 Plate count agar (PCA) medium

A.1.1 Ingredients

Tryptone (main nutrient). 5.0 g

Yeast extract (main nutrient). 2.5 g

Glucose (main nutrient). 1.0 g

Agar. 15.0 g

Distilled water. 1000 mL

A.1.2 Preparation method

Add the above ingredients to distilled water; boil to dissolve; adjust the pH to 7.0+/-0.2.

Dispense into suitable containers; sterilize by autoclaving at 121 °C for 15 min.

A.2 Sterile phosphate buffer

A.2.1 Ingredients

Potassium dihydrogen phosphate (KH2PO4). 34.0 g

Distilled water. 500 mL

A.2.2 Preparation method

Stock solution. Weigh 34.0 g of potassium dihydrogen phosphate and dissolve it in 500

mL of distilled water; use about 175 mL of 1 mol/L sodium hydroxide solution to adjust

the pH to 7.2; use distilled water to dilute to 1000 mL; store in the refrigerator.

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

divide it into suitable containers; sterilize it by autoclaving at 121 °C for 15 min.

A.3 Sterile normal saline

A.3.1 Ingredients

Sodium chloride. 8.5 g

Distilled water. 1000 mL

A.3.2 Preparation method

Weigh 8.5 g of sodium chloride; dissolve it in 1000 mL of distilled water; sterilize by

autoclaving at 121 °C for 15 min.

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