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GB 4789.2-2022 (GB4789.2-2022)

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GB 4789.2-2022: PDF in English

GB 4789.2-2022
National food safety standard - Microbiological examination
of food: Aerobic plate count
ISSUED ON: JUNE 30, 2022
Issued by: National Health Commission of the PRC;
State Administration for Market Regulation.
Table of Contents
Foreword ... 3 
1 Scope ... 4 
2 Terms and definitions ... 4 
3 Equipment and materials ... 4 
4 Medium and reagents ... 5 
5 Examination procedure ... 5 
6 Operation steps ... 6 
7 Result and report ... 8 
Appendix A Medium and reagents ... 10 
Appendix B Examples ... 12 
National food safety standard - Microbiological examination
of food: Aerobic plate count
1 Scope
This Standard specifies the method for the determination of aerobic plate count in food.
This Standard applies to the determination of aerobic plate count in food.
2 Terms and definitions
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,
3 Equipment and materials
In addition to the routine sterilization and culture equipment in the microbiology
laboratory, other equipment and materials are as follows:
a) Constant-temperature incubator: 36 ℃±1 ℃, 30 ℃±1 ℃.
b) Refrigerator: 2 ℃~5 ℃.
c) Thermostat: 48 ℃±2 ℃.
d) Balance: Sensitivity is 0.1 g.
e) Homogenizer.
f) Oscillator.
g) Sterile straw: 1 mL (with 0.01 mL scale), 10 mL (with 0.1 mL scale) or
micropipette and tip.
h) Sterile conical flask: Capacity is 250 mL and 500 mL.
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
46 °C~50 °C (which can be kept in a thermostat at 48 °C±2 °C) into a petri dish; rotate
the petri dish to make it evenly mixed.
6.2 Culture
6.2.1 Place horizontally until the agar solidifies; turn the plate over; incubate at
36 °C±1 °C for 48 h±2 h. Aquatic products are cultured at 30 °C±1 °C for 72 h±3 h. If
the sample may contain colonies that spread and grow on the surface of the agar
medium, it is possible to cover the surface of the solidified agar medium with a thin
layer of plate count agar medium (about 4 mL). After solidification, turn the plate over
and culture.
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
6.3.1 It is possible to use the naked eye to observe. If necessary, use a magnifying glass
or a colony counter, to record the dilution ratio and the corresponding number of
colonies. Colony counts are expressed in colony forming unit (CFU).
6.3.2 Select the plate with the colony number between 30 CFU and 300 CFU and no
spreading colony growth, to count the aerobic plate count. For plates with less than 30
CFU, record the specific number of colonies; those with more than 300 CFU can be
recorded as incalculable.
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 Result and report
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.
N - The number of colonies in the sample;
ΣC - The sum of the colony counts on the plates (containing plates with appropriate
range of colony counts);
n1 - Number of plates at the first dilution (low dilution ratio);
n2 - Number of plates at the second dilution (high dilution ratio);
d - Dilution factor (first dilution).
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
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
(Above excerpt was released on 2022-08-21, modified on 2022-08-21, translated/reviewed by: Wayne Zheng et al.)
Source: https://www.chinesestandard.net/PDF.aspx/GB4789.2-2022