Analysis of Changes in GB 4789.2-2022 “National Food Safety Standard - Microbiological Examination of Food - Determination of Total Bacterial Count“

In the determination of total bacterial count in food microbiology testing, due to the quantification of micro scale microorganisms that are invisible to the naked eye, there has always been a certain degree of uncertainty in the results of total bacterial count determination. Throughout the entire testing process, the accuracy of dilution, colony identification, and aseptic control are the key links that generate errors and lead to uncertainty in the results.

In order to improve the accuracy of the total bacterial count determination results, the Food Microbiological Examination Determination of Total Bacterial CountGB4789.2from2016Version to2022The version focuses on strengthening control over these aspects.
● In the liquid sample preparation process, the use of a tapping homogenizer has been added for preparation1:10Experimental steps for sample homogenization;
● In the sample dilution stage, change the repeated blowing and mixing method of the pipette to the oscillator oscillation mixing method;
● In the colony detection process, a detection method for total colony count test strips has been added;

Accuracy of dilution

Dilution is an important step in determining the total number of bacterial colonies. If the degree of mixing is not sufficient100%This indirectly generates dilution error, and the error increases geometrically with the increase of dilution ratio.

The tapping sample dilution method has been validated as an experimental method that can achieve good mixing uniformity, therefore2022The new national standard also requires the dilution of liquid samples to be carried out using a tapping mixing operation; The tapping type mixing operation can effectively promote the vibration between the sample and the diluent by applying external mechanical force, thereby achieving rapid and thorough mixing, effectively avoiding or reducing dilution errors caused by insufficient mixing.

SwitzerlandiNLABTEC Serial UCContinuous gradient diluter adopts a tapping mixing method, and the automatic tapping device can enhance the vibration of the diluted sample solution, requiring only4Reached in seconds100%The degree of mixing.

No need for straw blowing operation, in compliance with the requirements of the new national standard GB4789.2-2022;

● 4-second tapping mixing method, achieving sufficient and efficient mixing;

● Using aseptic technologyTipThe head, sterile dilution bag, and sterile bag opener fully comply with sterile control requirements.

Accuracy of colony recognition

The colonies on agar are visible bacterial clusters formed by the division and reproduction of bacterial cells or cell clusters that are not visible to the naked eye after cultivation. Due to the significant differences in colony morphology among different types of bacteria, there is still no standardized judgment for colony identification.

In order to ensure that the bacteria in the sample are not affected by the antagonistic effect of limited growth space, the pouring method is adopted to prepare sample petri dishes. But the growth of colonies in agar increases the complexity of colony recognition. Such as colony adhesion, colony overlap, agar bubbles, etc.

The 2022 version of the new national standard has added a detection method for total bacterial count test strips. The total bacterial count test strips only need to identify the color spots generated by the colonies to determine the colonies, effectively reducing the difficulty of agar recognition of colonies and providing a solution to reduce colony recognition errors. However, bacteria that can form color spots on colony test strips only meet the requirements of growing on the culture medium of the test paper and producing color reactions with substrates. Currently, there is no more comprehensive validation data available. Therefore, the entire standard only covers the testing paper strip method6.2.2The middle sentence is mentioned without detailed guidance or explanation.

The Punmicro ICON fully automatic colony counter innovatively introduces artificial intelligence technology, which has objective and accurate recognition ability for colonies on complex pouring method sample plates, effectively improving the accuracy of colony counting results.

● AI artificial algorithm technology, capable of autonomously learning colony determination rules, combined with high-resolution imaging systems, can achieve or even exceed manual counting levels, solving complex colony recognition and determination problems such as overlapping colonies, adherent colonies, and irregular colonies;

● Full dish counting, effectively distinguishing between refracted and reflected edges of the culture dish, solving the problem of automatic identification and counting of edge colonies;

● One click counting eliminates the need for manual parameter settings based on sample conditions, allowing for direct counting of samples and solving the practicality issue of fully automatic colony counters;

● Bacterial colony testing paper analysis can be performed, and with the support of a high-resolution imaging system, the identification of overlapping bacterial plaques that are difficult to recognize with the naked eye can be solved;

● It can comprehensively record images and data, and customize data statistical operations and detection report formats.

Aseptic control

Aseptic control is a fundamental and important requirement for the determination of total bacterial count, and avoiding external contamination and cross contamination directly affects the accuracy of detection data.

Attention paid to the 2022 version of the new national standard10During the dilution process of the multiple series, there is a great risk of external contamination when using a pipette to repeatedly blow and mix, so an oscillator is used for oscillation mixing.

But the vibration amplitude of the oscillator for the dilution system is far less than that of repeated blowing. If you want to achieve 100% mixing, the only way is to extend the oscillation time, which will significantly reduce the speed of experimental operation.