Introduction to Microbial Plate Counting Method and Flow Cytometry Detection Method

The importance of food safety is self-evident, and microbial detection is an important part of food safety inspection. When conducting microbial detection, it is first necessary to select a suitable microbial inspection method according to its nature and characteristics, and at the same time clarify the pollution status, hazard level of food, as well as specific inspection items and requirements.

Microbial detection methods include conventional detection methods (such as plate counting method, smear microscopy, isolation and culture, biochemical test, serological test, etc.), immunological methods, molecular biology methods, metabolomics technology, etc. In this issue, we will introduce 2 commonly used microbial detection methods: plate counting method and flow cytometry detection method.

1. Plate Counting Method

Three types of media are commonly used for microbial plate counting: Nutrient Broth Agar Medium, Gause's Medium No.Ⅰ, and Martin Agar Medium. For the preparation of common media used in food microbial detection, different preparation materials should be selected according to different inspection purposes. The common material ratios of media are shown in Table 1. In the actual process of food microbial inspection, preparation materials are selected according to different needs.

Nutrient Broth Agar Medium: Mainly used for the cultivation of bacteria in food microorganisms. To ensure the quality of the medium, it is necessary to perform sterilization at 125°C for 15 minutes continuously.

Gause's Medium No.Ⅰ: Mainly used for the cultivation of actinomycetes. During the preparation process, a small amount of cold water should first be added to starch, stirred to obtain pasty starch, then poured into 100°C water, and then heated and added with other preparation materials and stirred until dissolved. After preparation, it also needs to be sterilized at 125°C for 15 minutes continuously.

Martin Agar Medium: Mainly used for the isolation of fungi in microorganisms. After sterilization, 0.02% streptomycin dilution is added before use, usually 100~150mL, to ensure that each milliliter of medium contains 25μg of streptomycin.

However, the plate counting method has insufficient timeliness, is difficult to distinguish between live and dead cells, and cannot detect non-culturable microorganisms. The Viable but Non-Culturable (VBNC) state of microorganisms

2. Flow Cytometry Detection Method

Flow cytometer detection of microorganisms is also a commonly used detection technology in scientific research. Compared with eukaryotic cells, microorganisms such as bacteria have smaller volumes, and their scattered light is difficult to distinguish from particles of other substances. Therefore, detection by flow cytometer relies on fluorescent dyes bound to cells to identify real targets from the background.

According to different fluorescent substrates, fluorescent dyes suitable for flow cytometry detection mainly include: dyes that bind to nucleic acids, such as PI and 7-AAD; dyes that label antibody proteins, such as Cy5, FITC, and PE. Different fluorescent dyes have different excitation wavelengths and emission wavelengths. Flow cytometers are usually equipped with multiple lasers, such as 405nm, 488nm, 633nm, etc. Multiple lasers can realize multi-fluorescence channel analysis, reduce autofluorescence interference, reduce compensation between fluorescent signals, and improve detection sensitivity.

In addition, the selection of fluorescence labels for flow cytometry also needs to consider multiple factors such as the light source wavelength configured by the instrument, the matching of dye brightness with target expression level, dye spectral overlap, and sample autofluorescence.

3. Principle and Method of 7-AAD Flow Cytometry Detection

7-AAD is a nucleic acid dye that is released during cell apoptosis and cell death. When cells start to apoptose, the permeability of their plasma membrane to 7-AAD gradually increases. Under excitation at a specific wavelength, 7-AAD can emit bright red fluorescence. Through the intensity of this fluorescence, cells can be divided into three populations: those with strong 7-AAD signals represent dead cells, those with weak 7-AAD signals are apoptotic cells, and those with no detected 7-AAD signals are normal viable cells.

This operation procedure is applicable to most cells, but different cell types, cell densities, used media, and other factors may affect the staining effect. The following steps are for reference only.

1.       Preparation of Stock Solution: Add an appropriate amount of DMSO to the original 7-AAD vial to prepare a 1-10mM stock solution, aliquot and store at -20°C, which can be stably stored for 6 months;

2.       Select appropriate steps to fix cells according to your own samples. 7-AAD staining is generally performed after other staining is completed, and only stains dead cells;

3.       Collect cells by centrifugation, and resuspend the cells with an appropriate buffer solution or medium (pH=7.4);

      Note: Adherent cells can be subjected to in situ staining on coverslips or culture plates.

4.       Add an appropriate amount of 7-AAD staining solution. The recommended working concentration of this dye is 0.5-5µM, and incubate for 15-60min;

      Note: For the first experiment, it is recommended to set a concentration gradient within the recommended working concentration range for detection to explore the optimal staining concentration.

5.       Detect the fluorescence intensity on a flow cytometer (FL3 channel) according to its maximum excitation and emission wavelengths (Ex/Em=545nm/650nm).

References

[1] Wu F Y, Feng Q F, Lin L. Research on Preparation, Sterilization and Storage of Common Media for Food Microbial Inspection [J]. Food Safety Guide, 2020(36):1.

[2] Gao H M, Yan C R, Xu C X, et al. Research Progress of Flow Cytometry in Food Microbial Inspection [J]. Biological Disaster Science, 2023, 46(4): 549-556.

[3] Tian X X, Nanding K, Dai X Y, et al. Pattern recognition receptor mediated innate immune response requires a Rif-dependent pathway [J]. Journal of Autoimmunity, 134 (2023) 102975. Impact Factor: 14.511

Microbial Medium Raw Materials

Common Dyes for Flow Cytometry Detection

Common Tandem Dyes for Flow Cytometry Detection

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