flow cytometric data acquisition of microbial aggregates

使用成像流式细胞术定量分析微生物自聚集

Bacterial autoaggregation is considered a primary step in biofilm formation. In this process (sometimes also called autoagglutination or flocculation), bacteria of the same type form multicellular clumps that eventually settle at the bottom of culture tubes or attach to their target tissue or surface1.
Autoaggregation is a widely observed phenomenon and has been shown so far in Gram-negative pathogens such as the opportunistic pathogen Acinetobacter baumannii2, the dental pathogen Aggregobacter actinomycetemcomitans3, and the emerging pathogen Burkholderia pseudomallei4. Autoaggregation has also been described in several probiotic Gram-positive strains5,6,7,8. In Lactobacillus (L.) acidophilus, autoaggregation was partially mediated by S-layer proteins and correlated with an adhesion to xylan7. Similarly, we found a correlation between glucose-dependent autoaggregation and induction of the adhesive properties (as judged by adhesion to mucin) of the probiotic species Lacticaseibacillus rhamnosus GG, Lacticaseibacillus casei, L. acidophilus, Lacticaseibacillus paracasei, and Lactiplantibacillus plantarum5. The increased autoaggregation most likely reflected changes in the expression of cellular adhesins in response to glucose and its catabolites9. While the molecular mechanisms of autoaggregation remain to be determined, it has been shown that this process alters the phenotype of the aggregated bacteria and grants them enhanced tolerance to environmental stressors1, as well as increased sensitivity to quorum sensing molecules10.
Several approaches have been used to measure autoaggregation; one experimental approach is to let cultures stand statically in narrow culture tubes for a given time. Control cultures remain turbid, whereas autoaggregation cultures will settle at the bottom of the tube. A more quantitative approach measures autoaggregation by sedimentation or settling assay11.
Flow cytometry has also been increasingly employed in recent years to investigate bacterial autoaggregation. This method is appropriate for analyzing particles between approximately 0.5 and 1000 &#181;m in size. The single bacterium or formed aggregates are suspended in fluid, fed into a stream, and can be detected one by one11. Recording forward scattered light allows measuring the relative size of the cell or aggregate. It is relatively fast and straightforward but cannot detect several parameters, such as aggregate size or the average number of cells in aggregates. Therefore, this approach can be complemented microscopically, allowing more parameters to be checked12. However, traditional microscopy is time-consuming and thereby limits the number of tested samples and the statistical power of the analysis. In general, imaging flow cytometry provides several features compared to traditional flow cytometry, such as simultaneous analysis of cell morphology and phenotype, conducting image-based analysis, detecting rare events, and validating flow cytometry data13. These advantages enhance the capabilities of flow cytometry and facilitate a more detailed examination of cell populations.
This study provides a valuable protocol for imaging flow cytometry to monitor autoaggregation in lactic acid bacteria (LAB). These Gram-positive rods are facultative anaerobes and belong to the LAB group. These efficient glucose fermenters generate lactic acid as their main end-product of carbohydrate metabolism14. These bacteria are beneficial core members of the microbiome and are naturally found in the gastrointestinal tract (GIT) of humans and animals, as well as in the urogenital tract of females15. Therefore, the exact characterization of their autoaggregation properties is of high biotechnological and clinical interest.
Our previous findings indicated that the basal level of autoaggregation differs between different probiotic strains. This heterogeneity is affected by the different carbohydrates used as a carbon source5. To overcome this fundamental property of probiotic bacteria, the effects of carbohydrates from the diet were monitored on autoaggregation on the single-cell level using IFC. This IFC-based approach combines the power and speed of traditional flow cytometers with the microscope&#39;s resolution. Therefore, it allows high-rate complex morphometric measurements in a phenotypically defined way16,17. This approach can be extended to other probiotic and pathogenic bacteria, combined with fluorescent reporters to monitor gene expression, and fluorescently labeled strains to monitor the presence and abundance of specific bacterial species in heterogeneous aggregates.

作者聚焦：使用成像流式细胞术推进微生物自聚集的研究

成像流式细胞术研究微生物自聚集

This study utilized Lactobacillus species as a model to demonstrate the effectiveness of imaging flow cytometry analysis in studying microbial autoaggregation. The focus is on analyzing the response of autoaggregation of Lactobacillus species to simple carbohydrates from the diet. The most used methods for measuring autoaggregation are spectral photometry, which measures microbial suspensions, turbidity, or optical density, and traditional microscopy techniques, such as bright field, phase contrast, and fluorescent microscopy.When talking about hight-throughput imaging, one major experimental challenge is single event segmentation. In this protocol, it is a single aggregate. Imaging flow cytometry addresses this challenge elegantly and give researchers a powerful tool for studying autoaggregation.

微生物聚集体的流式细胞术数据采集

要开始流式细胞术分析，首先将 785 纳米激光器的功率设置为 5 毫瓦，用于暗场测量。使用数值孔径为 0.9 的 60X 镜头。然后选择高灵敏度设置并将速度设置为低。为确保校准磁珠的稳定性，请按下液流盒中的“播放”按钮并检查磁珠图像，该图像应看起来清晰锐利。通过选择“新建散点图”，在工作区框中生成新的散点图。然后，在明场通道中选择对应于SSC通道强度的区域，并排除样品中同时运行的校准磁珠。接下来，按下加载按钮并将装有乳酸杆菌样品的试管放入支架中。在“采集设置”框中，输入样本名称并指定数据存储位置。设置要收集的事件数，通常在 10， 000 到 20， 000 个事件的范围内。现在，通过单击位于“采集”框中的“记录”按钮开始采集过程。在达到指定的事件量后，该过程将停止。按下返回按钮卸下管子，然后按照弹出窗口中的提示将其从支架上取下。对所有样品重复该过程后，保存模板以保持数据采集的均匀性。对于数据分析，要将原始文件加载到分析软件中，请单击“文件”并打开 rif 文件。在打开的窗口中，选择“使用采集分析”，然后单击“确定”。接下来，要创建梯度均方根的直方图，请单击新建直方图按钮，然后从采集中选择要分析的总体。在 X 轴功能下，选择明场通道的梯度 RMS。接下来，通过单击分析区域中的“创建线区域”按钮来放置一个门以排除非聚焦事件。通过单击分析区域中的“新建散点图”按钮，创建面积与纵横比的散点图。在“新建散点图”窗口中选择重点总体。然后选择 X 轴特征的明场通道区域。选择 Y 轴特征的纵横比。使用“创建矩形”按钮根据聚合事件群体的面积值在散点图上绘制门。同样，为单身群体和小群体群体画另一个门。单击“文件”选项卡，选择“另存为模板”ast，然后打开同一模板下的下一个示例文件以创建数据分析文件，从而将数据分析另存为模板。要测量聚合大小分布，首先绘制聚合事件总体面积的直方图。通过单击文件并选择“另存为模板”选项来保存数据分析。保存后，在数据分析文件的同一模板下打开下一个示例文件。在LGG和副干酪乳杆菌中观察到单细胞、小聚集体、大聚集体和链，但无法区分链和大聚集体。观察到不同乳酸菌株在可发酵或不可发酵糖的聚集特性上存在显著差异。

Research

JoVE Journal

Biology

Flow Cytometric Data Acquisition of Microbial Aggregates

Imaging Flow Cytometry to Study Microbial Autoaggregation

Beneficial and probiotic bacteria play essential roles in their hosts, providing various health benefits, including immunity to infectious diseases. The ...

科研

生物学

Lactobacillaceae Sample Preparation for Flow Cytometric Analysis

乳酸杆菌科 Lactobacillaceae 流式细胞术分析的样品制备