这项工作得到了以色列科学基金会（Grant 119/16）和IMoh资助（3-15656）对IKG的支持。R.S. 由 Kreitman 奖学金支持。

.ast 文件以 鼠李糖乳杆菌 GG （LGG） 的模板为例，在 补充编码文件 1 中提供。
1. 培养基制备
<ol><li>按照制造商的说明（55 g 在 1 L 去离子水中）和 1.5% （w/v） 琼脂制备乳酸杆菌 MRS 琼脂平板（参见 材料表）。高压灭菌后，培养基可直接使用或可在4°C下储存。</li>
	<li>准备 50%（15 g 在 1 L 去离子水中）胰蛋白酶大豆肉汤 （TSB）（参见 材料表），TSB 补充有 1% （w/v） D-（+）-葡萄糖和 1% （w/v） D-（+）-棉子糖，然后高压灭菌以灭菌培养基。最好将其储存在4°C以避免污染。</li>
	<li>对于含有葡萄糖的缓冲培养基，制备补充有 1% （w/v） D-（+）-葡萄糖、5 mM 磷酸二氢钾和氢二钾和 100 mM MOPS（3-（N-吗啉基）丙烷磺酸，参见 材料表）pH 7 的 TSB。然后高压灭菌以对培养基进行灭菌。</li>
</ol>2. 样品的制备
注意：此步骤涉及评估细胞聚集对饮食中可发酵或不可发酵糖的反应。样品制备过程的示意图如图 1 所示。
<ol><li>在乳酸杆菌MRS肉汤板（1.5%琼脂）上从冷冻甘油原液（50%甘油）中划线乳酸杆菌科菌株。 注：在此介绍的示例实验中，使用了 嗜酸乳杆 菌 （ATCC 4356）、 干酪乳杆 菌 （ATCC 393）、干酪乳酪杆菌 副干酪亚种 （ATCC BAA-52）、 植物乳 杆菌 （ATCC 8014） 和 鼠李糖乳杆 菌 GG （ATCC 53103） （LGG） 益生菌菌株。为了将该方法扩展到厚壁菌门的其他益生菌成员，选择了 凝结芽孢杆 菌（ATCC 10545）。</li>
	<li>在静态条件下在37°C下生长细胞过夜。</li>
	<li>将5mL乳酸杆菌MRS肉汤与单个菌落接种，并在37°C的静态条件下生长过夜。</li>
	<li>在 3 mL 50% 胰蛋白酶大豆肉汤 （TSB） 中稀释上一步 （1：100） 的细胞培养物，TSB 补充有 1% （w/v） D-（+）-葡萄糖，或 TSB 补充有 1% （w/v） D-（+）-棉子糖。</li>
	<li>在静态条件下在37°C孵育过夜。</li>
	<li>通过在培养基中均匀混合细胞来制备样品，并从步骤 2.5 开始轻轻涡旋细胞培养管。轻轻地倒置试管，直到完全混合。将 200-300 μL 转移到 1.5 mL 微量离心管中。 注意：必须不要对样品进行超声处理，以免破坏聚集体。在从步骤 3.4 到 3.10 的数据采集过程中，可能会出现样本过于集中的情况。如果观察到样品运行非常缓慢或根本不运行，则可以使用适当的新鲜培养基稀释样品。</li>
</ol>3. 数据采集
<ol><li>根据制造商提供的说明设置成像流式细胞仪（参见 材料表）。</li>
	<li>将 785 nm 激光器设置为 5 mW 进行暗场测量（相当于传统流式细胞术中的侧向散射，缩写为 SSC）。使用数值孔径 （N.A） 为 0.9 的 60 倍镜头，选择 高灵敏度，并将速度设置为 低。</li>
	<li>在收集任何数据之前，请确保校准磁珠在 SSC 通道中的流动稳定性。单击“Fluidics”框中的 播放 按钮，然后检查磁珠图像的质量。珠子图像应看起来清晰明了。</li>
	<li>按下 加载 按钮，将装有样品的试管（来自步骤 2.6）放入支架中。</li>
	<li>在“工作区”框中创建一个新的散点图。单击 “新建散点图 ”，然后在明场通道中选择与 SSC 通道强度相对应的区域。排除与样品一起在仪器中运行的校准珠。 注意：可以通过仅加载带有缓冲液的样品并识别区域与上的磁珠 来排除磁珠。SSC图。然后使用 “创建多边形区域 ”按钮在珠子群周围绘制一个门。</li>
	<li>在“采集设置”框中，输入 样本名称，指定数据存储位置，选择要记录的群体（没有磁珠的所有细胞），并设置要收集的事件数。通常，10,000-20,000 个事件就足够了。</li>
	<li>单击位于“采集”框中的 “录制 ”按钮，一旦达到指定的事件数，采集过程将自动结束。</li>
	<li>单击 “返回 ”按钮卸下试管，并在小窗口中出现提示时从支架中取出试管。</li>
	<li>对每个样品重复步骤 3.5-3.9。</li>
	<li>保存模板以保持数据采集的均匀性，以便后续重复。</li>
</ol>4. 数据分析
<ol><li>测量来自总体的自动聚合的百分比 （%）。<ol><li>使用IDEAS软件分析在成像流式细胞仪上采集的数据（参见 材料表）。</li>
		<li>通过将 （.rif） 文件加载到分析软件中，从原始文件 （.rif） 创建数据分析文件 （.daf）。单击“ 文件 ”按钮，然后打开所需的 （.rif） 文件。在打开的窗口中，单击“ 使用采集分析 ”，然后单击“ 确定”。</li>
		<li>在明场通道中创建梯度 RMS（图像对比度和焦点的测量）直方图，以识别聚焦事件。在分析区域中，单击 “新建直方图 ”按钮。在“新建直方图”窗口中，从采集中选择要分析的总体，然后在“x 轴特征”中，选择明场通道的 梯度 RMS 。<ol><li>通过单击分析区域中的 “创建线区域 ”按钮来放置门，以排除非聚焦事件（图2A）。 注意：通常，“聚焦”线区域应包括 80%-90% 具有最高梯度 RMS 分数的事件，但应为每个实验设置确定“聚焦”单元格的特定阈值。</li>
		</ol></li>
		<li>创建面积 （μm2） 与明场通道的纵横比（宽度除以最佳拟合椭圆的长度）的散点图。在分析区域中按 新建散点图 按钮。在“新建散点图”窗口中，从步骤 4.1.3 中选择 聚焦 人口。<ol><li>为 x 轴特征选择明场通道的 面积 ，为 y 轴特征选择明场通道的 纵横比 。该散点图允许对聚焦群体进行门控，以区分单峰和小聚集体事件与较大的微生物聚集体和链。</li>
		</ol></li>
		<li>在此散点图上，根据聚合事件群体的面积值，使用 “创建矩形区域 ”按钮（或 “创建多边形区域”）绘制一个门，并为单峰和小聚合群体绘制另一个门（图 2B）。 注意：在某些情况下，从单峰中分离小聚集体可能具有挑战性，因此它们可以合并到同一个浇口中。这同样适用于较大的聚集体和链（图 3）。</li>
		<li>手动查看和评估每个闸门内的事件图像，以验证闸门策略的可靠性。如果需要，请调整闸门的面积值。通过从“人口”下拉菜单中选择已审查的人口来执行此操作。 注意：设门没有特定的面积值，因为它可能会根据被分析细菌的特性而变化。</li>
		<li>将数据分析另存为模板。单击 “文件 ”选项卡，选择“ 另存为 Template.ast”，然后打开同一模板下的下一个示例 （.rif） 文件，以创建数据分析文件 （.daf）（支持 .ast 文件作为 LGG 的示例）。</li>
		<li>创建一个统计信息表以枚举关键事件以供进一步分析。单击“ 报告 ”选项卡，然后单击“ 定义统计信息报告”。</li>
		<li>在新窗口中，单击“ 添加列”。添加单峰/聚合计数和 %门控统计信息。在“统计信息”下，选择“ %门控/计数”，然后在所选总体下，选择 单峰/聚合。单击“ 添加统计信息 ”以将统计信息添加到列表中并将其另存为模板。</li>
		<li>单击“ 生成统计报告 ”，然后选择统计模板和 （.daf） 文件进行分析。</li>
	</ol></li>
	<li>测量聚集体的尺寸分布。<ol><li>要分析聚集事件的平均大小，请绘制步骤4.1.5中聚集事件群体的面积（μm2）直方图（图2C）。</li>
		<li>将数据分析另存为模板。单击“ 文件 ”选项卡，选择“ 另存为模板”，然后在数据分析文件 （.daf） 的同一模板下打开下一个示例 （.rif） 文件。</li>
		<li>重复步骤 4.1.7-4.19。对于聚合的大小，请在统计数据下选择 均值 ，然后在所选总体下选择 聚合 。在“要素”下，选择“明场通道 的区域 ”。单击“ 添加统计信息 ”以将统计信息添加到列表中并将其另存为模板。</li>
		<li>重复步骤 4.1.9 以生成统计报告。</li>
	</ol></li>
</ol>

使用成像流式细胞术定量分析微生物自聚集

<ol>
	<li>Trunk, T., Khalil, H. S., Leo, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+autoaggregation.">Bacterial autoaggregation.</a> AIMS Microbiology. 4 (1), 140-164 (2018).</li><li>Ishikawa, M., Nakatani, H., Hori, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AtaA,+a+new+member+of+the+trimeric+autotransporter+adhesins+from+Acinetobacter+sp.+Tol+5+mediating+high+adhesiveness+to+various+abiotic+surfaces.">AtaA, a new member of the trimeric autotransporter adhesins from Acinetobacter sp. Tol 5 mediating high adhesiveness to various abiotic surfaces.</a> PLoS One. 7, e48830 (2012).</li><li>Inoue, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characterization+of+low-molecular-weight+component+protein,+Flp,+in+Actinobacillus+actinomycetemcomitans+fimbriae.">Molecular characterization of low-molecular-weight component protein, Flp, in Actinobacillus actinomycetemcomitans fimbriae.</a> Medical Microbiology and Immunology. 42 (4), 253-258 (1998).</li><li>Boddey, J. A., Flegg, C. P., Day, C. J., Beacham, I. R., Peak, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-regulated+microcolony+formation+by+Burkholderia+pseudomallei+requires+pilA+and+enhances+association+with+cultured+human+cells.">Temperature-regulated microcolony formation by Burkholderia pseudomallei requires pilA and enhances association with cultured human cells.</a> Infection and Immunity. 74 (9), 5374-5381 (2006).</li><li>Suissa, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Context-dependent+differences+in+the+functional+responses+of+Lactobacillaceae+strains+to+fermentable+sugars.">Context-dependent differences in the functional responses of Lactobacillaceae strains to fermentable sugars.</a> Frontiers in Microbiology. 13, 949932 (2022).</li><li>Isenring, J., Geirnaert, A., Lacroix, C., Stevens, M. J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bistable+auto-aggregation+phenotype+in+Lactiplantibacillus+plantarum+emerges+after+cultivation+in+in+vitro+colonic+microbiota.">Bistable auto-aggregation phenotype in Lactiplantibacillus plantarum emerges after cultivation in in vitro colonic microbiota.</a> BMC Microbiology. 21, 268 (2021).</li><li>Kos, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesion+and+aggregation+ability+of+probiotic+strain+Lactobacillus+acidophilus+M92.">Adhesion and aggregation ability of probiotic strain Lactobacillus acidophilus M92.</a> Journal of Applied Microbiology. 94 (6), 981-987 (2003).</li><li>Zommiti, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+assessment+of+the+probiotic+properties+and+bacteriocinogenic+potential+of+Pediococcus+pentosaceus+MZF16+isolated+from+artisanal+tunisian+meat+&amp;#34;Dried+Ossban&amp;#34;.">In vitro assessment of the probiotic properties and bacteriocinogenic potential of Pediococcus pentosaceus MZF16 isolated from artisanal tunisian meat &amp;#34;Dried Ossban&amp;#34;.</a> Frontiers in Microbiology. 9, 2607 (2018).</li><li>Suissa, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+rewiring+of+the+probiotic+bacterium+rhamnosus&lt;/em&gt;+GG+contributes+to+cell-wall+remodeling+and+antimicrobials+production.">Metabolic rewiring of the probiotic bacterium rhamnosus&lt;/em&gt; GG contributes to cell-wall remodeling and antimicrobials production.</a> bioRxiv. , (2023).</li><li>Connell, J. L., Kim, J., Shear, J. B., Bard, A. J., Whiteley, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+monitoring+of+quorum+sensing+in+3D-printed+bacterial+aggregates+using+scanning+electrochemical+microscopy.">Real-time monitoring of quorum sensing in 3D-printed bacterial aggregates using scanning electrochemical microscopy.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (51), 18255-18260 (2014).</li><li>Misawa, N., Blaser, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+characterization+of+autoagglutination+activity+by+Campylobacter+jejuni.">Detection and characterization of autoagglutination activity by Campylobacter jejuni.</a> Infection and Immunity. 68 (11), 6168-6175 (2000).</li><li>Sherlock, O., Schembri, M. A., Reisner, A., Klemm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+roles+for+the+AIDA+adhesin+from+diarrheagenic+Escherichia+coli:+cell+aggregation+and+biofilm+formation.">Novel roles for the AIDA adhesin from diarrheagenic Escherichia coli: cell aggregation and biofilm formation.</a> Journal of Bacteriology. 186 (23), 8058-8065 (2004).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+flow+cytometry.">Imaging flow cytometry.</a> Nature Reviews Methods Primers. 2, 87 (2022).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+characteristics+of+lactic+acid+bacteria+and+the+expanding+applications+in+food+industry.">Metabolism characteristics of lactic acid bacteria and the expanding applications in food industry.</a> Frontiers in Bioengineering and Biotechnology. 9, 612285 (2021).</li><li>Turroni, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+dialogue+between+the+human+gut+microbiota+and+the+host:+a+Lactobacillus+and+Bifidobacterium+perspective.">Molecular dialogue between the human gut microbiota and the host: a Lactobacillus and Bifidobacterium perspective.</a> Cellular and Molecular Life Sciences. 71, 183-203 (2014).</li><li>Zuba-Surma, E. K., Kucia, M., Abdel-Latif, A., Lillard, J. W., Ratajczak, M. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ImageStream+System:+a+key+step+to+a+new+era+in+imaging.">The ImageStream System: a key step to a new era in imaging.</a> Folia Histochemica et Cytobiologica. 45, 279-290 (2007).</li><li>Dashkova, V., Malashenkov, D., Poulton, N., Vorobjev, I., Barteneva, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+flow+cytometry+for+phytoplankton+analysis.">Imaging flow cytometry for phytoplankton analysis.</a> Methods. 112, 188-200 (2017).</li><li>Maan, H., Gilhar, O., Porat, Z., Kolodkin-Gal, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacillus+subtilis+colonization+of+arabidopsis+thaliana+roots+induces+multiple+biosynthetic+clusters+for+antibiotic+production.">Bacillus subtilis colonization of arabidopsis thaliana roots induces multiple biosynthetic clusters for antibiotic production.</a> Frontiers in Cellular and Infection Microbiology. 11, 722778 (2021).</li><li>Maan, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+flow+cytometry+reveals+a+dual+role+for+exopolysaccharides+in+biofilms:+To+promote+self-adhesion+while+repelling+non-self-community+members.">Imaging flow cytometry reveals a dual role for exopolysaccharides in biofilms: To promote self-adhesion while repelling non-self-community members.</a> Computational and Structural Biotechnology Journal. 20, 15-25 (2022).</li><li>Konieczny, M., Rhein, P., Czaczyk, K., Bialas, W., Juzwa, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+flow+cytometry+to+study+biofilm-associated+microbial+aggregates.">Imaging flow cytometry to study biofilm-associated microbial aggregates.</a> Molecules. 26 (23), 7096 (2021).</li><li>Niederdorfer, R., Peter, H., Battin, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attached+biofilms+and+suspended+aggregates+are+distinct+microbial+lifestyles+emanating+from+differing+hydraulics.">Attached biofilms and suspended aggregates are distinct microbial lifestyles emanating from differing hydraulics.</a> Nature Microbiology. 1, 16178 (2016).</li></ol>

流式细胞术是一种广泛使用的方法，用于量化真核细胞中的荧光强度，但由于细菌细胞的尺寸较大或聚集体较小，它可能无法为细菌细胞提供准确的测量结果。这些因素会显著影响自聚集的精确定量和不同条件下聚集体形成的基础水平。为了解决这个问题，采用成像流式细胞术 （IFC） 来更好地解析碳水化合物如何影响益生菌聚集5。IFC 通过收集每个样本的大量数字图像并提取基...

细菌自聚集被认为是生物膜形成的主要步骤。在此过程中（有时也称为自凝集或絮凝），相同类型的细菌形成多细胞团块，最终沉淀在培养管底部或附着在其靶组织或表面1。
自聚集是一种广泛观察到的现象，迄今为止已在革兰氏阴性病原体中得到证实，例如机会性病原体鲍曼不动杆菌 2、牙科病原体放线菌聚集杆菌 3 和新出现的病原体 Burkholderia pseudomallei4。在几种益生菌革兰氏阳性菌株中也描述了自聚集 5,6,7,8。在嗜酸乳杆菌 （L.） 中，自聚集部分由 S 层蛋白介导，并与与木聚糖7 的粘附相关。同样，我们发现葡萄糖依赖性自聚集与益生菌物种 Lacticaseibacillus rhamnosus GG、Lacticaseibacillus casei、L. acidophilus、Lacticaseibacillus paracasei 和 Lactiplantibacillus plantarum5 的粘附特性（通过粘蛋白粘附来判断）之间存在相关性.自聚集的增加很可能反映了细胞粘附素表达对葡萄糖及其分解代谢物的反应变化9。虽然自聚集的分子机制仍有待确定，但已经表明，该过程改变了聚集细菌的表型，并赋予它们对环境压力源1 的增强耐受性，并增加了对群体感应分子的敏感性10。
已经使用了几种方法来测量自聚合;一种实验方法是让培养物在狭窄的培养管中静态放置给定时间。对照培养物保持浑浊，而自聚集培养物将沉淀在试管底部。一种更定量的方法通过沉降或沉降测定法测量自聚集11。
近年来，流式细胞术也越来越多地用于研究细菌自聚集。该方法适用于分析大小约为 0.5 至 1000 μm 的颗粒。将单个细菌或形成的聚集体悬浮在液体中，送入液流中，并且可以逐个检测11。记录前向散射光可以测量细胞或聚集体的相对大小。它相对快速和直接，但无法检测多个参数，例如聚集体大小或聚集体中的平均单元格数。因此，这种方法可以在显微镜上进行补充，允许检查更多的参数12。然而，传统的显微镜检查非常耗时，因此限制了测试样品的数量和分析的统计能力。一般来说，与传统流式细胞术相比，成像流式细胞术具有多种功能，例如同时分析细胞形态和表型、进行基于图像的分析、检测罕见事件以及验证流式细胞术数据13。这些优点增强了流式细胞术的能力，并有助于对细胞群进行更详细的检查。
这项研究为成像流式细胞术监测乳酸菌 （LAB） 中的自聚集提供了一种有价值的方案。这些革兰氏阳性杆菌是兼性厌氧菌，属于乳酸菌组。这些高效的葡萄糖发酵罐产生乳酸作为碳水化合物代谢的主要最终产物14.这些细菌是微生物组的有益核心成员，天然存在于人类和动物的胃肠道 （GIT） 以及女性的泌尿生殖道中15。因此，对其自聚集特性的准确表征具有高度的生物技术和临床意义。
我们之前的发现表明，不同益生菌菌株之间的自聚集基础水平不同。这种异质性受到用作碳源的不同碳水化合物的影响5.为了克服益生菌的这一基本特性，使用IFC监测饮食中碳水化合物对单细胞水平自聚集的影响。这种基于 IFC 的方法将传统流式细胞仪的功率和速度与显微镜的分辨率相结合。因此，它允许以表型定义的方式进行高速率复杂的形态测量16,17。这种方法可以扩展到其他益生菌和致病菌，结合荧光报告基因来监测基因表达，并结合荧光标记菌株来监测异质聚集体中特定细菌物种的存在和丰度。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>14 mL culture tubes</td>
			<td>Falcon</td>
			<td>352051</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL centrifuge tube</td>
			<td>Falcon</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Agar</td>
			<td>Baeton,Dickinson and Company</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Typtic Soy Broth</td>
			<td>Baeton,Dickinson and Company</td>
			<td>211825</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma</td>
			<td>G7021-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Raffinose pentahydrate</td>
			<td>Sigma</td>
			<td>83400-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Difco Lactobacilli MRS broth</td>
			<td>Baeton,Dickinson and Company</td>
			<td>288130</td>
			<td></td>
		</tr>
		<tr>
			<td>EASY-LOCK MICROPR. 1.5 mL (Eppendorf)</td>
			<td>FL medical</td>
			<td>23053</td>
			<td></td>
		</tr>
		<tr>
			<td>IDEAS Software</td>
			<td>Amnis/EMD Millipore</td>
			<td>N/A</td>
			<td>&#160;Details available at: https://www.merckmillipore.com/INTL/en/20150212_144049?ReferrerURL=https%3A%2F%2Fwww.google.com%2F&#38;bd=1</td>
		</tr>
		<tr>
			<td>ImageStream X Mark II</td>
			<td>Amnis/EMD Millipore</td>
			<td>N/A</td>
			<td>&#160;Details available at: https://www.merckmillipore.com/INTL/en/20150121_205948?ReferrerURL=https%3A%2F%2Fwww.google.com%2F</td>
		</tr>
		<tr>
			<td>MOPS, 3-(N-morpholino)propanesulfonic acid</td>
			<td>Fisher bioreagents</td>
			<td>BP308-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic</td>
			<td>Fisher Scientific, 174.18 g/mol</td>
			<td>BP363-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma, 136.09 g/mol</td>
			<td>P0662-500G</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging flow cytometry to study microbial autoaggregation

有益菌和益生菌在其宿主中发挥着至关重要的作用，提供各种健康益处，包括对传染病的免疫力。乳酸杆菌科由革兰氏阳性菌组成，具有已证实的益生菌特性。本研究以乳酸杆菌科物种为模型，证明了单细胞高通量分析在研究细胞聚集方面的有效性。重点是分析这些有益物种对饮食中简单碳水化合物的反应。
该研究展示了成像流式细胞术 （IFC） 如何克服益生菌在存在和不存在碳水化合物的情况下组装的根本差异。IFC 将传统流式细胞术的功率和速度与显微镜的空间分辨率相结合，能够在有益细菌菌株和条件库中以表型定义的方式进行高速率复杂形态测量。该协议提供了对乳酸杆菌科物种自动聚集的见解，并揭示了它们对膳食碳水化合物的反应，有助于理解这些益生菌有益作用背后的机制。

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.

结果表明，该方法可以很容易地测量乳酸菌中膳食糖分自聚集的差异。通过将个体与聚集体分离，该方法允许计算聚集事件的群体占所有事件的百分比，以响应饮食中的可发酵或不可发酵糖。此外，还可以测量处理之间总人口的平均大小是否存在差异。
图 3 中的代表性图像展示了每种 LAB 细菌的门控策略。在 LGG 和副干酪乳杆菌中，检测到单细胞?...

Read this Scientific Article Protocol about Author Spotlight: Advancing Research in Microbial Auto Aggregation Using Imaging Flow Cytometry at JoVE.com

该协议描述了一种使用成像流式细胞术测量微生物自聚集的定量方法。

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

本研究以乳酸菌属为模型，证明了成像流式细胞术分析在研究微生物自聚集方面的有效性。重点是分析乳酸菌物种的自聚集对饮食中简单碳水化合物的反应。测量自聚集最常用的方法是光谱光度法（测量微生物悬浮液、浊度或光密度）和传统显微镜技术（如明场、相衬和荧光显微镜）。在谈到高通量成像时，一个主要的实验挑战是单粒子分割。在此协议中，它是单个聚合。成像流式细胞术巧妙地解决了这一挑战，并为研究人员提供了研究自聚集的强大工具。

imaging-flow-cytometry-to-study-microbial-autoaggregation

Research

JoVE Journal

Biology

Imaging Flow Cytometry to Study Microbial Autoaggregation

498 次观看.  Ben-Gurion University of the Negev. 本研究以乳酸菌属为模型，证明了成像流式细胞术分析在研究微生物自聚集方面的有效性。重点是分析乳酸菌物种的自聚集对饮食中简单碳水化合物的反应。测量自聚集最常用的方法是光谱光度法（测量微生物悬浮液、浊度或光密度）和传统显微镜技术（如明场、相衬和荧光显微镜）。在谈到高通量成像时，一个主要的实验挑战是单粒子分割。在此协议中，它是单个聚?...

视频: 作者聚焦：使用成像流式细胞术推进微生物自聚集的研究

Beneficial and probiotic bacteria play essential roles in their hosts, providing various health benefits, including immunity to infectious diseases. The Lactobacillaceae family consists of Gram-positive bacteria with confirmed probiotic properties. This study utilizes Lactobacillaceae species as a model to demonstrate the effectiveness of single-cell high throughput analysis in studying cellular aggregation. The focus is on analyzing the response of these beneficial species to simple carbohydrates from the diet.
The study showcases how Imaging Flow Cytometry (IFC) can overcome the fundamental differences in the assembly of probiotic bacteria in the presence and absence of carbohydrates. IFC combines the power and speed of conventional flow cytometry with the spatial resolution of microscopy, enabling high-rate complex morphometric measurements in a phenotypically defined manner across a library of beneficial bacterial strains and conditions. This protocol provides insights into the autoaggregation of Lactobacillaceae species and sheds light on their response to dietary carbohydrates, contributing to understanding the mechanisms behind the beneficial effects of these probiotic bacteria.

This protocol describes a quantitative approach to measure microbial autoaggregation using imaging flow cytometry.

科研

生物学

Lactobacillaceae Sample Preparation for Flow Cytometric Analysis

Start by inoculating five milliliters of lactobacilli MRS broth with a single colony of the lactobacilli strain, incubated at 37 degrees Celsius under static conditions overnight. Dilute the cell culture in a one-to-100 ratio in three milliliters of 50%supplemented tryptic soy broth, vortex the culture, and incubate it. Then invert the tube to mix the cells in the medium before transferring 200 to 300 microliters of the sample into a 1.5 milliliter micro centrifuge tube.

乳酸杆菌科 Lactobacillaceae 流式细胞术分析的样品制备

Flow Cytometric Data Acquisition of Microbial Aggregates

To begin the flow cytometric analysis, first set the 785-nanometer laser to a power of five milliwatts for dark field measurements. Use a 60X lens with a numerical aperture of 0.9. Then select the high-sensitivity setting and set the speed to low.To ensure calibration bead stability, press the Play button in the fluidics box and examine the bead images, which should appear sharp and clear. Generate a new scatterplot in the workspace box by selecting New Scatterplot. Then select the area in the Brightfield channel that corresponds to the SSC channel intensity and exclude the calibration beads that are running concurrently within the sample.Next, press the Load button and position a tube containing the Lactobacilli sample in the holder. In the Acquisition Settings box, enter the sample name and specify the location for data storage. Set the number of events to be collected, typically within the range of 10, 000 to 20, 000 events.Now, begin the acquisition process by clicking the Record button located in the Acquisition box. The process will halt after reaching the specified volume of events. Press the Return button to unload the tube and remove it from the holder as prompted in the popup window.After repeating the process for all samples, save the template to maintain data acquisition uniformity. For data analysis, to load the raw file into the analysis software, click on File and open the rif file. In the opened window, select Use Acquisition Analysis and then click OK.Next, to create a histogram of the gradient root mean square, click the New Histogram button and choose the population from the acquisition to analyze.Under the X Axis feature, select Gradient RMS of the Brightfield channel. Next, place a gate to exclude non-focused events by clicking the Create Line Region button in the analysis area. Create a scatterplot of the area versus the aspect ratio by clicking the New Scatterplot button in the analysis area.Select the focused population in the New Scatterplot window. Then select the area of the Brightfield channel for the X-Axis feature. Choose the aspect ratio for the Y-Axis feature.Draw a gate on the scatterplot using the Create Rectangle button based on the area value for the aggregate events population. Similarly, draw another gate for the singles and small aggregates population. Save your data analysis as a template by clicking the File tab, choosing Save As Template ast, and then open the next sample file under the same template to create the data analysis file.To measure the aggregate size distribution, start by plotting a histogram of the area of the aggregation events population. Save the data analysis by clicking on the file and selecting the Save As Template option. After saving, open the next sample file under the same template for the data analysis file.Single cells, small aggregates, large aggregates, and chains were observed in LGG and L.paracasei but it was not possible to distinguish between chains and larger aggregates. Significant variations in the aggregation characteristics of different LAB strains were observed in response to fermentable or non-fermentable sugars.