Name: visualizing bacterial motility based on a color reaction
Uploaded: 2022-02-15T15:00:00
Description: Watch this Scientific Journal Video about Visualizing Bacterial Motility Based on a Color Reaction at JoVE.com

本研究得到了江苏省高校优先学术项目开发（PAPD）和中国药科大学教学改革研究项目（2019XJYB18）的支持。

1. 半固体培养基的制备
<ol><li>传统半固体琼脂
	<ol><li>根据细菌运动测试培养基配方，使用基本成分制备传统的半固体琼脂11。将 10 g 色糖、15 g NaCl、4 g 琼脂溶解在足够的蒸馏水中，将 pH 值调节至 7.2 ± 0.2，并将最终体积补足至 1，000 mL。</li>
		<li>将琼脂在121°C高压灭菌20分钟，并将其作为3cm高的半固体培养基分配到10mL试管中。</li>
	</ol></li>
	<li>含...

<ol>
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J., 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=Spatial+arrangement+of+several+flagellins+within+bacterial+flagella+improves+motility+in+different+environments.">Spatial arrangement of several flagellins within bacterial flagella improves motility in different environments.</a> Nature Communication. 9 (1), 5369 (2018).</li><li>Mitchell, A. J., Wimpenny, J. W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+agar+concentration+on+the+growth+and+morphology+of+submerged+colonies+of+motile+and+non-motile+bacteria.">The effects of agar concentration on the growth and morphology of submerged colonies of motile and non-motile bacteria.</a> Journal of Applied Microbiology. 83 (1), 76-84 (2010).</li><li>Xu, A., Zhang, M., Du, W., Wang, D., Ma, L. 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半固体培养基法细菌运动的检测受许多因素13，14的影响。细菌生长条件，例如氧气（琼脂表面有氧，半固体培养基在管底部无氧），pH和温度，会影响细菌鞭毛的活力，从而导致运动能力降低甚至运动力丧失15。此外，一些粘液型细菌作为其运动性会受到鬼臼结合物产生的影响。 
将TTC添加到半固体介质中有助于观...

细菌运动在细菌致病性、生物膜形成和耐药性中起关键作用1。细菌运动性与致病性密切相关，是宿主细胞早期感染期间细菌定植所必需的2。生物膜的形成与细菌运动密切相关，细菌通过运动粘附在固体培养基的表面。长期以来，人们一直认为细菌运动与生物膜形成呈正相关。由于生物膜引起的高度细菌耐药性可导致对人类健康构成威胁的持续感染3，4，5。因此，检测细菌运动非常重要。细菌运动性测试主要用于检查不同形式细菌在生活状态下的运动性，可以间接确定鞭毛的存在与否，因此对细菌的鉴定具有重要作用。
有直接和间接的方法来检测细菌运动6。由于带有鞭毛的细菌表现出运动性，因此可以通过检测是否存在鞭毛来间接检测细菌是否具有运动性。例如，可以通过电子显微镜和鞭毛染色间接检测运动性，以表明细菌是运动的。也可以通过直接方法进行检测，例如悬浮液滴和半固体穿刺方法。 
本科微生物学实验室常用的半固体穿刺法检测细菌运动性，根据细菌生长方向，将细菌接种到含有0.4-0.8%琼脂的半固体琼脂培养基中穿刺中。如果细菌沿着穿刺线生长到周围扩散，则会出现云状（刷状）生长痕迹，表明存在鞭毛，因此存在运动性。如果没有穿刺线生长痕迹，则细菌既不鞭毛也不运动。 
然而，这种方法有其缺点：细菌无色透明，鞭毛活性受活细菌的生理特性和其他因素的影响，以及琼脂的浓度和试管的小直径。而且，需氧菌只适合在琼脂表面生长，影响细菌运动的观察。因此，为了改进该实验，将2，3，5-三苯基氯化四唑（TTC）（无色）添加到培养基中，以建立比目前使用细胞内脱氢酶催化形成TTC7，8，9，10红色产物的直接穿刺方法更可靠，更直观的细菌运动性测定方法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bacto Agar</td>
			<td>Difco</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Escherichia coli</td>
			<td>ATCC</td>
			<td>ATCC25922</td>
			<td>Positive control</td>
		</tr>
		<tr>
			<td>Pseudomonas aeruginosa</td>
			<td>ATCC</td>
			<td>ATCC27853</td>
			<td>Positive control</td>
		</tr>
		<tr>
			<td>Salmonella typhimurium</td>
			<td>ATCC</td>
			<td>ATCC14028</td>
			<td>Positive control</td>
		</tr>
		<tr>
			<td>Staphylococcus aureus</td>
			<td>ATCC</td>
			<td>ATCC25923</td>
			<td>Negative nonmotile control</td>
		</tr>
		<tr>
			<td>Tryptose</td>
			<td>&#160;OXOID</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TTC</td>
			<td>Sigma</td>
			<td>298-96-4</td>
			<td></td>
		</tr>
		<tr>
			<td>VITEK 2 automated microbial identification system</td>
			<td>Bio M&#233;rieux</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

visualizing bacterial motility based on a color reaction

细菌运动性对细菌致病性、生物膜形成和耐药性至关重要。细菌运动对于许多致病物种的入侵和/或传播至关重要。因此，检测细菌运动非常重要。细菌生长条件，如氧气、pH和温度，会影响细菌的生长和细菌鞭毛的表达。这可能导致运动能力降低甚至失去运动能力，导致对细菌运动性的评估不准确。基于2，3，5-三苯基氯化四唑（TTC）与活细菌细胞内脱氢酶的显色反应，将TTC添加到传统的半固体琼脂中用于细菌运动检测。结果表明，该TTC半固体琼脂法检测细菌运动性简单，操作方便，不涉及大型且昂贵的仪器。结果还表明，在用0.3%琼脂制备的半固体培养基中观察到最高的运动性。与传统的半固体介质相比，结果更容易评估，也更准确。

比较标准菌株和分离菌株的运动检测，结果如 表1所示。由于没有鞭毛， 金黄色葡萄球菌 和 肺炎克雷伯菌 仅在传统和TTC半固体培养基上沿接种线生长。相比之下， 铜绿假单胞菌、 大肠杆菌 和 鼠伤寒沙门氏菌 在TTC半固体培养基上培养24 h后，在接种系周围各方向均显示生长。培养48小时后，这一点更加明显（图1）。尽管细菌?...

Watch this Scientific Journal Video about Visualizing Bacterial Motility Based on a Color Reaction at JoVE.com

Visualizing Bacterial Motility Based on a Color Reaction

在这里，我们提出了一种基于颜色反应检测细菌运动的方案。这种方法的主要优点是易于评估且更准确，并且不需要专门的设备。

基于颜色反应可视化细菌运动

Bacterial motility is crucial for bacterial pathogenicity, biofilm formation, and drug resistance. Bacterial motility is crucial for the invasion and/or ...

Detection of bacterial motility is important because motility is crucial for pathogenicity. The protocol described here is a reliable method for the detection of bacterial motility, because it is based on the color reaction of TTC with intracellular dehydrogenases of living bacteria. The TTC semi-solid agar method for detecting bacterial motility is simple, easy to operate and does not involve large and expensive instruments.The method can be used by clinical microbiologists or researchers to investigate pathogenic bacteria. Demonstrating the procedure will be Xiyi Zhuang, a senior experimentalist from my laboratory. To begin, prepare the traditional semi-solid medium by adding 10 grams of Tryptose, 15 grams of sodium chloride and four grams of agar in an adequate volume of distilled water.Adjust the pH to 7.2 and make up the final volume to 1, 000 milliliters. Autoclave the medium at 121 degrees Celsius for 20 minutes and dispense the medium into 10 milliliter test tubes to a height of three centimeters. To prepare the TTC medium, cool the autoclaved traditional medium to 50 degrees Celsius then add five milliliters of sterile 1%TTC solution to 100 milliliters of medium.Mix and dispense the medium into 10 milliliter test tubes to a height of three centimeters. Pick single colonies of bacteria from agar plates and inoculate them into both the semi-solid media by puncture using inoculating needles. Remember to include non-modal strains as negative controls and modal strains as positive controls.To observe the effect of agar concentration on bacterial motility, prepare semi-solid media containing different agar concentrations and inoculate them by puncture. Incubate the tubes at 37 degrees Celsius and observe the results after 24 to 48 hours of incubation. If only the puncture line is red, characterize the bacteria as non-modal.If the red color spreads outward along the puncture line, characterize the bacteria as modal. Bacterial motility was compared for various strains on traditional and TTC semi-solid media and summarized in this table. After culturing for 24 hours on TTC semi-solid medium, Staphylococcus aureus showed growth along the inoculated line.Whereas, Escherichia coli showed growth in all directions around the inoculated line. Highest motility for Escherichia coli was observed in TTC semi-solid medium prepared with 0.3%agar in which the medium turned almost entirely red. In contrast, the area of red diffusion decreased in 0.5%agar and the diffusion was prolonged with increasing agar concentration to 0.8%It is important to adjust the pH to 7.2 and cool the autoclaved medium to 50 degrees Celsius before inoculation using the needles.Bacteria should be inoculated by puncture into the semi-solid medium containing TTC. It is a direct method for detecting bacterial motility. It is also possible to detect motility indirectly by electronic microscope and

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