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TTC的传统半固体琼脂
	<ol><li>将常规半固体培养基高压灭菌后，冷却至50°C，将5mL无菌1%TTC溶液加入100mL培养基中，混合，并将其分配到10 mL试管中，形成3cm高的半固体培养基。</li>
	</ol></li>
</ol>2. 细菌菌株
注意：从水生环境中分离出80株菌株，并使用自动细菌鉴定仪器（见材料表）进行鉴定，包括大肠杆菌，铜绿假单胞菌，沙门氏菌属，弧菌属，肺炎克雷伯菌和嗜水气单胞菌（表1）。 金黄色葡萄球菌（见材料表）被用作阴性非运动对照;大肠杆菌、铜绿假单胞菌和鼠伤寒沙门氏菌（见材料表）用作阳性对照菌株。
<ol><li>确定用于运动分析的测试细菌菌株。</li>
	<li>包括阴性非运动对照和运动阳性对照菌株。</li>
</ol>3. TTC增强细菌运动观察
<ol><li>从琼脂平板中挑选测试细菌的单个菌落，并使用接种针穿刺将它们接种到上述两种半固体培养基中（步骤1.1.2和1.2.1）。</li>
	<li>将管在培养箱中于37°C培养24-48小时以观察结果。</li>
	<li>观察生长状态：如果只有穿刺线是红色的，则将细菌表征为非运动（-）。如果红色沿穿刺线12 向外轻微扩散，则将细菌表征为运动 （+）。</li>
</ol>4. 不同琼脂浓度对细菌运动的影响
<ol><li>如上所述，制备含有0.3%，0.5%和0.8%琼脂的半固体培养基，并通过穿刺接种它们。孵育24-48小时后观察结果。</li>
</ol>

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作者没有利益冲突需要披露。

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 ...

visualizing-bacterial-motility-based-on-a-color-reaction

Research

JoVE Journal

Biology

3.6K 次观看.  China Pharmaceutical University. 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。

视频: Visualizing Bacterial Motility Based on a Color Reaction

Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is largely due to the difficulty with which one can create a cell-compatible and steady microenvironment using conventional microfabrication techniques and materials. We address this problem via the introduction of a novel microfabrication material, agarose gel, as the base material for the microfluidic device. Agarose gel is highly malleable, and permeable to gas and nutrients necessary for cell survival, and thus an ideal material for cell-based assays. We have shown previously that agarose gel based devices have been successful in studying bacterial and neutrophil cell migration2. In this report, three parallel microfluidic channels are patterned in an agarose gel membrane of about 1mm thickness. Constant flows with media/buffer are maintained in the two side channels using a peristaltic pump. Cells are maintained in the center channel for observation. Since the nutrients and chemicals in the side channels are constantly diffusing from the side to center channel, the chemical environment of the center channel is easily controlled via the flow along the side channels. Using this device, we demonstrate that the movement of neural stem cells can be monitored optically with ease under various chemical conditions, and the experimental results show that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells. Motility of neural stem cells is an important biomarker for assessing cells aggressiveness, thus tumorigenic factor3. Deciphering the mechanism underlying NSC motility will yield insight into both disorders of neural development and into brain cancer stem cell invasion.

We demonstrate that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells(NSCs) using a novel agarose gel based microfluidic device. This technology can be readily adaptable to other mammalian cell systems where cell sources are scarce, such as human neural stem cells, and the turn around time is critical.

使用琼脂糖凝胶为基础的微流体装置的评估神经干细胞的运动

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is ...

科研

生物学

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

可视化的单分子荧光显微镜下的DNA复制

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

可视化的RNA中的本地化爪蟾卵母细胞

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

Gastrointestinal Motility Monitor (GIMM)

The Gastrointestinal Motility Monitor (GIMM; Catamount Research and Development; St. Albans, VT) is an in vitro system that monitors propulsive motility in isolated segments of guinea pig distal colon. The complete system consists of a computer, video camera, illuminated organ bath, peristaltic and heated water bath circulating pumps, and custom GIMM software to record and analyze data. Compared with traditional methods of monitoring colonic peristalsis, the GIMM system allows for continuous, quantitative evaluation of motility. The guinea pig distal colon is bathed in warmed, oxygenated Krebs solution, and fecal pellets inserted in the oral end are propelled along the segment of colon at a rate of about 2 mm/sec. Movies of the fecal pellet proceeding along the segment are captured, and the GIMM software can be used track the progress of the fecal pellet. Rates of propulsive motility can be obtained for the entire segment or for any particular region of interest. In addition to analysis of bolus-induced motility patterns, spatiotemporal maps can be constructed from captured video segments to assess spontaneous motor activity patterns. Applications of this system include pharmacological evaluation of the effects of receptor agonists and antagonists on propulsive motility, as well as assessment of changes that result from pathophysiological conditions, such as inflammation or stress. The guinea pig distal colon propulsive motility assay, using the GIMM system, is straightforward and simple to learn, and it provides a reliable and reproducible method of assessing propulsive motility.

Gastrointestinal Motility Monitor GIMM

Evaluation of colonic motility in the guinea pig distal colon with the Gastrointestinal Motility Monitor (GIMM) is a straightforward and simple to learn approach to quantitatively evaluate propulsive motility in the gastrointestinal tract.

肠胃蠕动监视器（GIMM）

The Gastrointestinal Motility Monitor (GIMM; Catamount Research and Development; St. Albans, VT) is an in vitro system that monitors propulsive motility ...

Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar and sometimes inclusion of specific nutrients in the growth medium. The preparation of such explicit media and surface growth conditions serves to provide the favorable conditions that allow not just bacterial growth but coordinated motility of bacteria over these surfaces within thin liquid films. Reproducibility of swarm plate and other surface motility plate assays can be a major challenge. Especially for more &#8220;temperate swarmers&#8221; that exhibit motility only within agar ranges of 0.4%-0.8% (wt/vol), minor changes in protocol or laboratory environment can greatly influence swarm assay results. &#8220;Wettability&#8221;, or water content at the liquid-solid-air interface of these plate assays, is often a key variable to be controlled. An additional challenge in assessing swarming is how to quantify observed differences between any two (or more) experiments. Here we detail a versatile two-phase protocol to prepare and image swarm assays. We include guidelines to circumvent the challenges commonly associated with swarm assay media preparation and quantification of data from these assays. We specifically demonstrate our method using bacteria that express fluorescent or bioluminescent genetic reporters like green fluorescent protein (GFP), luciferase (lux operon), or cellular stains to enable time-lapse optical imaging. We further demonstrate the ability of our method to track competing swarming species in the same experiment.

Swarming motility is influenced by physical and environmental factors. We describe a two-phase protocol and guidelines to circumvent the challenges commonly associated with swarm assay preparation and data collection. A macroscopic imaging technique is employed to obtain detailed information on swarm behavior that is not provided by current analysis techniques.

准备，成像和定量的细菌表面运动性测定

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar ...

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic levels. Tight control over ER calcium is imperative for protein folding, modification and trafficking. Perturbations to ER calcium can result in the activation of the unfolded protein response, a three-prong ER stress response mechanism, and contribute to pathogenesis in a variety of diseases. The ability to monitor ER calcium alterations during disease onset and progression is important in principle, yet challenging in practice. Currently available methods for monitoring ER calcium, such as calcium-dependent fluorescent dyes and proteins, have provided insight into ER calcium dynamics in cells, however these tools are not well suited for in vivo studies. Our lab has demonstrated that a modification to the carboxy-terminus of Gaussia luciferase confers secretion of the reporter in response to ER calcium depletion. The methods for using a luciferase based, secreted ER calcium monitoring protein (SERCaMP) for in vitro and in vivo applications are described herein. This video highlights hepatic injections, pharmacological manipulation of GLuc-SERCaMP, blood collection and processing, and assay parameters for longitudinal monitoring of ER calcium.

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

Endoplasmic reticulum calcium homeostasis is disrupted in diverse pathologies. A secreted ER calcium monitoring protein (SERCaMP) reporter can be used to detect disruptions in the ER calcium store. This protocol describes the use of a Gaussia luciferase SERCaMP to examine ER calcium homeostasis in vitro and in vivo.

监测内质网钙稳态使用<em&gt; Gaussia</em&gt;荧光素酶SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

测量外产酸和分析，以确定糖酵解率

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

SA-&#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell culture, termed senolytics, can reduce the senescent cell burden in vivo and extend healthspan. Multiple classes of senolytics have been identified to date including HSP90 inhibitors, Bcl-2 family inhibitors, piperlongumine, a FOXO4 inhibitory peptide and the combination of Dasatinib/Quercetin. Detection of SA-&#946;-Gal at an increased lysosomal pH is one of the best characterized markers for the detection of senescent cells. Live cell measurements of senescence-associated &#946;-galactosidase (SA-&#946;-Gal) activity using the fluorescent substrate C12FDG in combination with the determination of the total cell number using a DNA intercalating Hoechst dye opens the possibility to screen for senotherapeutic drugs that either reduce overall SA-&#946;-Gal activity by killing of senescent cells (senolytics) or by suppressing SA-&#946;-Gal and other phenotypes of senescent cells (senomorphics). Use of a high content fluorescent image acquisition and analysis platform allows for the rapid, high throughput screening of drug libraries for effects on SA-&#946;-Gal, cell morphology and cell number.

SA-&amp;#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cellular senescence is the key factor in the development of chronic age-related pathologies. Identification of therapeutics that target senescent cells show promise for extending healthy aging. Here, we present a novel assay to screen for the identification of senotherapeutics based on measurement of senescence associated &#946;-Galactosidase activity in single cells.

SA-β-加拉基沙酶筛查检测,用于识别治疗药物

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell ...

Ex Vivo Method for Assessing the Mouse Reproductive Tract Spontaneous Motility and a MATLAB-based Uterus Motion Tracking Algorithm for Data Analysis

Dysmenorrhea, or painful cramping, is the most common symptom associated with menses in females and its severity can hinder women&#39;s everyday lives. Here, we present an easy and inexpensive method that would be instrumental for testing new drugs decreasing uterine contractility. This method utilizes the unique ability of the entire mouse reproductive tract to exhibit spontaneous motility when maintained ex vivo in a Petri dish containing oxygenated Krebs buffer. This spontaneous motility resembles the wave-like myometrial activity of the human uterus, referred to as endometrial waves. To demonstrate the effectiveness of the method, we employed a well-known uterine relaxant drug, epinephrine. We demonstrate that the spontaneous motility of the entire mouse reproductive tract can be quickly and reversibly inhibited by 1 &#181;M epinephrine in this Petri dish model. Documenting the changes of uterine motility can be easily done using an ordinary smart phone or a sophisticated digital camera. We developed a MATLAB-based algorithm allowing motion tracking to quantify spontaneous uterine motility changes by measuring the rate of uterine horn movements. A major advantage of this ex vivo approach is that the reproductive tract remains intact throughout the entire experiment, preserving all intrinsic intrauterine cellular interactions. The major limitation of this approach is that up to 10-20% of uteri may exhibit no spontaneous motility. Thus far, this is the first quantitative ex vivo method for assessing spontaneous uterine motility in a Petri dish model.

Uterine contractions are important for the well-being of females. However, pathologically increased contractility may result in dysmenorrhea, especially in younger females. Here, we describe a simple ex vivo preparation allowing quick assessment of the efficacy of smooth muscle relaxants that may be used for treating dysmenorrhea.

用于评估小鼠生殖道自发性的方法和用于数据分析的基于 MATLAB 的 Uterus 运动跟踪算法

Dysmenorrhea, or painful cramping, is the most common symptom associated with menses in females and its severity can hinder women&#39;s everyday lives. ...

A Capsule-Based Model for Immature Hard Tick Stages Infestation on Laboratory Mice

Ticks are obligatory blood feeding parasites at all stages of development (except eggs) and are recognized as vectors of various pathogens. The use of mouse models in tick research is critical for understanding their biology and tick-host-pathogen interactions. Here we demonstrate a non-laborious technique for the feeding of immature stages of hard ticks on laboratory mice. The benefit of the method is its simplicity, short duration, and the ability to monitor or collect ticks at different time points of an experiment. In addition, the technique allows attachment of two individual capsules on the same mouse, which is beneficial for a variety of experiments where two different groups of ticks are required to feed on the same animal. The non-irritating and flexible capsule is made from easily accessible materials and minimizes the discomfort of the experimental animals. Furthermore, euthanasia is not necessary, mice recover completely after the experiment and are available for re-use.

In this study, a feeding system for nymphal and larval stages of hard tick was developed using a capsule attached to laboratory mouse. The feeding capsule is made from flexible materials and remains firmly attached to the mouse for at least one week and allows comfortable monitoring of tick feeding.

基于胶囊的模型，用于在实验鼠身上的不成熟硬滴答阶段侵扰

Ticks are obligatory blood feeding parasites at all stages of development (except eggs) and are recognized as vectors of various pathogens. The use of ...