这项研究得到了韩国食品研究所（KFRI）的主要研究计划（E0210702-03）的支持，该计划由科学和信息通信技术部资助。

1.细菌接种物的制备
<ol><li>取出储存在-80°C的甘油储备瓶。</li>
	<li>使用无菌移液器吸头从小瓶中取出细菌菌株（2-10μL）。 注： 不动杆菌 菌株， 布维 曲霉（13-1-1）， 朱氏 曲霉（13-1-2）， 皮蒂 曲霉（13-2-5）， 鲍曼 曲霉（13-2-9）， 放射性曲霉 （20-1）和 乌辛曲 霉（24-1）用于本协议。</li>
	<li>用细菌接种市售的血琼脂平板（补充有5%羊血的胰蛋白酶大豆琼脂，见 材料表）。 注意：也可以使用其他培养基，例如营养琼脂或MacConkey琼脂。</li>
	<li>使用间歇性燃烧的无菌环在琼脂表面划线以使其变薄。</li>
	<li>将琼脂平板倒置在培养箱中，并在30°C下孵育20-24小时。</li>
	<li>用无菌针头挑选细菌培养物的单个菌落，并用培养物接种5mL无菌脑心脏输注液（BHI，参见 材料表）。</li>
	<li>将接种的肉汤在30°C的振荡培养箱中以约150rpm孵育20-24小时。</li>
	<li>将 1 mL 过夜培养物转移到无菌微量离心管中，并在室温 （RT） 下以 6,000 × g 离心过夜培养物 10 分钟。</li>
	<li>用移液管除去上清液。</li>
	<li>使用涡旋，将沉淀重悬于 1 mL 无菌磷酸盐缓冲盐水 （PBS） 中。</li>
	<li>在室温下以6,000× g 离心10分钟，除去上清液。</li>
	<li>在600nm处使用涡旋至约0.1的光密度，将沉淀重悬于无菌的10倍稀释BHI中。 注：约 0.1 的光密度相当于约 107 CFU/mL。</li>
</ol>2. 使用结晶紫进行生物膜定量
<ol><li>向无菌聚苯乙烯 96 孔板的每个孔中加入 200 μL 接种物（参见 材料表），并向最外层的每个孔中加入 200 μL 去离子水以防止内孔变干15.</li>
	<li>向同一板的每个孔中加入 200 μL 10 倍稀释的 BHI 作为阴性对照。</li>
	<li>将板在25°C孵育24小时。</li>
	<li>用移液管除去上清液。 注意：多通道移液器通常更方便用于多个样品。</li>
	<li>小心地向每个孔中加入300μL PBS，并用移液管除去上清液。 注意：多通道移液器通常更方便用于多个样品。</li>
	<li>再重复步骤 2.5 两次。</li>
	<li>向每个孔中加入 200 μL 结晶紫溶液 （1%）（参见 材料表），并在室温下孵育 30 分钟。</li>
	<li>重复步骤 2.4-2.6。</li>
	<li>向每个孔中加入 200 μL 无水乙醇，并在室温下孵育 15 分钟，通过上下移液数次彻底混合。</li>
	<li>将 100 μL 洗脱液从每个孔转移到新的 96 孔板中。</li>
	<li>使用酶标仪测量595nm处的吸光度（参见 材料表）。</li>
	<li>当吸光度超过2.0时，将样品在无水乙醇中适当稀释至2.0以下的吸光度，并按步骤2.11进行测量。然后，通过将观察值乘以稀释因子来计算样品的吸光度（最终值）。</li>
	<li>通过从同一孔板上测试样品的每个孔的值中减去阴性对照的平均值来计算样品的真实吸光度。</li>
</ol>3. 生物膜活力计数
<ol><li>向无菌聚苯乙烯 12 孔板15 的每个孔中加入 1 mL 接种物（步骤 1）。将板在25°C孵育24小时。用移液管除去上清液。</li>
	<li>小心地向每个孔中加入 1.5 mL 无菌 PBS，并用移液管除去上清液。向每个孔中加入 1 mL 无菌 PBS。</li>
	<li>使用安装的细胞刮刀刮掉孔的底部和壁面。</li>
	<li>将收获的细胞悬液转移到含有 9 mL 盐水 （0.85% NaCl） 和 10-20 个玻璃珠的无菌塑料管 （10-1） 中（参见 材料表）。 注意：要收集残留在孔底面上的任何残留生物膜碎片，可以将其重新悬浮并使用10-1 管中的生理盐水通过移液管转移。</li>
	<li>以最大速度涡旋10-1 管60秒。</li>
	<li>通过将 1 mL 转移到下一个含有 9 mL 盐水 （0.85 % NaCl） 至 10-7 的无菌管 （10-2） 中，进行 10 倍的连续稀释。</li>
	<li>将每种稀释液中的100μL涂布在不动杆菌琼脂上（参见 材料表），并将琼脂平板在30°C下孵育24-42小时。</li>
	<li>手动计算每个板上典型红色菌落的数量，范围在 10 到 250 之间。</li>
	<li>使用以下公式计算每个孔的生物膜中的活细胞数： 活细胞 = 菌落数 × 稀释因子 × 10</li>
</ol>4. 使用共聚焦激光扫描显微镜（CLSM）进行生物膜可视化
<ol><li>向用于显微镜分析的无菌 96 孔板的每个孔中加入 200 μL 接种物。</li>
	<li>将板在25°C孵育24小时。</li>
	<li>用移液管除去上清液。</li>
	<li>小心地向每个孔中加入 300 μL 过滤灭菌的去离子水，并用移液管除去上清液。</li>
	<li>重复步骤 4.4。</li>
	<li>制备在过滤灭菌的去离子水中稀释至最终浓度分别为10μM和60μM的SYTO 9和碘化丙啶（见 材料表）的混合物。</li>
	<li>将混合物以 200 μL 加入每个孔中，并在室温下在黑暗中孵育板 20-30 分钟。</li>
	<li>重复步骤 4.3-4.4</li>
	<li>使用激发波长分别为 488 nm 和 561 nm，发射波长范围为 499-535 nm 和 568-712 nm 的 CLSM 可视化底表面附着的生物膜。</li>
</ol>

不动杆菌生物膜形成的评估和生物膜的表征

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作者没有什么可透露的。

使用所描述的方案，测量、可视化不同程度的不动杆菌分离株的生物膜形成，并估计生物膜中的活细胞（图1、图2和图3）。
在该方案中，使用两种不同的温度，30°C用于生长，25°C用于不动杆菌的生物膜形成。使用30°C是因为许多研究使用超过30°C的不动杆菌的最佳生长，这对于获得足够的...

已知不动杆菌会引起医院感染，其人类感染，尤其是在医疗机构中，越来越多的报道1.它广泛存在于医院、医疗机构和食品相关环境中 2,3,4。它可以在包括医院表面（如床栏、床头柜、呼吸机表面和水槽）在内的环境中长期存活4.这种在环境表面的持久性可能是导致不动杆菌4 院内感染的重要因素之一。
生物膜是微生物生命的一种形式，是由活的微生物细胞和来自细胞的细胞外聚合物物质（EPS）组成的微生物基质5。微生物细胞嵌入基质中，通常对环境压力（如热、盐、干燥、抗生素、消毒剂和剪切力）具有很强的抵抗力6,7。
不动杆菌可以在表面上形成生物膜，这表明它可能有助于延长在环境表面（包括医院表面）的存活时间，并增强对抗生素治疗的耐药性8,9。此外，不动杆菌的生物膜形成可能与人类临床结果高度相关8。因此，不动杆菌菌株的生物膜形成性可能是预测环境生存和人类感染的指标之一8,10。
表面附着的生物膜可以被量化和可视化，以评估生物膜的形成性。为了量化表面附着的生物膜，通常用生物膜染色染料（如结晶紫）对生物膜进行染色，并将染料在溶液中洗脱并测量光密度11。生物膜的可视化是评估生物膜形成性的另一种好方法11。与其他技术（如 SEM12,13）相比，使用荧光染料的特异性共聚焦激光扫描显微镜 （CLSM） 可视化方法可能更有助于表征生物膜形态。
可以对生物膜中的活细胞进行计数，以估计生物膜中的活细胞数量11。将嵌入生物膜中的活细胞分离、稀释、铺在琼脂平板上、孵育和计数。由于较高的细胞数可能满足感染剂量，因此可以提供有关生物膜的更详细的信息，例如与细胞数相关的感染电位13,14。
本文介绍了 （1） 量化表面附着的生物膜，（2） 对生物膜中的活细胞进行计数，以及 （3） 使用 Acinetobacter 的 CLSM 可视化生物膜的分步方案。所提出的方案描述了评估 不动杆菌 分离株的生物膜形成性和表征其生物膜的方法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96-well cell culture plate</td>
			<td>SPL</td>
			<td>30096</td>
			<td>Polystyrene 96-well plate</td>
		</tr>
		<tr>
			<td>BHI (Brain Heart Infusion) broth</td>
			<td>Merck KGaA</td>
			<td>1.10493.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Blood Agar Base Plate</td>
			<td>KisanBio</td>
			<td>MB-B1005-P50</td>
			<td>Growth media for Acinetobacter</td>
		</tr>
		<tr>
			<td>CHROMagar Acinetobacter</td>
			<td>CHROMagar</td>
			<td>AC092</td>
			<td>Selective plate for Acinetobacter</td>
		</tr>
		<tr>
			<td>Crystal violet solution</td>
			<td>Sigma-Aldrich</td>
			<td>V5265</td>
			<td></td>
		</tr>
		<tr>
			<td>Filmtracer LIVE/DEAD biofilm viability kit</td>
			<td>Invitrogen</td>
			<td>L10316</td>
			<td>SYTO9 and propidium iodide</td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>Tecan</td>
			<td>Infinite M200 PRO NanoQuant</td>
			<td>Biofilm measurement</td>
		</tr>
		<tr>
			<td>RBC Glass Plating Beads</td>
			<td>RBC</td>
			<td>RG001</td>
			<td>Glass beads</td>
		</tr>
		<tr>
			<td>&#956;-Plate 96 Well Black</td>
			<td>ibidi</td>
			<td>89621</td>
			<td>Microplate intended for CLSM</td>
		</tr>
	</tbody>
</table>

quantification, viability assessment, and visualization strategies for acinetobacter biofilms

不动杆菌 会引起院内感染，其生物膜的形成有助于在医院环境等干燥表面上存活。因此，生物膜定量和可视化是评估 不动杆菌菌 株引起医院感染潜力的重要方法。在微孔板表面形成的生物膜可以根据体积和细胞数量进行量化。生物膜体积可以通过使用结晶紫染色、洗涤、使用乙醇脱色，然后使用酶标仪测量溶解染料来定量。为了量化嵌入生物膜中的细胞数量，使用细胞刮刀将生物膜刮掉，在盐水中收获，在玻璃珠存在下剧烈搅拌，并涂抹在不动杆菌琼脂上。然后，将板在30°C孵育24-42小时。孵育后，计数红色菌落以估计生物膜中的细胞数。这种可行的计数方法也可用于计数混合物种生物膜中的 不动杆菌 细胞。 不动杆菌 生物膜可以使用荧光染料进行可视化。使用设计用于显微分析的市售微孔板来形成生物膜。然后，用SYTO9和碘化丙啶染料染色底表面附着的生物膜，洗涤，然后用共聚焦激光扫描显微镜观察。

Acinetobacter is known to cause nosocomial infections, and its human infection, especially in healthcare facilities, is increasingly reported1. It is widespread in hospitals, healthcare facilities, and food-associated environments2,3,4. It can survive for a long period in environments including hospital surfaces such as bed rails, bedside tables, the surface of ventilators, and sinks4. Such persistence on environmental surfaces may be one of the significant factors contributing to the nosocomial infections of Acinetobacter4.
Biofilm is a form of microbial life and is a microbial matrix composed of live microbial cells and extracellular polymeric substances (EPS) from the cells5. The microbial cells are embedded in the matrix and are often highly resistant to environmental stresses such as heat, salts, dryness, antibiotics, disinfectants, and shear forces6,7.
Acinetobacter can form biofilms on surfaces, suggesting that it may contribute to extended survival on environmental surfaces, including hospital surfaces, and enhanced resistance to antibiotic treatment8,9. In addition, the biofilm formation of Acinetobacter could be highly associated with human clinical outcomes8. Therefore, the biofilm formability of Acinetobacter strains could be one of the indicators in predicting environmental survival and human infections8,10.
The surface-attached biofilms can be quantified and visualized to assess biofilm formability. To quantify surface-attached biofilms, the biofilms are normally stained by biofilm-staining dyes such as crystal violet, and the dyes are eluted in solution and measured for optical density11. Visualization of biofilms is another good approach to assess biofilm formability11. Confocal Laser Scanning Microscopy (CLSM) visualization method employing specificity using fluorescent dyes could be more useful to characterize biofilm morphology compared to other techniques such as SEM12,13.
The viable cells in biofilms can be counted to estimate the number of viable cells in biofilms11. The viable cells embedded in biofilms are detached, diluted, spread on agar plates, incubated, and enumerated. Because the higher number of cells is likely to meet the infectious dose, it can provide more detailed information on biofilms, such as infection potentials associated with the number of cells13,14.
This article presents step-by-step protocols to (1) quantify surface-attached biofilms, (2) count viable cells in the biofilms, and (3) visualize the biofilms using CLSM of Acinetobacter. The presented protocols describe the methods to assess the biofilm formability of Acinetobacter isolates and characterize their biofilms.

作者聚焦：不动杆菌生物膜定量、评估和可视化的综合方案 

不动杆菌生物膜的定量、活力评估和可视化策略

Biofilms are composed of live microbial cells and are difficult to remove from the surfaces. Acinetobacter strains commonly found in our environment, causing nosocomial infections, are also known to form biofilms on surfaces. So, we aim to develop methods to both quantify and visualize Acinetobacter biofilms.Our protocol provides a simple and easy method to quantify and visualize the biofilms of Acinetobacter. The viable number of cells inside the biofilms is quantified by using the viable count method. Our research findings reveal that the naturally-existing Acinetobacter vector strains have quite varying potentials for biofilm formation.Also, there will exist distinct morphological differences within the various biofilms. This paved the way for research focusing on the causes of such a large variation among Acinetobacter strains.

按照该方案，最初从厨房表面分离的 不动杆菌 分离株的生物膜在聚苯乙烯96孔板上形成，用结晶紫染色，并将染料溶解在乙醇中并测量生物膜质量（图1）。生物膜的数量因菌株而异，范围从OD 0.04到1.69（图1）。根据 Stepanović 等人 16 建立的标准，除 A. bouvetii 外，所有分离株都形成了生物膜。 A.放射性抗性剂 形成?...

Watch this Scientific Journal Video about A Comprehensive Protocol for Acinetobacter Biofilm Quantification, Assessment, and Visualization at JoVE.com

该方案描述了接种物的制备，使用结晶紫染料在微量滴定板上的生物膜定量，生物膜中的活菌计数以及 不动杆菌生物膜的可视化。

Acinetobacter causes nosocomial infections and its biofilm formation can contribute to the survival on dry surfaces such as hospital environments. Thus, ...

生物膜由活的微生物细胞组成，很难从表面去除。不动杆菌菌株常见于我们的环境中，引起医院感染，也已知会在表面形成生物膜。因此，我们的目标是开发量化和可视化不动杆菌生物膜的方法。我们的方案提供了一种简单易行的方法来量化和可视化不动杆菌的生物膜。通过使用活菌计数方法量化生物膜内细胞的活菌数。我们的研究结果表明，天然存在的不动杆菌载体菌株具有相当不同的生物膜形成潜力。此外，各种生物膜内将存在明显的形态差异。这为研究不动杆菌菌株之间如此大差异的原因铺平了道路。

quantification-viability-assessment-visualization-strategies-for

Research

JoVE Journal

Biology

Quantification, Viability Assessment, and Visualization Strategies for Acinetobacter Biofilms

1.4K 次观看.  Korea Food Research Institute. 生物膜由活的微生物细胞组成，很难从表面去除。不动杆菌菌株常见于我们的环境中，引起医院感染，也已知会在表面形成生物膜。因此，我们的目标是开发量化和可视化不动杆菌生物膜的方法。我们的方案提供了一种简单易行的方法来量化和可视化不动杆菌的生物膜。通过使用活菌计数方法量化生物膜内细胞的活菌数。我们的研究结果表明，天然存在的不动杆菌载体?...

视频: 作者聚焦：不动杆菌生物膜定量、评估和可视化的综合方案 

Acinetobacter causes nosocomial infections and its biofilm formation can contribute to the survival on dry surfaces such as hospital environments. Thus, biofilm quantification and visualization are important methods to assess the potential of Acinetobacter strains to cause nosocomial infections. The biofilms forming on the surface of the microplate can be quantified in terms of volume and cell numbers. Biofilm volumes can be quantified by staining using crystal violet, washing, destaining using ethanol, then measuring the solubilized dye using a microplate reader. To quantify the number of cells embedded in the biofilms, the biofilms are scrapped off using cell scrapers, harvested in the saline, vigorously agitated in the presence of glass beads, and spread on Acinetobacter agar. Then, the plates are incubated at 30 &#176;C for 24-42 h. After incubation, the red colonies are enumerated to estimate the number of cells in biofilms. This viable count method can also be useful for counting Acinetobacter cells in mixed-species biofilms. Acinetobacter biofilms can be visualized using fluorescent dyes. A commercially available microplate designed for microscopic analysis is employed to form biofilms. Then, the bottom-surface attached biofilms are stained with SYTO9 and propidium iodide dyes, washed, then visualized with confocal laser scanning microscopy.

This protocol describes the preparation of the inoculum, the biofilm quantification on microtiter plates using crystal violet dye, the viable count in biofilms, and the visualization of biofilms of Acinetobacter.

科研

生物学

Preparation of Bacterial Inoculum for Acinetobacter Biofilm Formation

Preparation of Bacterial Inoculum for Acinetobacter Biofilm Formation  

To begin the process of bacterial inoculum preparation, collect 2 to 10 microliters of the bacterial strain from the vial, using a sterile pipette tip, and inoculate a commercially-available blood agar plate with it. Using a sterile loop, streak the agar surface, thinning it out with intermittent flaming. Incubate the prepared agar plate in an upside-down position at 30 degrees Celsius for about 20 to 24 hours.Next, select a single colony of the bacterial culture with a sterile needle and inoculate five milliliters of sterile BHI broth with this culture. Incubate the inoculated broth in a shaking incubator at 30 degrees Celsius at a speed of 150 rotations per minute for about 20 to 24 hours. Then transfer one milliliter of the culture to a sterile micro-centrifuge tube and centrifuge at 6, 000 g and room temperature for 10 minutes.Following centrifugation, discard the supernatant. Add one milliliter of sterile phosphate-buffered saline, or PBS, to the pellet, and re-suspend it using a vortex. Centrifuge the re-suspended pellet at 6, 000 g for 10 minutes at room temperature and discard the supernatant.This time re-suspend the resulting pellet in sterile, tenfold-diluted BHI broth using vortex. Ensure to achieve an optical density of around 0.1 at 600 nanometers.

用于 不动杆菌 生物膜形成的细菌接种物的制备

Acinetobacter Biofilm Quantification Using Crystal Violet

Acinetobacter Biofilm Quantification Using Crystal Violet  

To begin, add 200 microliters of the bacterial inoculum to each well of a sterile polystyrene 96-well plate. Then add 200 microliters of deionized water into the outermost wells to keep the inner wells from drying out. Incubate the 96-well plate at 25 degrees Celsius for 24 hours.Following incubation, discard the supernatant in each well using a pipette. Carefully add 300 microliters of PBS to each well and remove the PBS again by using the pipette. Add 200 microliters of 1%crystal violet solution to each well before incubating the plate at room temperature for 30 minutes.Then after dispensing 200 microliters of absolute ethanol in each well, leave the plate for 15 minutes at room temperature. Ensure thorough mixing by pipetting the contents in each well up and down repeatedly. Using a microplate reader, measure the absorbance of the wells at 595 nanometers to determine the biofilm mass.The number of biofilms varied greatly depending on the strains, ranging from an optical density or OD value of 0.04 to 1.69 All of the Acetobacter isolates, except for A.bouvetii, formed biofilms, while A.radioresistens formed a weak biofilm. A.junii and A.baumannii formed moderate biofilms, and A.pittii and A.ursingii formed strong biofilms.

不动杆菌 使用Crystal Violet进行生物膜定量

Assessing Acinetobacter Biofilm Viability Count

Assessing Acinetobacter Biofilm Viability Count  

Begin by adding one milliliter of the bacterial inoculum to each well of a sterile polystyrene 12 well plate. Incubate the plate at 25 degrees Celsius for 24 hours. Then, remove the supernatant using a pipette.Carefully add 1.5 milliliters of sterile phosphate buffered saline or PBS to each well, and again, remove the supernatant with the pipette. After adding one milliliter of sterile PBS to each well once more, use fitted cell scrapers to scrape off the bottom and wall surfaces of the wells. Transfer the harvested cell suspension to a sterile plastic tube marked as 10 to the negative one containing nine milliliters of saline and 10 to 20 glass beads.Vortex the tube for 60 seconds at maximum speed. Next, perform a tenfold serial dilution by transferring one milliliter of the cell suspension from the previous tube to another sterile tube marked 10 to the negative two containing nine milliliters of saline solution. Continue this process of serial dilution up to tube 10 to the negative seven.Spread 100 microliters from each dilution onto separate acinetobacter agar plates. Incubate the agar plates at 30 degrees Celsius for 24 to 42 hours. Then count the number of typical red colonies on each plate manually, considering those within the range between 10 and 250.All the acinetobacter isolates except for A.bouvetii had cells equivalent to seven to eight log CFU per well in their biofilms. While A.bouvetii had a much lower cell number at 4.4 Log CFU.

评估 不动杆菌 生物膜活力计数

Acinetobacter Biofilm Visualization by Confocal Laser Scanning Microscopy (CLSM)

Begin by adding 200 microliters of bacterial inoculum into each well of a sterile 96-well plate prepared for microscopic analysis. Incubate the plate at 25 degrees Celsius for 24 hours before removing the supernatant with the pipette. Next, carefully add 300 microliters of filter-sterilized deionized water into each well.Remove the supernatant using a pipette once again. In another tube, prepare a diluted mixture of SYTO 9 and propidium iodide and filter-sterilized deionized water to the respective desired final concentrations. Then add 200 microliters of the mixture into each well and incubate the plate for 20 to 30 minutes at room temperature in a dark setting.Confocal laser scanning microscopy revealed that biofilm formation significantly varied depending on the strains. A substantial amount of biofilm was found in Acinetobacter junii, Acinetobacter baumannii, and Acinetobacter ursingii with distinct biofilm morphologies.