作者希望通过2017-231-001UCV 和2018-231-001UCV 赠款, 感谢瓦伦西亚天主教大学 Mártir 为这项工作提供财政支助。

1. 样品准备
<ol><li>将材料样品切成10毫米直径的圆盘, 直径为10毫米圆柱形冲床。 注: 脆性材料可在合适的无菌溶剂中软化1小时, 然后切成圆盘。例如, 海藻酸锌等亲水材料经过本协议测试, 并在蒸压水中湿润, 以避免试样破裂。然而, 其他疏水材料, 如聚 (3-羟基-3-hydroxyvalerate) 不需要任何一种以前的肿胀, 以便适当削减。</li>
	<li>在真空烘箱 (< 10-2乇) 中, 将样品材料盘干燥60摄氏度, 24 小时。 注: 某些材料可能需要较高的干燥温度。然而, 重要的是不要达到温度, 可以热降解的材料。</li>
	<li>用数字卡尺测量材料膜厚度。 注: 本议定书建议在比较不同材料的抗菌活性时, 使用具有恒定和相似厚度的材料薄膜。</li>
	<li>通过浸泡在乙醇 70% 10 分钟和随后的紫外线 (紫外线) 辐射每一侧1小时消毒每个标本。 注意: 紫外线辐射可以被执行放置在一个无菌的培养皿内的一个层流罩与12.0 瓦特的紫外线辐射灯的样品。 注意: 研究人员不应该暴露在紫外线照射下, 因为它是诱变。</li>
	<li>执行步骤1.1、1.2、1.3 和1.4。与控制材料盘。 注: 聚对苯二甲酸乙二醇酯 (PET) 或替代非抗菌材料可作为控制盘在扩散和接触方法。此外, 在表征纳米复合材料或处理材料时, 基材应作为控制材料使用。</li>
</ol>2. 推荐的微生物
注: 我们建议使用3种不同的微生物, 以研究对各种微生物的试验材料的抗菌能力。
<ol><li>采用纯培养3种微生物: 革兰氏阳性菌金黄色葡萄球菌、革兰阴性菌大肠杆菌、酵母白色念珠菌。 注: 其他微生物种类也可通过本议定书进行测试, 如有必要, 修改孵化条件。 注意: 根据本议定书所使用的微生物类型, 必须遵守所需的生物安全措施。</li>
	<li>使用未灭菌或蒸压材料, 在微生物操作的整个过程中或在生物安全柜 (如有必要时) 用本生燃烧器确保无菌条件。 注: 推荐的高压釜条件为121摄氏度, 在15分钟内培养培养基和121°c 在20分钟内为工作材料和生物残渣。</li>
</ol>3. 琼脂盘扩散试验 (扩散法)
注: 当抗菌化合物的液体扩散可能是先进材料的主要抗菌机理时, 扩散法可为这些材料的抗菌能力提供非常有用的信息。位于琼脂盘中心的物质盘可以形成一个透明环区 (光晕), 在 24 h 的培养后, 微生物的生长抑制 (见图 1)。
<ol><li>
扩散试验程序
	<ol>
<li>根据制造商的指示, 准备和蒸胰蛋白酶大豆琼脂 (TSA)。</li>
		<li>使用本生燃烧器或层流罩将 TSA 倒入无菌培养皿中。 注意: TSA 板必须第4-6 毫米厚。</li>
		<li>培养不同的微生物, 测试有氧 18–24 h 在培养皿与 TSA 在一个孵化器在37摄氏度。</li>
		<li>根据制造商的指示, 准备和蒸胰蛋白酶大豆汤。</li>
		<li>使用本生燃烧器或层流罩, 在无菌条件下用预灭菌的血清吸管在50毫升预灭菌的离心管中倒入。</li>
		<li>并用重悬几个殖民地从步骤3.1.3 在25毫升的在一个不育的离心机管, 使用不育棉拭子和涡旋他们1分钟, 以达到统一混合。</li>
		<li>调整吸收 (在540毫微米) 的文化与分光光度计, 以适当数量的菌落形成单位 (CFU) 每毫升: 大约 1.5 x 108 CFU/毫升为细菌和从 1 x 106到 5 x 106 CFU/毫升酵母。 注: 根据所使用的分光光度计的种类, 必须选择培养和试管量来测量吸光度。</li>
		<li>将微生物液涡旋5秒钟, 以改善微生物的分散性, 并在微生物悬浮液中简单地将无菌棉签浸泡。将多余的液体从拭子上按在含有该文化的管壁上。</li>
		<li>用不育棉拭子均匀地将微生物汤悬浮在3平面上的 TSA 板表面上, 用微生物覆盖其整个表面, 并在接种后使其干燥5分钟。 注: 要去除任何水分, TSA 板必须放在37摄氏度的倒置位置打开, 在接种前10–15分钟。</li>
		<li>用乙醇96% 浸泡在烧杯中, 然后用本生燃烧器或酒精燃烧器将一双镊子消毒。</li>
		<li>使用双不育镊子将样品盘置于 TSA 板中心的测试和控制盘上。</li>
		<li>孵化有氧的 TSA 板块在37摄氏度的倒置位置为24小时。 注: 为了避免任何污染风险, 建议将 TSA 板孵化成倒位。但是, 如果示例磁盘与 TSA 板分离, 则不要在反向位置执行此步骤。在这种情况下, 建议在层流罩中烘干板材。这种抗菌试验必须至少在 quadriplicate 中进行, 以确保重现性。</li>
	</ol>
</li>
	<li>
微生物悬浮液浓度和纯度的检测 注: 为了检查分光光度计在步进3.1.7 中测定的微生物浓度是否正确, 确保没有微生物环境污染, 需要采取以下步骤。在24小时的培养后, 各种微生物菌落的出现, 可以很容易地检测到微生物的环境污染。此外, 在十进制串行稀释的操作过程中, 必须确保使用负的控制板, 以保证对该介质的微生物污染。<ol>
<li>在无菌条件下, 用适当的蒸压小贴士, 将无菌的 3.1.4 (在步骤中制备) 成无菌离心管。其中一人将被用作消极的控制。</li>
		<li>在含有稀释的无菌离心管中, 使用在步骤3.1.9 中使用的微生物液悬浮液进行十进制串行。</li>
		<li>使用预灭菌的 Drigalski 刮刀或替代仪器, 在 TSA 板上分散100µL 的稀释。还传播 100 ul 的无微生物 (在步骤 3.2.1) 在 TSA 板作为一个负的控制板。</li>
		<li>孵化有氧 TSA 板有氧在37°c 24 小时。</li>
		<li>计算菌落数以检查 CFU/mL 是否与步骤3.1.7 中确定的相同。</li>
		<li>检查 TSA 板上没有微生物环境污染, 只有一种类型的菌落。</li>
		<li>检查无菌落的无菌控制板上没有微生物污染</li>
	</ol>
</li>
</ol>4. 材料表面抗菌活性的测定 (接触法)
 注: 当表面接触可能是一些先进材料的主要抗菌机理时, 接触方法可以提供有关这些材料的抗菌能力的非常有用的信息。在该方法中, 微生物直接放在材料表面上, 在一定的时间后可以确定其生长抑制。
<ol><li>
初始过程
	<ol>
<li>根据制造商的指示, 准备和蒸压。</li>
		<li>使用本生燃烧器或层流罩, 在无菌条件下用预灭菌的血清吸管在50毫升预灭菌的离心管中倒入。</li>
		<li>培养不同的微生物将被测试有氧一夜之间在管与在一个轨道振动筛 (140 rpm) 在37°c。</li>
		<li>在50毫升预灭菌的离心管中稀释20毫升的一夜文化, 浓度约为 106 CFU/毫升 (用分光光度计测定 540 nm)。 注: 根据所使用的分光光度计的种类, 必须选择培养和试管量来测量吸光度。</li>
		<li>在含有稀释的无菌离心管中执行此种培养的十进制串行。传播100µL 的文化在 TSA 板和孵化它有氧在37°c 24 小时。</li>
		<li>计算菌落数以确保初始细胞浓度约为 1 x 106 CFU/毫升</li>
		<li>放置4控制盘为微生物计数在 24 h 和4样品盘以后每类被测试的材料为微生物计数在 24 h 以后在分离井的一个无菌48井板材。</li>
		<li>吸管150µL 的微生物悬浮在每个圆盘表面上。</li>
	</ol>
</li>
	<li>
微生物计数在控制和被测试的材料 (在 24 h 以后)
	<ol>
<li>在步4.1.6 之后, 有氧孵化4个样品盘剩余在48井板材在37°c 为 24 h。 注意: 这24小时的潜伏期可以修改, 以研究生长抑制在较短或更长的时间。</li>
		<li>经过24小时的孵化, 吸管850µL 不育 PBS 的表面上的4样品盘, 并与150µL 的微生物悬浮液混合。</li>
		<li>收集 PBS/微生物悬浮混合物和每个磁盘从48井板和转移到10毫升预灭菌管。</li>
		<li>漩涡的 PBS/微生物悬浮混合物和每个磁盘1分钟, 油脂实验它在50赫兹5分钟, 并涡旋它再次为1分钟, 以确保没有可行的微生物保持在物质表面。</li>
		<li>在含稀释的无菌离心管中执行每种微气泡培养的十进制串行, 并在 TSA 板上传播100µL 的养殖, 并将其孵化有氧37摄氏度, 24 小时。</li>
		<li>计算菌落数, 这是每个样本和控制磁盘表面上可行微生物的数量。在 (CFU/毫升) 中表达这个数目的活细胞。</li>
		<li>拍摄样品和控制盘的最后微生物培养照片。</li>
	</ol>
</li>
</ol>5. 抗菌结果分析
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57710/57710fig1.jpg"> 图 1: 测量抗菌 "晕" 的规范化宽度.此面板显示抑制区 (d) 和磁盘直径 (d) 的直径。<a href="/files/ftp_upload/57710/57710fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<ol><li>
扩散法结果分析
	<ol>
<li>用数字卡尺测量抑制区 (d)和圆盘直径 (d) 的直径 (见图 1)。 注: 抑制区或抗菌 "晕" 是由抗菌物质盘产生的微生物生长抑制的结果形成的 (见图 1)。有可能观察到, 与样品比较, 有一个透明环区, 与其它微生物生长正常 (不透明区域) 的板块相比。</li>
		<li>应用方程 (1) 确定每个圆盘的抗菌 "晕" (nw晕) 的归一化宽度。 (1)<img alt="Equation 1" src="/files/ftp_upload/57710/57710eq1.jpg">
</li>
		<li>确定抗菌 "晕" 的规范化宽度的平均值和标准偏差, 并测定每个样品的4个确定的nw晕值。</li>
		<li>拍摄最后的微生物文化与物质磁盘的照片。 注: 通过使用合适的图像处理软件, 可以从步骤5.1.4 中拍摄的照片来测量抑制区 (d) 和圆盘直径 (d) 的直径。</li>
	</ol>
</li>
	<li>
联系方式结果分析
	<ol>
<li>根据方程式 (2) 确定可存活微生物的数量。 (2)<img alt="Equation 2" src="/files/ftp_upload/57710/57710eq2.jpg"> 注: 在这里, N是每个试验标本中每 cm2恢复的活菌数量;C是盘数;D是稀释因子;A是测试标本的表面积在 cm2确定了以样品盘直径。</li>
		<li>通过应用方程 (3) 确定生存能力的丧失 (LV) 以反映细胞生长的抑制。 (3)<img alt="Equation 3" src="/files/ftp_upload/57710/57710eq3v3.jpg"> 注: 在这里, C 是在 CFU/mL•cm2中存活的微生物的平均数量 (N), 从对照标本中恢复24小时;S 是在 CFU/mL•cm2中存活的微生物数量 (N), 在24小时后从试验标本中恢复。</li>
		<li>用每个样品的4确定的LV (%)值确定生存能力丧失的平均值和标准偏差。</li>
	</ol>
</li>
</ol>

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作者没有什么可透露的。

新的先进材料的抗菌活性可以通过这个易于遵循的协议来分析, 它由2个补充程序组成, 根据2现有的方法: 琼脂盘扩散试验38和测定的抗菌活性材料表面根据 ISO 22196:2007 规范39。
在这一研究领域中, 许多文献中报告的抗菌试验都是高度依赖于检测的。因此, 在实验室中制定详细和一致的协议是非常重要的。这篇文章是朝这个方向迈出的一步。此外, 这可能是非常有益的许多研究员在这一领域经验较少, 并要求深入, 循序渐进的程序, 以获得准确的结果。
该协议可与多种类型的材料切割成10毫米直径的圆盘形状。脆性材料可以在合适的溶剂中膨胀1小时, 使切割过程更容易。因此, 海藻酸盐等亲水性物质可以在蒸压蒸馏水中水合。其他溶剂, 如乙醇, 酮, 和二氯甲烷, 可用于膨胀疏水性材料1小时前切割它们。然而, 一些材料, 如聚 (3-羟基-3-hydroxyvalerate) 不需要肿胀, 他们可以直接削减。之后, 在真空烤箱中干燥样品材料盘, 用乙醇和紫外线照射1小时以避免任何污染风险是非常重要的。
该议定书建议 TSA 和传播媒介作为培养基和使用纯文化的3种微生物, 以达到广泛的微生物: 革兰氏阳性细菌金黄色葡萄球菌, 革兰阴性菌大肠杆菌, 和酵母白色念珠菌。然而, 其他需要不同孵化条件的培养基和其他微生物也可与本议定书一起使用。有时, 只有1种微生物被测试, 对新材料的抗菌活性有初步的认识。
对于推荐的3种不同类型的微生物, 还应对耐药性致病菌 (如耐甲氧西林金黄色葡萄球菌(MRSE)) 进行试验, 以证明对这种抗菌活性强的材料,已成功地用于该协议。引起人们关注的其他重要的耐药性微生物有革兰阳性耐甲氧西林金黄色葡萄球菌 (MRSA) 和万古霉素耐药肠球菌(VRE), 革兰阴性铜绿假单胞菌40,41。
生物膜抑制和材料对其他类型的微生物 (如病毒和寄生虫) 的抗菌活性不能用这个协议来检验。然而, 这一议定书提供了一个非常有用的出发点, 对新的先进材料的抗菌研究。
在抗菌琼脂盘扩散试验中, 当样品盘必须放在板的中心时, 就会出现一个关键步骤, 因为有些材料一旦接触到琼脂介质就会折叠起来。在这种情况下, 建议使用无菌对镊子仔细展开样品。另一方面, 在接触方法中, 非常重要的是要清洗的控制和样品盘很好地与 PBS 通过吹打他们的四倍, 其次是一个强有力的涡流和超声波, 以确保没有可行的微生物继续坚持材料表面。
该视频协议可用于许多生物工程应用, 如生物工程, 组织工程, 控制药物交付, 包装材料, 废水处理和农业, 其中使用生物材料与高度理想的抗菌能力。
该协议所得到的结果是定性的 (图像) 和定量 (抗菌 "晕" 的归一化宽度和生存能力的丧失), 并对其重现性 (平均标准偏差) 进行了较好的分析。在对不同材料进行比较时, 利用扩散和接触法结果分析得出的这些平均值必须采用单向方差分析, 其次是土耳其的事后分析, 以便研究它们在统计学上是否显著不同 (p < 0.01)。

种植失败往往是微生物感染的结果, 尽管抗菌预防和无菌工作条件。这个问题造成了很高的医疗成本, 在1患者中令人痛心。重要的细菌, 如金黄色葡萄球菌目前被认为是非常危险的病原体与导管和其他医疗植入物相关的医院感染, 是医疗器械的主要污染物2。因此, 迫切需要制定新的抗菌策略, 以供日常和医疗使用。
抗菌剂包括抗生素3, 季铵盐化合物4, 金属离子/氧化物5, 抗菌肽 (安培)6。由于抗生素过量使用8, 抗生素的效率正逐渐降低, 因为细菌耐药性7。季铵盐化合物是非常有效的短期使用, 因为微生物抗药性9。金属离子/氧化物长期以来被用作非常有效的抗菌剂, 并用于许多常见的商业产品, 包括绷带, 水过滤器, 油漆等10,11,12。然而, 已经证明, 这些类型的化合物可能对某些类型的哺乳动物细胞有毒13。
安培显示优秀的抗菌和免疫调节性能14,15, 和细菌似乎发现很难对他们的抵抗16。然而, 生产纯安培的过程是昂贵的;所以, 大规模生产是行不通的。因此, 制定了对付生产安培的问题的策略 (例如, 小分子抗菌 peptoid 模仿17, peptoids18, α肽19和β肽20)。对21的抗菌和防污涂料合成了甲基丙烯酸酯-端多肽和 polypeptoids。
新型抗菌剂的研制, 如先进材料的纯或混合形式, 能够预防和治疗多药耐药性感染, 越来越有必要。在过去数十年中, 通过几种方法改进了各种生物工程领域的新材料, 如组织和生物工程, 通过多种途径进行了化学和物理性质的改良: 等离子体聚合接枝到疏水性衬底22,23,24, 交联密度25,26, 聚合在溶液27,28,29,30, porogen 溶解31,32, 并且由纳米材料的并网例如石墨烯氧化物 (去)33,34,35,36和碳纳米纤维 (生长)37。
对这些新材料的抗菌能力的研究可以成倍地提高其潜在的生物工程适用性, 因此成为必要的。我们提出了一个易于遵循的协议, 以量化的抗菌活性, 这种新的先进材料。在样品制备后, 采用两种互补的方法: 第一个是基于琼脂盘扩散试验38 (扩散法), 第二种是基于 ISO 22196:2007 规范39来测量抗菌活性的材料表面 (接触法)。

<table><tbody><tr><td>Cylindrical punch&amp;nbsp;</td><td></td><td></td><td>10 mm diameter</td></tr><tr><td>Petri dishes</td><td>soria genlab</td><td>P101</td><td>90 mm diameter, sterile</td></tr><tr><td>Tryptic soy agar (TSA)</td><td>Liofilchem</td><td>610052</td><td>Dehydrated medium 500 g (powder)</td></tr><tr><td>Tryptic soy broth (TSB)</td><td>Liofilchem</td><td>610053</td><td>Dehydrated medium 500 g (powder)</td></tr><tr><td>Sterile cotton swab</td><td>EUTOTUBO</td><td>300200</td><td></td></tr><tr><td>Centrifuge tubes</td><td>VIDRA FOC, SA</td><td>429900</td><td>50 mL, sterile</td></tr><tr><td>Ethanol</td><td>VWR</td><td>83813360</td><td>Absolute ethanol</td></tr><tr><td>Sterile 48-wells plate</td><td>COSTAR</td><td>3548</td><td>Flat bottom with lid, tissue culture treated, non-pyrogenic, polystyrene</td></tr><tr><td>A pair of tweezers</td><td>BRAUN</td><td>24612036</td><td>Toothless</td></tr><tr><td>Sterile phosphate buffered saline (PBS).</td><td>VWR</td><td>E404-100TAPBS</td><td></td></tr><tr><td>Vaccum oven with a connected vacuum pump</td><td>JP Selecta, SA</td><td>5900620</td><td></td></tr><tr><td>Laminar flow hood</td><td>TELSTAR Technologies, SL</td><td>TELSTAR AH-100</td><td>12.0 W lamp of UV-C radiation</td></tr><tr><td>Class II Biological safety cabinet</td><td>LABOGENE</td><td>MARS 1200</td><td></td></tr><tr><td>Incubator</td><td>ASTEC CO, LTD</td><td>SCA-165DR</td><td></td></tr><tr><td>Vortex mixer</td><td>Biosan</td><td>V-1 Plus</td><td></td></tr><tr><td>Spectrophotometer</td><td>Macherey-Nagel, Germany</td><td>Nanocolor UV/VIS II</td><td></td></tr><tr><td>Bunsen burner</td><td>JP Selecta, SA</td><td>7001539</td><td></td></tr><tr><td>Alcohol burner</td><td>VIDRA FOC, SA</td><td>1658/20</td><td>In case sterilisation is necessary to be performed inside class II biological safety cabinet</td></tr><tr><td>Orbital shaker</td><td>sartorius stedim</td><td>8864845</td><td></td></tr><tr><td>Sonicator</td><td>SELECTA</td><td>3000617</td><td>50/60 Hz</td></tr><tr><td>Digital calliper</td><td>ACHA</td><td>17-260</td><td>0-150 mm</td></tr><tr><td>Serological pipette</td><td>Fisherbrand</td><td>13-678-11</td><td>25 mL, sterile</td></tr><tr><td>Serological pipette</td><td>VWR</td><td>612-4950</td><td>5 mL, sterile</td></tr><tr><td>Serological pipette</td><td>VWR</td><td>612-5541</td><td>10 mL, sterile</td></tr><tr><td>Micropipette</td><td>GILSON</td><td>FA10005P</td><td>Pipetman L P200L, plastic 20-200 &amp;micro;L</td></tr><tr><td>Micropipette</td><td>GILSON</td><td>F123602</td><td>Pipetman P1000, 200-1000 &amp;micro;L</td></tr><tr><td>Micropipette&amp;nbsp;</td><td>GILSON</td><td>FA10016</td><td>Pipetman L P12X300L, 20-300 &amp;micro;L</td></tr><tr><td>Micropipette tips</td><td>LABBOX</td><td>TIBP-200-960</td><td>2-200 &amp;micro;L</td></tr><tr><td>Micropipette tips</td><td>LABBOX</td><td>TIBP-1K0-480</td><td>100-1000 &amp;micro;L</td></tr><tr><td>Pre-sterilized tube&amp;nbsp;</td><td>INSULAB</td><td>301402</td><td>10 mL&amp;nbsp;</td></tr><tr><td>Photo camera</td><td>Canon EOS 5D</td><td></td><td>Any camera with high resolution can also be utilized</td></tr><tr><td>&lt;em&gt;Gram-positive bacteria Staphylococcus aureus&lt;/em&gt;</td><td></td><td>&amp;nbsp;strain V329</td><td>Cucarella et al. &lt;em&gt;J Bacteriol&lt;/em&gt; &lt;strong&gt;183 (9)&lt;/strong&gt;,&amp;nbsp; 2888&amp;ndash;2896 (2001)</td></tr><tr><td>Gram-negative bacteria &lt;em&gt;Escherichia coli&lt;/em&gt;</td><td>Colecci&amp;oacute;n Espa&amp;ntilde;ola de Cultivos Tipo CECT</td><td>CECT 101</td><td></td></tr><tr><td>Yeast &lt;em&gt;Candida albicans&lt;/em&gt;</td><td>Colecci&amp;oacute;n Espa&amp;ntilde;ola de Cultivos Tipo CECT</td><td>CECT 1394</td><td></td></tr><tr><td>Microcentrifuge tubes&amp;nbsp;</td><td>DASLAB</td><td>175508</td><td>1,5 mL</td></tr><tr><td>Autoclave</td><td>JP Selecta, SA</td><td>4002136</td><td></td></tr><tr><td>Spectrophotometer-cuvettes</td><td>UVAT Bio CB</td><td>F-0902-02</td><td>4,5 mL</td></tr><tr><td>Drigalski spatula</td><td>LABBOX</td><td>SPRP-L05-1K0</td><td>Sterile, disposable&amp;nbsp;</td></tr><tr><td>glass balls (2 mm diameter)</td><td>Hecht Karl</td><td>1401/2</td><td>Autoclavable, alternative device to the Drigalski spatula</td></tr><tr><td>Autoclave bags</td><td>DELTALAB</td><td>200318</td><td>To sterilize microbiological residues or contaminated material</td></tr><tr><td>Electronic pipette filling device</td><td>JetPip</td><td>JET BIOFIL</td><td></td></tr><tr><td>Laboratory bottle with ISO thread, graduated, borosilicate 3.3</td><td>LABBOX</td><td>SBG3-100-010</td><td>100 mL, for autoclaving culture media</td></tr><tr><td>Laboratory bottle with ISO thread, graduated, borosilicate 3.3</td><td>LABBOX</td><td>SBG3-250-010</td><td>250 mL, for autoclaving culture media</td></tr><tr><td>Laboratory bottle with ISO thread, graduated, borosilicate 3.3</td><td>LABBOX</td><td>SBG3-500-010</td><td>500 mL, for autoclaving culture media</td></tr><tr><td>Laboratory bottle with ISO thread, graduated, borosilicate 3.3</td><td>LABBOX</td><td>SBG3-1K0-010</td><td>1000 mL, for autoclaving culture media</td></tr><tr><td>Latex gloves</td><td>DENIA</td><td>2278000000</td><td></td></tr><tr><td>Indicator tape for sterilization</td><td>LABBOX</td><td>STAP-A55-001</td><td>Self-adhesive tape with impregnated paper turning to colour when exposed to sterilization process.</td></tr><tr><td>Universal test tube rack</td><td>LABBOX</td><td>MTSP-001-001</td><td>To hold centrifuge tubes</td></tr><tr><td>Microcentrifuge tube rack</td><td>VWR</td><td>211-0210</td><td>To hold microcentrifuge tubes</td></tr><tr><td>Sterile loop</td><td>ACEFE S.A.</td><td>100140055</td><td>10 &amp;micro;L of capacity for microbial culture&amp;nbsp;</td></tr><tr><td>Material M1</td><td>Universidad Cat&amp;oacute;lica de Valencia San Vicente M&amp;aacute;rtir (UCV)</td><td></td><td>Material type 1&amp;nbsp;</td></tr><tr><td>Material M2</td><td>Universidad Cat&amp;oacute;lica de Valencia San Vicente M&amp;aacute;rtir (UCV)</td><td></td><td>Material type 2&amp;nbsp;</td></tr><tr><td>Material M3</td><td>Universidad Cat&amp;oacute;lica de Valencia San Vicente M&amp;aacute;rtir (UCV)</td><td></td><td>Material type3</td></tr><tr><td>Material M4</td><td>Universidad Cat&amp;oacute;lica de Valencia San Vicente M&amp;aacute;rtir (UCV)</td><td></td><td>Material type 4&amp;nbsp;</td></tr><tr><td>Material C</td><td>Universidad Cat&amp;oacute;lica de Valencia San Vicente M&amp;aacute;rtir (UCV)</td><td></td><td>Control material</td></tr></tbody></table>

antimicrobial characterization of advanced materials for bioengineering applications

在各种生物工程应用中, 开发具有增强性能的新型新材料越来越重要。因此, 许多新的生物材料正在设计, 以模拟生物医学应用所需的特定环境, 如组织工程和控制药物交付。在生物工程中, 对细胞或酶的固定化性能改善的材料的开发也是目前研究的课题。然而, 在这些应用中, 材料的最可取的属性之一是抗菌能力, 以避免任何不良感染。为此, 我们提出了基于 (i) 琼脂盘扩散试验 (扩散法) 和 (ii) ISO 22196:2007 规范测量材料表面抗菌活性的材料抗菌性能的易于遵循的协议 (接触方法)。该协议必须使用革兰氏阳性和革兰阴性菌和酵母, 以涵盖广泛的微生物。以4种不同化学性质的材料为例, 对金黄色葡萄球菌、大肠杆菌和白色念珠菌进行了试验. 这些试验的结果显示, 第一种材料的非抗菌活性和增加抗菌活性的革兰阳性和革兰阴性菌的其他3种材料。然而, 4 种材料中没有一个能抑制白色念珠菌的生长。

以此为例, 以4种不同化学性质的材料对3推荐微生物的抗菌能力进行了测试:金黄色葡萄球菌、大肠杆菌和白色念珠菌 .琼脂盘扩散试验 (扩散法) 的结果为第一材料 (M1) 在控制盘 (C、图像未显示) 和增加抗菌活性的革兰氏阳性和革兰阴性时的非抗菌活性。其他3材料的细菌 M2、M3 和 M4 (见图 2)。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57710/57710fig2.jpg"> 图 2: 抗菌扩散方法结果.该面板显示了4种材料 (M1、M2、M3 和 M4) 盘 (10 毫米直径 x 1 毫米厚度) 的抗菌扩散方法, 对金黄色葡萄球菌和大肠杆菌进行了24小时的孵化。<a href="/files/ftp_upload/57710/57710fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
图 3显示了不同的示例材料 M1、M2、M3 和 M4 的不同规范化宽度的抗菌 "晕" (nw晕), 对革兰氏阳性和革兰阴性菌的计算公式 (1)。然而, 4 种材料中没有一个能抑制酵母白色念珠菌的生长 (图像没有显示)。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57710/57710fig3v2.jpg"> 图 3: 抗菌扩散 "晕" 结果.这个面板显示了标准化的 "晕" (nw晕) 为每个材料 (M1, M2, M3 和 M4) 磁盘 (10 毫米直径 x 1 毫米厚度) 对S. 金黄色葡萄球菌和大肠杆菌后24小时的孵化。差异有统计学意义 (p < 0.01)。然而, 样品 M1 没有表现出抗菌活性。<a href="/files/ftp_upload/57710/57710fig3v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
接触方法的结果还显示了第一材料 (M1) 在控制盘 (C) 中的非抗菌活性, 并增加了对其他3种材料的 Grampositive 和革兰阴性菌的抗菌活性 (见图 4)。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57710/57710fig4v3.jpg"> 图 4: 抗菌接触方法结果.该面板显示了4材料 (M1、M2、M3 和 M4) 表面抗菌活性的各自90毫米板, 根据 ISO 22196:2007 后的24小时的孵化为S. 金黄色葡萄球菌和大肠杆菌(稀释因子 10-4)。C是在24小时孵化后从控制盘中恢复的活菌。<a href="/files/ftp_upload/57710/57710fig4v3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
生存能力的损失 (%) 是由等式 (2) 和 (3) 确定的, 如本议定书所示 (见图 5)。
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57710/57710fig5v2.jpg"> 图 5: 通过接触方法丧失生存能力.此面板显示了 M1、M2、M3 和 M4 对材料表面上的金黄色葡萄球菌和大肠杆菌的生存能力 (%) 的丧失。样品 M1 无抗菌活性。<a href="/files/ftp_upload/57710/57710fig5v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
然而, 4 种材料中没有一个能通过接触法抑制酵母念珠菌的生长 (图像未显示)。因此, 这4种先进材料中的3对革兰氏阳性和革兰阴性菌有积极的抗菌效果, 因此对许多具有高抗菌活性要求的生物工程应用非常有用。然而, 4 种材料中没有一个能抑制酵母的生长。

Watch this Scientific Journal Video about Antimicrobial Characterization of Advanced Materials for Bioengineering Applications at JoVE.com

Antimicrobial Characterization of Advanced Materials for Bioengineering Applications

我们提出了一种先进材料的抗菌性能的协议。在这里, 材料表面的抗菌活性通过两种相互补充的方法来测量: 一是基于琼脂盘扩散试验, 另一种是基于 ISO 22196:2007 规范的标准程序。

生物工程应用先进材料的抗菌性能研究

The development of new advanced materials with enhanced properties is becoming more and more important in a wide range of bioengineering applications. ...

antimicrobial-characterization-advanced-materials-for-bioengineering

Research

JoVE Journal

Bioengineering

22.0K 次观看.  Universidad Católica de Valencia San Vicente Mártir. 我们提出了一种先进材料的抗菌性能的协议。在这里, 材料表面的抗菌活性通过两种相互补充的方法来测量: 一是基于琼脂盘扩散试验, 另一种是基于 ISO 22196:2007 规范的标准程序。

视频: Antimicrobial Characterization of Advanced Materials for Bioengineering Applications

High-throughput Identification of Bacteria Repellent Polymers for Medical Devices

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as contributing to antimicrobial resistance. One possible avenue for the reduction of device-associated infections is the identification of bacteria-repellent polymer coatings for these devices, which would prevent bacterial binding at the initial attachment step. A method for the identification of such repellent polymers, based on the parallel screening of hundreds of polymers using a microarray, is described here. This high-throughput method resulted in the identification of a range of promising polymers that resisted binding of various clinically relevant bacterial species individually and also as multi-species communities. One polymer, PA13 (poly(methylmethacrylate-co-dimethylacrylamide)), demonstrated significant reduction in attachment of a number of hospital isolates when coated onto two commercially available central venous catheters. The method described could be applied to identify polymers for a wide range of applications in which modification of bacterial attachment is important.

A high-throughput microarray method for the identification of polymers which reduce bacterial surface binding on medical devices is described.

医疗器械细菌高通量鉴定驱蚊聚合物

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as ...

科研

生物工程

Biomimetic Materials to Characterize Bacteria-host Interactions

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The biochemical and functional characterization of adhesins mediating these initial bacteria-host interactions is often compromised by the presence of other bacterial factors, such as cell wall components or secreted molecules, which interfere with the analysis. This protocol describes the production and use of biomimetic materials, consisting of pure recombinant adhesins chemically coupled to commercially available, functionalized polystyrene beads, which have been used successfully to dissect the biochemical and functional interactions between individual bacterial adhesins and host cell receptors. Protocols for different coupling chemistries, allowing directional immobilization of recombinant adhesins on polymer scaffolds, and for assessment of the coupling efficiency of the resulting &#8220;bacteriomimetic&#8221; materials are also discussed. We further describe how these materials can be used as a tool to inhibit pathogen mediated cytotoxicity and discuss scope, limitations and further applications of this approach in studying bacterial - host interactions.

Bacterial attachment to host cells is a key step during host colonization and infection. This protocol describes the generation of polymer-coupled recombinant adhesins as biomimetic materials which allow analysis of the contribution of individual adhesins to these processes, independent of other bacterial factors.

仿生材料表征细菌宿主相互作用

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The ...

免疫与感染

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

定性和定量测定为检测和蛋白质抗菌药物的表征

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority of laboratory work is conducted with planktonic cells. Here, we describe a peg plate biofilm assay as performed with Staphylococcus aureus. Bacterial biofilms are grown on pegs attached to a 96-well microtiter plate lid, washed through gentle submersion in buffer, and placed in a drug challenge plate. After subsequent incubation they are again washed and moved to a final recovery plate, in which the fluorescent dye resazurin serves as a viability indicator. This assay offers greatly increased ease-of-use, reliability, and reproducibility, as well as a wealth of data when conducted as a kinetic read. Moreover, this assay can be adapted to a medium-throughput drug screening approach by which an endpoint fluorescent readout is taken instead, offering a path for drug discovery efforts.

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Most bacterial infections produce a biofilm. By virtue of their environment, biofilm associated bacteria are often phenotypically drug resistant. Novel antibacterial molecules that kill bacteria in biofilms are thus a high priority. We establish an assay to quickly screen for antimicrobial compounds that are effective at eradicating biofilms.

针对相关生物膜<em&gt;金黄色葡萄球菌</em&gt;使用基于刃天青药物敏感性测定

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority ...

A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of antibiotic-tolerant infections. Biofilms have displayed an exceptionally high tolerance for chemotherapy, which is thought to be multifactorial. For instance, the matrix provides a physical barrier that decreases the penetration of antibiotics into the biofilm. Also, cells within the biofilms are phenotypically diverse. Likely, biofilm resilience arises from a combination of these and other, yet unknown, mechanisms. All of the currently existing antibiotics have been developed against single-cells (planktonic) bacteria. Therefore, so far, a very limited repertoire of molecules exists that can selectively act on mature biofilms. This situation has driven a progressive paradigm shift in drug discovery, in which searching for anti-biofilms has been urged to occupy a more prominent place. An additional challenge is that there are a very limited number of standardized methods for biofilm research, especially those that can be used for large-throughput screening of chemical libraries. Here, an experimental anti-biofilm platform for chemical screening is presented. It uses three assays to measure biofilm viability (with resazurin staining), total biomass (with crystal violet staining), and biofilm matrix (using a wheat germ agglutinin, WGA-fluorescence-based staining of the poly-N-acetyl-glucosamine, PNAG, fraction). All the assays were developed using Staphylococcus aureus as the model bacteria. Examples of how the platform can be used for primary screening as well as for functional characterization of identified anti-biofilm hits are presented. This experimental sequence further allows for the classification of the hits based upon the measured end-points. It also provides information on their mode of action, especially on long-term versus short-term chemotherapeutic effects. Thus, it is very advantageous for the quick identification of high-quality hit compounds that can serve as starting points for various biomedical applications.

Biofilm infections show high tolerance towards chemotherapy. No single assay captures the complexity of biofilms. Instead, complementary assays are needed. We present a screening platform (developed for S. aureus) that combines assays for viability, biomass, and biofilm matrix. It allows anti-biofilm drug discovery, including the assessment of long-term chemotherapeutic effects.

适合与天然化合物库的探索抗生物试验的平台

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of ...

Synthesis of Multi-walled Carbon Nanotubes Modified with Silver Nanoparticles and Evaluation of Their Antibacterial Activities and Cytotoxic Properties

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver MWCNTs were prepared by the reductive deposition of silver from an aqueous solution of AgNO3 on the oxidized MWCNTs. Given the unique color of the CNTs, it was not possible to apply them to the minimum inhibitory concentration or mitochondrial toxicity assays to evaluate the toxicity and antibacterial properties, since they would interfere with the assays. The inhibition zone and minimum bactericidal concentration for the Ag-MWCNTs were measured and Live/Dead and Trypan Blue assays were used to measure the toxicity and antibacterial properties without interfering with the color of the CNTs.

In this study, antimicrobial nanomaterials were synthesized by acidic oxidation of multiwalled carbon nanotubes and subsequent reductive deposition of silver nanoparticles. Antimicrobial activity and cytotoxicity tests were performed with the as-prepared nanomaterials.

银纳米粒子改性多壁碳纳米管的合成及其抗菌活性和细胞毒性的评价

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver ...

Evaluation of Antimicrobial Activities of Nanoparticles and Nanostructured Surfaces In Vitro

The antimicrobial activities of nanoparticles and nanostructured surfaces, such as silver, zinc oxide, titanium dioxide, and magnesium oxide, have been explored previously in clinical and environmental settings and in consumable food products. However, a lack of consistency in the experimental methods and materials used has culminated in conflicting results, even amongst studies of the same nanostructure types and bacterial species. For researchers who wish to employ nanostructures as an additive or coating in a product design, these conflicting data limit their utilization in clinical settings. 
To confront this dilemma, in this article, we present four different methods to determine the antimicrobial activities of nanoparticles and nanostructured surfaces, and discuss their applicability in different scenarios. Adapting consistent methods is expected to lead to reproducible data that can be compared across studies and implemented for different nanostructure types and microbial species. We introduce two methods to determine the antimicrobial activities of nanoparticles and two methods for the antimicrobial activities of nanostructured surfaces. 
For nanoparticles, the direct co-culture method can be used to determine the minimum inhibitory and minimum bactericidal concentrations of nanoparticles, and the direct exposure culture method can be used to assess real-time bacteriostatic versus bactericidal activity resulting from nanoparticle exposure. For nanostructured surfaces, the direct culture method is used to determine the viability of bacteria indirectly and directly in contact with nanostructured surfaces, and the focused-contact exposure method is used to examine antimicrobial activity on a specific area of a nanostructured surface. We discuss key experimental variables to consider for in vitro study design when determining the antimicrobial properties of nanoparticles and nanostructured surfaces. All these methods are relatively low cost, employ techniques that are relatively easy to master and repeatable for consistency, and are applicable to a broad range of nanostructure types and microbial species.

Evaluation of Antimicrobial Activities of Nanoparticles and Nanostructured Surfaces In Vitro

We introduce four methods to evaluate the antimicrobial activities of nanoparticles and nanostructured surfaces using in vitro techniques. These methods can be adapted to study the interactions of different nanoparticles and nanostructured surfaces with a broad range of microbial species.

体外评估纳米颗粒和纳米结构表面的抗菌活性

The antimicrobial activities of nanoparticles and nanostructured surfaces, such as silver, zinc oxide, titanium dioxide, and magnesium oxide, have been ...

Generation of Greater Bacterial Biofilm Biomass using PCR-Plate Deep Well Microplate Devices

Bacterial biofilms are difficult to eradicate from surfaces using conventional antimicrobial interventions. High-throughput 96-well microplate methods are frequently used to cultivate bacterial biofilms for rapid antimicrobial susceptibility testing to calculate minimal biofilm eradication concentration (MBEC) values. Standard biofilm devices consist of polystyrene pegged-lids fitted to 96-well microplates and are ideal for measuring biofilm biomass and MBEC values, but these devices are limited by available peg surface area for biomass accumulation and cost. Here, we outline a protocol to use self-assembled polypropylene 96-well deep well PCR-plate pegged-lid device to grow Escherichia coli BW25113 and Pseudomonas aeruginosa PAO1 biofilms. A comparison of 24-hour biofilms formed on standard and deep well devices by each species using crystal violet biomass staining and MBEC determination assays are described. The larger surface area of deep well devices expectedly increased overall biofilm formation by both species 2-4-fold. P. aeruginosa formed significantly greater biomass/mm2 on deep well pegs as compared to the standard device. E. coli had greater biomass/mm2 on standard polystyrene devices as compared the deep well device. Biofilm eradication assays with disinfectants such as sodium hypochlorite (bleach) or benzalkonium chloride (BZK) showed that both compounds could eliminate E. coli and P. aeruginosa biofilms from both devices but at different MBEC values. BZK biofilm eradication resulted in variable E. coli MBEC values between devices, however, bleach demonstrated reproducible MBEC values for both species and devices. This study provides a high throughput deep well method for growing larger quantities of biofilms on polypropylene devices for downstream studies requiring higher amounts of static biofilm.

This protocol presents methodology to perform biofilm growth and biomass measurements using self-assembled deep well PCR-plate devices for high-throughput 96-well pegged lid static biofilm screening.

使用PCR板深孔微孔设备生成更大的细菌生物膜生物质

Bacterial biofilms are difficult to eradicate from surfaces using conventional antimicrobial interventions. High-throughput 96-well microplate methods are ...

<meta charset="utf8"></head><body>用于耐药细菌的抗菌氧化石墨烯/铜纳米复合材料

Fabrication of Antibacterial Graphene Oxide/Copper Nanocomposites

Antibiotics are currently the most used antibacterial treatment for killing bacteria. However, bacteria develop resistance when continually overexposed to antibiotics. Developing&#160;antimicrobial agents that can replace existing antibiotics is essential because antibiotic-resistant bacteria have resistance mechanisms for all current antibiotics and can promote nosocomial infections. To address this challenge, in this study, we propose graphene oxide/copper (GO/Cu) nanocomposites as antibacterial materials that can replace the existing antibiotics. GO/Cu nanocomposites are characterized by transmission electron microscopy and scanning electron microscopy. They show that copper (Cu) nanoparticles are well-grown on the graphene oxide sheets. Additionally, a microdilution broth method is used to confirm the efficacy of the antimicrobial substance against methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (P. aeruginosa), which are frequently implicated in nosocomial infections. Specifically, 99.8% of MRSA and 84.7% of P. aeruginosa are eliminated by 500 &#181;g/mL of GO/Cu nanocomposites. Metal nanocomposites can eradicate antibiotic-resistant bacteria by releasing ions, forming reactive oxygen species, and physically damaging the bacteria. This study demonstrates the potential of antibacterial GO/Cu nanocomposites in eradicating antibiotic-resistant bacteria.

<meta charset="utf8"></head><body>作者聚焦:消除抗生素耐药细菌的金属纳米复合材料

Herein, we introduce graphene oxide/copper (GO/Cu) nanocomposites as an antibacterial nanomaterial. The antibacterial effectiveness of the GO/Cu nanocomposites was evaluated against both antibiotic-resistant gram-positive and gram-negative bacteria.

抗菌氧化石墨烯/铜纳米复合材料的制备

Antibiotics are currently the most used antibacterial treatment for killing bacteria. However, bacteria develop resistance when continually overexposed to ...

<meta charset="utf8"></head><body> 氧化锌纳米颗粒的合成及其抗菌作用分析

Preparation of Zinc Oxide Nanoparticles and the Evaluation of their Antibacterial Effects

Nosocomial bacterial infections have become increasingly challenging due to their inherent resistance to antibiotics. The emergence of multidrug-resistant bacterial strains in hospitals has been attributed to the extensive and varied use of antibiotics, further exacerbating the problem of antibiotic resistance. Metal nanomaterials have been widely studied as an alternative solution for eradicating antibiotic-resistant bacterial cells. Metallic nanoparticles attack bacterial cells through various mechanisms, such as the release of antibacterial ions, generation of reactive oxygen species, or physical disruption, against which bacteria cannot develop resistance. Among the actively researched antimicrobial metal nanoparticles, zinc oxide nanoparticles, which are FDA-approved, are known for their biocompatibility and antibacterial properties. In this study, we focused on successfully developing a precipitation method for synthesizing zinc oxide nanoparticles, analyzing the properties of these nanoparticles, and conducting antimicrobial tests. Zinc oxide nanoparticles were characterized using transmission electron microscopy (TEM), dynamic light scattering (DLS), ultraviolet/visible spectroscopy, and X-ray diffraction (XRD). Antibacterial tests were conducted using the broth microdilution test with the multidrug-resistant strains of methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa. This study demonstrated the potential of zinc oxide nanoparticles in inhibiting the proliferation of antibiotic-resistant bacteria.

<meta charset="utf8"></head><body>作者聚焦：探索氧化锌纳米颗粒在克服抗生素耐药性中的抗菌作用

In this study, zinc oxide nanoparticles were synthesized using a precipitation method. The antibacterial effect of the synthesized particles was tested against multidrug-resistant methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa bacterial strains.

氧化锌纳米颗粒的制备及其抗菌效果评价

Nosocomial bacterial infections have become increasingly challenging due to their inherent resistance to antibiotics. The emergence of multidrug-resistant ...