JCM由BBSRC-IPA奖与联合利华公司（BB / L023040 / 1）资助。 HAG由高等教育博士助学金的沙特阿拉伯教育部资助。

下面的方法适合用7,8抑制剂生产菌株，以测试应变竞争对手。 注:该协议涉及气溶胶的产生与细菌培养。这只能在包含的空间与执行本地认可的安全方面的考虑来执行。一个2级安全柜内到琼脂平板培养物喷洒将最小化的琼脂平板的污染和周围环境。这也将防止喷射细菌雾化吸入的研究者，这是一个主要问题，如果研究者使用包含级别2的生物体。适当的个人防护设备必须佩戴。围绕培养皿的区域最好与一次性吸湿组织覆盖。使用UV光喷雾消毒后建议。 1.准备琼脂平板<ol><li>准备脑心脏浸液（BHI）琼脂按照给马努厂家的说明。 </li><li>高压灭菌的BHI琼脂在121℃，15根据高压釜制造商的建议psi的20分钟，或者，进行消毒。 </li><li>倾灭菌BHI琼脂15毫升分装到无菌培养皿中，并允许它设置。 注:确保要使用的每一个琼脂平板内测定从琼脂的相同库存始发，并且有在每个板中的相同量的琼脂。这限制了速效养分和板材允许可比性的抑制剂扩散的可变性。 </li></ol> 2.准备无菌肉汤<ol><li>根据制造商的说明准备BHI肉汤。 </li><li> BHI添加10毫升分装到28毫升玻璃奶瓶普遍。 </li><li> ，15磅20分钟，或根据高压釜制造商的建议高压釜在121℃的BHI液体培养基，灭菌。 注:确保在试验中使用的所有肉汤同一批肉汤AUT的起源同时以限制的营养成分，它可能导致结果变异变异oclaved。 </li></ol> 3.准备无菌蒸发器瓶（S） <ol><li>拆开vapourizer瓶。 </li><li>喷雾5％的表面活性清洗剂通过vapourizer，消除内部的污染物，泡vapourizer瓶子，盖子和vapourizer在5％的表面活性清洗剂过夜。 注:手套和适当的个人防护装备应处理的表面活性剂清洗时使用。 </li><li>在干燥无菌空气流罩蒸发器，盖子和瓶子。 </li><li>把瓶子装满70％的乙醇，重新连接vapourizer并通过vapourizer喷些乙醇。 </li><li>放在蒸发器盖子，里面直到使用乙醇储存。 </li><li>使用，无菌冲洗瓶子用无菌BHI肉汤，并通过vapourizer喷汤前夕。 注意:这可以防止乙醇AFF阿拉斯加入到瓶子中6.3节接种。 </li></ol> 4.准备细菌过夜培养（S） <ol><li>条纹的竞争者和抑制剂株上的无菌BHI琼脂平板上，并在37°C孵育需氧大约18小时（过夜）。 注意:此板产生时保存在4℃下，可以被用于在同一周内多次重复细菌的库存。 </li><li>接种10毫升无菌BHI肉汤的与所需的竞争者细菌菌株的28毫升普遍瓶子内的单个菌落。 </li><li>通过在轨道摇床以250rpm摇动孵育在37℃的普遍瓶需氧过夜。 </li></ol> 5.抑制剂生产隔离现货文化<ol><li>在第4节准备过夜培养（S）。 </li><li>确保琼脂平板完全干燥后，盖上盖子不结露，在接种前（ 例如 ，通过培育解放军德在37℃下60分钟）;这防止了接种物从接种部位穿过该板传播。 </li><li>吸移管25微升过夜培养物到琼脂平板的中央。避免完全放开吸管柱塞，防止气泡横跨琼脂喷文化。 </li><li>允许培养在室温下干燥，以限制文化的传播。在37℃下孵育琼脂平板需氧过夜。 </li></ol> 6.准备测试竞争者应变的喷雾接种（S） <ol><li>如第4稀释的过夜培养10倍的BHI肉汤中所述，得到约4×10 6 CFU毫升-1制备过夜培养，并悬浮液的OD 600 0.3±0.05的。 注:10倍稀释不会放弃4×106 CFU毫升-1所有的细菌种类，通 ​​过传播板之间1×10系列稀释计算所需的稀释倍数<s达&gt; -1 -1×10 -6。 </li><li>倒入稀释文化带入无菌塑料香水瓶喷雾器。 注:总体积必须更对于被喷琼脂板的数量比是必要的。大约每板250μl的是适合于9厘米直径的陪替氏培养皿。 </li></ol> 7.递延生长抑制试验<ol><li>通过各蒸发器的瓶子的喷雾的培养，以确保培养物在喷雾机构喷涂到琼脂之前加载。 </li><li>从约15厘米的琼脂上面的距离，喷约250稀释的培养的微升（典型50毫升汽化器3喷雾剂）在整个琼脂表面。在37℃下孵育琼脂平板需氧过夜。重复具有相同数量的喷雾剂的其它板。 </li></ol> 8.递延解读生长抑制试验结果<ol><li>测量喷雾competi的生长抑制区器菌株（ 图1A - C，"X"为"X"）以毫米为单位，减去抑制剂生产者的中央点的直径（ 图1A - C，"Y"为"Y"）。 注: 图1E表示这个数据是如何被呈现</li><li>记录清晰度得分抑制区。 注:这表示观察到抑制程度。实例示于图1A - D，可用于标准化的菌株中的代表性结果部分的建议。以下尺度可用于清晰度评分:0清晰度评分来描述竞争者菌株的生长的完全抑制的区域。 1分来形容的增长几乎完全抑制的区域，与目前在空地的区域只有个别殖民地。 2分来形容降低经济增长的可见区域，这个区域内的增长是骗子流利或接近汇合但&gt; 50％密度减少。 3分来形容只有最小的增长减少，生长汇合并低于50％降低，区域仍然是可见和可衡量的。 4分来形容生长没有明显的抑制作用。将5得分来描述的竞争者菌株的生长在测试抑制剂生产菌株的接近而增加。参见图1中的例子。这些分数是主观的实验者。 </li></ol> 9.重复<ol><li>至少在生物一式三份重复试验，以确定获得的结果的可重复性。 注意:如果重复在不同的天进行的，进行测量，并在4℃下从温育中取出后立即得分板，然后或者商店板或将其废弃之前采取板的高品质的照片。存储或板的照片，使不同天之间的结果易于比较，T他是整个天/实验证实可重复性的得分尤为重要。注意，在板的存储具有改变的结果的可能性。 注:对于清晰的照片代替板直立没有他们在漫射光的面积的哑光黑色的背景盖，使用高分辨率相机（≥8MP）能够专注于物体靠近镜头（微距模式）。或者，用反式照明部分凝胶成像仪设置为白色光可以生产出高品质，高重现性的图像。 </li></ol>

<ol>
	<li>Bolnick, D. I., 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=Why+intraspecific+trait+variation+matters+in+community+ecology.">Why intraspecific trait variation matters in community ecology.</a> Trends Ecol Evol. 26 (4), 183-192 (2011).</li><li>Christensen, G. J. 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=Antagonism+between+Staphylococcus+epidermidis+and+Propionibacterium+acnes+and+its+genomic+basis.">Antagonism between Staphylococcus epidermidis and Propionibacterium acnes and its genomic basis.</a> BMC Genomics. 17 (152), (2016).</li><li>Faust, K., 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=Microbial+co-occurrence+relationships+in+the+human+microbiome.">Microbial co-occurrence relationships in the human microbiome.</a> PloS Comput Biol. 8 (7), (2012).</li><li>Frank, D. N., Feazel, L. M., Bessesen, M. T., Price, C. S., Janoff, E. N., Pace, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Human+nasal+microbiota+and+Staphylococcus+aureus+carriage.">The Human nasal microbiota and Staphylococcus aureus carriage.</a> PLoS One. 5 (5), e10598 (2010).</li><li>Fredheim, E. G., Flaegstad, T., Askarian, F., Klingenberg, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colonisation+and+interaction+between+S.+epidermidis+and+S.+aureus+in+the+nose+and+throat+of+healthy+adolescents.">Colonisation and interaction between S. epidermidis and S. aureus in the nose and throat of healthy adolescents.</a> Eur J Clin Microbiol Infect Dis. 34, 123-129 (2015).</li><li>Hibbing, M. E., Fuqua, C., Parsek, M. R., Peterson, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+competition:+surviving+and+thriving+in+the+microbial+jungle.">Bacterial competition: surviving and thriving in the microbial jungle.</a> Nat Rev Microbiol. 8, 15-25 (2010).</li><li>Libberton, B., Coates, R. E., Brockhurst, M. A., Horsburgh, 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=Evidence+that+intraspecific+trait+variation+among+nasal+bacteria+shapes+the+distribution+of+Staphylococcus+aureus.">Evidence that intraspecific trait variation among nasal bacteria shapes the distribution of Staphylococcus aureus.</a> Infect Immun. 82 (9), 3811-3815 (2014).</li><li>Libberton, B., Horsburgh, M. J., Brockhurst, M. A. <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+spatial+structure,+frequency+dependence+and+resistance+evolution+on+the+dynamics+of+toxin-mediated+microbial+invasions.">The effects of spatial structure, frequency dependence and resistance evolution on the dynamics of toxin-mediated microbial invasions.</a> Evol appl. 8, 738-750 (2015).</li><li>Liu, C. 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=Staphylococcus+aureus+and+the+ecology+of+the+nasal+microbiome.">Staphylococcus aureus and the ecology of the nasal microbiome.</a> Sci Adv. 1, e1400216 (2015).</li><li>Nascimento, J. S., Ceotto, H., Nascimento, S. B., Giambiagi-deMarval, M., Santos, K. R., Bastos, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteriocins+are+alternative+agents+for+control+of+multiresistant+staphylococcal+strains.">Bacteriocins are alternative agents for control of multiresistant staphylococcal strains.</a> Lett Appl Microbiol. 42, 215-221 (2006).</li><li>Park, B., Iwase, T., Liu, G. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intranasal+application+of+S.+epidermidis+prevents+colonization+by+methicillin-resistant+Staphylococcus+aureus+in+mice.">Intranasal application of S. epidermidis prevents colonization by methicillin-resistant Staphylococcus aureus in mice.</a> PLoS One. 6 (10), e25880 (2011).</li><li>Uehara, 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=Bacterial+interference+among+nasal+inhabitants:+eradication+of+Staphylococcus+aureus+from+nasal+cavities+by+artificial+implantation+of+Corynebacterium+sp.">Bacterial interference among nasal inhabitants: eradication of Staphylococcus aureus from nasal cavities by artificial implantation of Corynebacterium sp.</a> J Hosp Infect. 44, 127-133 (2000).</li><li>Wos-Oxley, M. 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作者什么都没有透露。

从其他人的区别就这个细菌物种竞争协议的一个关键步骤，是抑制剂的孵育过夜除了竞争对手分离的前隔离开来。抑制剂应变需要这种增长时期产生的抑制化合物。作为竞争者菌株在相同介质的顶部重叠，此方法允许研究人员观察到的全部潜力 抑制剂菌株，干扰竞争者菌株的生长。其结果是，该测定反映了与自然发生的情况;一个孤立完全建立在一个利基，另一个必须能够抵消任何抑制变量能够成功入侵的利基。 有两个部分，以该协议最有可能执行的，如果不正确会导致错误的结果。首先，蒸发器瓶杀菌不完全可以引进污染细菌进入检测。 INCU用于冲洗汽化器瓶在步骤3.6在无菌容器中的肉汤bation可以用来确认瓶完全灭菌。在消毒液，如卫可附加洗涤，可先于表面活性清洁剂洗涤，在步骤3.2中所述，如果需要额外的杀菌使用。 可能导致错误的结果的第二步骤是竞争者菌株（步骤6.1）的稀释液。如果稀释并非最佳，抑制的明确测量可能不容易确定。该测定是使用不同的鼻菌株作为抑制剂生产在大范围的革兰氏阳性和革兰氏阴性物种与链球菌菌株优化金黄色葡萄球菌 SH1000作为竞争对手的应变7,8。它后来被用各种凝固酶阴性和凝固酶阳性葡萄球菌作为竞争对手和抑制剂生产菌株进行测试。当测试除上述作为竞争者其他属应变，对于接种稀释的一些优化可能是必要的。 如果清晰度分数重复之间显著变化，可能有必要通过将其设置为特定的光学密度进行标准化竞争者菌株接种物。然而，这将增加该测定需要来设置，并有可能降低菌株的量是可能每个实验进行处理的时间。在视觉上调整文化，用麦氏标准可以提供快速的替代适当的OD。 这种技术的一个限制是，它只能适用于培养细菌。这就是为什么许多研究使用的16S rRNA测序预测竞争排斥相互作用3,4,13。然而，遗传和宏基因组技术只能社区成员区分的物种水平，和/或不考虑特定种内性状的变化。能够筛选一个particul的关联AR基因，该基因编码具有细菌物种5的存在或不存在的性状，但是这需要可能与一个物种的排斥相关联的基因的预先知识。 这种技术的安全性的考虑是，由于气雾产生的细菌，它只能在实验室使用具有2级安全柜，或具有类似的安全预防措施。有替代技术测试为占种内性状变异，不产生细菌菌株气溶胶之间的竞争。一种这样的方法是同时拮抗试验2。在该方法中，一个分离株的接种物涂布在琼脂平板和第二分离接种物点样或刺伤顶部一旦第一已干燥。这将产生其中菌株直接竞争（同时对抗）的环境中，既竞争产生的抑制化合物和第一清除营养素。该advantag递延生长抑制试验在同时对抗法的e是该抑制剂分离应该总是有竞争对手分离的入侵之前被完全建立竞争优势。这是更加反射性的环境中，会发生什么，因为在大多数龛，加入前另一个应变，而不是在同一时间8被加入了两个分离一种隔离群将被建立。 另一种替代，以避免气溶胶的产生菌的是添加竞争者隔离在顶层琼脂，这可能是倾而不是喷洒在抑制剂分离物。然而，上层琼脂中加入的体积将需要约3毫升至确保即使板的覆盖范围。在该卷中，竞争者分离物将不为相同的介质作为抑制剂分离物上生长，所以将不从减少营养物受损。竞争对手也不会与任何UNT抑制化合物直接接触IL它们扩散到顶层琼脂。这样一个试验不能被描述为递延抑制或同时拮抗作用。 类似的方法以前已经用来评估之间临床S.杆菌肽抗性水平金黄色葡萄球菌菌株10。由10描述这里描述的协议和之间的主要区别，是后者只分类竞争者菌株为敏感（完成inhibition-清晰度评分0）或抗性（部分无inhibition-清晰度分数1-5）。延迟生长抑制试验可以因此还可以用于筛选对特定抗微生物剂，或者甚至是生产者的抗性水平来搜索对耐多药病原体活性新颖抗微生物剂。 有人建议，该宿主微生物可以被操纵以消除细菌群落9的不希望的成员。棒状杆菌或金黄色葡萄球菌中的应用ylococcus表皮已经被证明消除S.葡萄球菌定植在人口11,12的一个显著比例鼻孔。因此，研究为特定菌株能竞争排除主机菌群的不良成员，通过技术如这里所描述的人，可能会导致未来的治疗。

Bacteria living in communities compete at the interspecific and intraspecific level for space and nutrients 6. Competition between microbes can often lead to exclusion of particular species or strains within a community. Exclusion can be due to competition for a particular receptor or nutrient, or the production of an antimicrobial compound by a member of the community 6.
Studies investigating competitive exclusion frequently examine the presence and absence of species using 16S rRNA sequencing 3,4,13. However, intraspecific trait variation, for example the presence or absence of antimicrobial peptide production, can hugely impact the ability of one species to inhibit another 1. Since 16S rRNA sequencing cannot distinguish between strains of the same species, intraspecific trait variation cannot be assessed. The technique described here can be used to assess the potential of individual strains to exclude other bacteria. This method has previously been used to show a positive correlation between the presence of Staphylococcus aureus inhibitory bacteria and the absence of S. aureus in a nasal community 7.
The deferred growth inhibition (competition) assay can identify production of antimicrobial compounds or nutrient competition by the inhibitor strain. It can also detect positive interactions, whereby the presence of the inhibitor strain actually improves the growth of the competitor strain. This assay can be used to create quantitative data (zone of clearing size) and qualitative data (degree of inhibition), both of which can be overlaid onto data of the presence or absence of a species within a community to find correlations between phenotype (trait) and competitive exclusion 7.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Brain heart infusion broth</td>
			<td style="width:71px;">Lab M</td>
			<td style="width:119px;">lab049</td>
			<td style="width:167px;">rich general purpose media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">agar-agar</td>
			<td style="width:71px;">Merck Milipore</td>
			<td align="right" style="width:119px;">101614</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">petri dishes</td>
			<td style="width:71px;">Sarstedt</td>
			<td style="width:119px;">82.1473.001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">plastic perfume vaporizer</td>
			<td style="width:71px;">not known</td>
			<td style="width:119px;">-</td>
			<td>10 cm high with 3.5 cm diameter clear plastic spray bottle with lid, purchased from the travel section of a supermarket</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Universal bottles</td>
			<td style="width:71px;"></td>
			<td style="width:119px;">no longer in production, alternative SLS BOT5006</td>
			<td>28 ml glass cylindrical bottle (approx 280 mm diameter, 850 mm height), flat bottomed with plastic screw cap</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Decon 90</td>
			<td style="width:71px;">Decon&#160;</td>
			<td style="width:119px;"></td>
			<td>surface active cleaning agent&#160;</td>
		</tr>
	</tbody>
</table>

deferred growth inhibition assay to quantify the effect of bacteria-derived antimicrobials on competition

Competitive exclusion can occur in microbial communities when, for example, an inhibitor-producing strain outcompetes its competitor for an essential nutrient or produces antimicrobial compounds that its competitor is not resistant to. Here we describe a deferred growth inhibition assay, a method for assessing the ability of one bacterium to inhibit the growth of another through the production of antimicrobial compounds or through competition for nutrients. This technique has been used to investigate the correlation of nasal isolates with the exclusion of particular species from a community. This technique can also be used to screen for lantibiotic producers or potentially novel antimicrobials. The assay is performed by first culturing the test inhibitor-producing strain overnight on an agar plate, then spraying over the test competitor strain and incubating again. After incubation, the extent of inhibition can be measured quantitatively, through the size of the zone of clearing around the inhibitor-producing strain, and qualitatively, by assessing the clarity of the inhibition zone. Here we present the protocol for the deferred inhibition assay, describe ways to minimize variation between experiments, and define a clarity scale that can be used to qualitatively assess the degree of inhibition.

该试验的结果应当是观察到的作为在靠近中央斑点抑制剂生产菌株（ 图1）叠加的竞争者应变的差。这些差异可以解释为充分抑制（ 图1A），部分抑制（ 图1B和1C），无抑制（ 图1D）或正相关。在广泛使用的乳酸菌菌株S.表皮 Tü3298和S.表皮 Rp62A和S.表皮 Rp62A和S.金黄色葡萄球菌 Newman是在图1B和图1D分别使用。我们建议，这些菌株可用于测试此协议。 根据测试种类，充分抑制和部分抑制最有可能抗菌肽，抗生素或其他扩散性增长inhibito的结果由抑制剂生产菌株生产的RS。部分抑制也被链接到降低的养分可用性由抑制剂应变6由于竞争者应变摄取或营养物的螯合剂。 针对同一竞争者应变测试了不同的抑制剂生产菌株之间抑制区域的大小的比较表明在任一以下的差异:通过抑制剂生产菌株产生的抗微生物的量;抑制剂通过媒体扩散的能力;抑制剂的类型产生和正在生产的竞争者应变的抑制剂的抗性程度。针对同一抑制剂生产菌株测试不同竞争者菌株之间抑制区域的大小的比较是表示竞争者菌株所生产的抑制剂的抗性水平的。 如果已经发生的细菌的不一致应用不应使用所述板。例如，如果测试竞争者培养是太稀抑制区域将出现大的并用比通常所观察到的（ 图2A）不太明确界定边缘;这会影响任何比较区测量的可靠性。如果该抑制剂生产菌株点没有充分孵育前干燥过夜，抑制剂菌株是可能传播，而不是形成一个明确定义的点（ 图2B），这通常会影响区的测量，但也可以影响透明度得分。如果参赛者应变不均匀喷洒这可能会导致细菌的不均匀扩散（ 图2C）。如果参赛者应变过于集中可能的是所述竞争者菌株可以在抑制剂菌株（ 图2C）的顶部生长，在极端情况下，这可能会导致竞争者/抑制剂应变角色的一些反转。 <img alt="图1" src="/files/ftp_upload/54437/54437fig1.jpg" /> 图 1: 可从延迟生长抑制试验中获得的的定性和定量结果范围结果生长抑制的范围可以从（A）的完全抑制（0分），（B）的部分抑制（评分为1） （ 三 ）部分抑制（2分）至（D）没有抑制（4分）。标签指示抑制剂菌株（Y）的增长的边缘和抑制的最外边缘区（X）的。抑制范围的区域的从0毫米到大于10mm的大小，如图（E）所示，其示出对A拍摄到的板的测量结果，B，C和D的结果示于最近葡萄球菌分离株（A ，C）或普通实验室分离株金黄色葡萄球菌S.表皮 Tü3298（抑制剂）和S.表皮 Rp62A（竞争者）（B）和S.表皮 Rp62A（抑制剂）和S.金黄色葡萄球菌 Newman（竞争对手）（D）。 <a href="https://www.jove.com/files/ftp_upload/54437/54437fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54437/54437fig2.jpg" /> 图 2: 由于技术不佳的结果不一致的例子 ，结果可能会有所不同，如果比预期的（A。 </stroNG&gt;）竞争对手菌株太稀，（B）抑制剂菌株没有完全干的孵化/板前不干燥或（C）如果参赛者菌株并不均匀喷洒并过于集中， <a href="https://www.jove.com/files/ftp_upload/54437/54437fig2large.jpg" target="_blank">请点击这里查看大图这一数字。</a>

Watch this Scientific Journal Video about Deferred Growth Inhibition Assay to Quantify the Effect of Bacteria-derived Antimicrobials on Competition at JoVE.com

Deferred Growth Inhibition Assay to Quantify the Effect of Bacteria-derived Antimicrobials on Competition

The deferred growth inhibition assay can be used to assess the competition effect by one bacterial isolate on another. Inhibition is quantified by measuring the zone of clearing around the inhibitor-producing isolate, or qualitatively assessed by determining the visible extent of inhibition.

递延生长抑制试验来量化竞争性细菌来源的抗菌剂的影响

Competitive exclusion can occur in microbial communities when, for example, an inhibitor-producing strain outcompetes its competitor for an essential ...

deferred-growth-inhibition-assay-to-quantify-effect-bacteria-derived

Nanyang Technological University

Research

JoVE Journal

Biology

14.5K 次观看.  University of Liverpool. The deferred growth inhibition assay can be used to assess the competition effect by one bacterial isolate on another. Inhibition is quantified by measuring the zone of clearing around the inhibitor-producing isolate, or qualitatively assessed by determining the visible extent of inhibition.

视频: Deferred Growth Inhibition Assay to Quantify the Effect of Bacteria-derived Antimicrobials on Competition

Growth Factor-Coated Bead Placement on Dorsal Forebrain Explants

This video demonstrates two methods for preparing and placing beads, which have been coated with growth factor, on explants of the developing cerebral cortex. These beads can be used to induce spatially restricted gene expression on developing neural tissue such as forebrain explants. Methods are given for using both Affi-Gel beads and heparin acryllic beads.

涂层生长因子-珠放在背的前脑植

科研

生物学

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within the cell, type III effectors subvert host cellular processes to suppress immune responses and promote pathogen growth. Numerous type III effectors of plant and animal bacterial pathogens have been identified to date, yet only a few of them are well characterized. Understanding the functions of these effectors has been undermined by a combination of functional redundancy in the effector repertoire of a given bacterial strain, the subtle effects that they may exert to increase virulence, roles that are possibly specific to certain infection stages, and difficulties in genetically manipulating certain pathogens. Expression of type III effectors in the budding yeast Saccharomyces cerevisiae may allow circumventing these limitations and aid to the functional characterization of effector proteins. Because type III effectors often target cellular processes that are conserved between yeast and other eukaryotes, their expression in yeast may result in growth inhibition phenotypes that can be exploited to elucidate effector functions and targets. Additional advantages to using yeast for functional studies of bacterial effectors include their genetic tractability, information on predicted functions of the vast majority of their ORFs, and availability of numerous tools and resources for both genome-wide and small-scale experiments. Here we discuss critical factors for designing a yeast system for the expression of bacterial type III effector proteins. These include an appropriate promoter for driving expression of the effector gene(s) of interest, the copy number of the effector gene, the epitope tag used to verify protein expression, and the yeast strain. We present procedures to induce expression of effectors in yeast and to verify their expression by immunoblotting. In addition, we describe a spotting assay on agar plates for the identification of effector-induced growth inhibition phenotypes. The use of this protocol may be extended to the study of pathogenicity factors delivered into the host cell by any pathogen and translocation mechanism.

In this video, we describe a procedure for the expression of bacterial type III effectors in yeast and the identification of effector-induced growth inhibition phenotypes. Such phenotypes can be subsequently exploited to elucidate effector functions and targets.

细菌III型效应，在酵母中的表达诱导的生长抑制的表型鉴定

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within ...

Effect of Male Accessory Gland Products on Egg Laying in Gastropod Molluscs

In internally fertilizing animals, seminal fluid is usually added to the spermatozoa, together forming the semen or ejaculate. Besides nourishing and activating sperm, the components in the seminal fluid can also influence female physiology to augment fertilization success of the sperm donor. While many studies have reported such effects in species with separate sexes, few studies have addressed this in simultaneously hermaphroditic animals. This video protocol presents a method to study effects of seminal fluid in gastropods, using a simultaneously hermaphroditic freshwater snail, the great pond snail Lymnaea stagnalis, as model organism. While the procedure is shown using complete prostate gland extracts, individual components (i.e., proteins, peptides, and other compounds) of the seminal fluid can be tested in the same way. Effects of the receipt of ejaculate components on egg laying can be quantified in terms of frequency of egg laying and more subtle estimates of female reproductive performance such as egg numbers within each egg masses. Results show that seminal fluid proteins affect female reproductive output in this simultaneous hermaphrodite, highlighting their importance for sexual selection.

This video protocol demonstrates a method to study effects of seminal fluid in gastropods, using the hermaphroditic freshwater snail Lymnaea stagnalis.

男性附属性腺产品对腹足软体动物下蛋的影响

In internally fertilizing animals, seminal fluid is usually added to the spermatozoa, together forming the semen or ejaculate. Besides nourishing and ...

Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard approach to determining intracellular protein degradation relies on biochemical assays for following the kinetics of protein decline. Such methods are often laborious and time consuming and therefore not amenable to experiments aimed at assessing multiple substrates and degradation conditions. As an alternative, cell growth-based assays have been developed, that are, in their conventional format, end-point assays that cannot quantitatively determine relative changes in protein levels.
Here we describe a method that faithfully determines changes in protein degradation rates by coupling them to yeast cell-growth kinetics. The method is based on an established selection system where uracil auxotrophy of URA3-deleted yeast cells is rescued by an exogenously expressed reporter protein, comprised of a fusion between the essential URA3 gene and a degradation determinant (degron). The reporter protein is designed so that its synthesis rate is constant whilst its degradation rate is determined by the degron. As cell growth in uracil-deficient medium is proportional to the relative levels of Ura3, growth kinetics are entirely dependent on the reporter protein degradation.
This method accurately measures changes in intracellular protein degradation kinetics. It was applied to: (a) Assessing the relative contribution of known ubiquitin-conjugating factors to proteolysis (b) E2 conjugating enzyme structure-function analyses (c) Identification and characterization of novel degrons. Application of the degron-URA3-based system transcends the protein degradation field, as it can also be adapted to monitoring changes of protein levels associated with functions of other cellular pathways.

Here we describe a robust biological assay for quantifying the relative rate of proteolysis by the ubiquitin-proteasome system. The assay readout is yeast growth rate in liquid culture, which is dependent on the cellular levels of a reporter protein comprising a degradation signal fused to an essential metabolic marker.

记者的增长分析的蛋白质降解的系统分析

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard ...

Comparing the Affinity of GTPase-binding Proteins using Competition Assays

In this protocol we demonstrate a method for comparing the competition between GTPase-binding proteins. Such an approach is important for determining the binding capabilities of GTPases for two reasons: The fact that all interactions involve the same face of the GTPases means that binding events must be considered in the context of competitors, and the fact that the bound nucleotide must also be controlled means that conventional approaches such as immunoprecipitation are unsuitable for GTPase biochemistry. The assay relies on the use of purified proteins. Purified Rac1 immobilized on beads is used as the bait protein, and can be loaded with GDP, a non-hydrolyzable version of GTP or left nucleotide free, so that the signaling stage to be investigated can be controlled. The binding proteins to be investigated are purified from mammalian cells, to allow correct folding, by means of a GFP tag. Use of the same tag on both proteins is important because not only does it allow rapid purification and elution, but also allows detection of both competitors with the same antibody during elution. This means that the relative amounts of the two bound proteins can be determined accurately.

This protocol compares the relative affinities of binding partners for Rho-family GTPases, including Rac1. In vivo, Rac1-binding proteins compete for a single binding interface, the conformation of which is dictated by a bound nucleotide. The nucleotide is both important and difficult to control experimentally, due to the high hydrolysis rate.

比较GTP酶结合蛋白的使用竞争性试验的亲和

In this protocol we demonstrate a method for comparing the competition between GTPase-binding proteins. Such an approach is important for determining the ...

Techniques to Induce and Quantify Cellular Senescence

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed cellular senescence. Accumulation of senescent cells occurs with organismal aging and through continual culturing in vitro. Senescent cells influence many biological processes, including embryonic development, tissue repair and regeneration, tumor suppression, and aging. Hallmarks of senescent cells include, but are not limited to, increased senescence-associated &#946;-galactosidase activity (SA-&#946;-gal); p16INK4A, p53, and p21 levels; higher levels of DNA damage, including &#947;-H2AX; the formation of Senescence-associated Heterochromatin Foci (SAHF); and the acquisition of a Senescence-associated Secretory Phenotype (SASP), a phenomenon characterized by the secretion of a number of pro-inflammatory cytokines and signaling molecules. Here, we describe protocols for both replicative and DNA damage-induced senescence in cultured cells. In addition, we highlight techniques to monitor the senescent phenotype using several senescence-associated markers, including SA-&#946;-gal, &#947;-H2AX and SAHF staining, and to quantify protein and mRNA levels of cell cycle regulators and SASP factors. These methods can be applied to the assessment of senescence in various models and tissues.

Cellular senescence, the irreversible state of cell-cycle arrest, can be induced by various cellular stresses. Here, we describe protocols to induce cellular senescence and methods to assess markers of senescence.

技术诱导和量化细胞衰老

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed ...

An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm

Staphylococcus aureus (S. aureus) is a common human pathogen, which causes pyogenic and systemic infections. S. aureus infections are difficult to eradicate not only due to the emergence of antibiotic-resistant strains but also its ability to form biofilms. Recently, photodynamic therapy (PDT) has been indicated as one of the potential treatments for controlling biofilm infections. However, further studies are required to improve our knowledge of its effect on bacterial biofilms, as well as the underlying mechanisms. This manuscript describes an in vitro model of PDT with 5-aminolevulinic acid (5-ALA), a precursor of the actual photosensitizer, protoporphyrin IX (PpIX). Briefly, mature S. aureus biofilms were incubated with ALA and then exposed to light. Subsequently, the antibacterial effect of ALA-PDT on S. aureus biofilm was quantified by calculating the colony forming units (CFUs) and visualized by viability fluorescent staining via confocal laser scanning microscopy (CLSM). Representative results demonstrated a strong antibacterial effect of ALA-PDT on S. aureus biofilms. This protocol is simple and can be used to develop an in vitro model to study the treatment of S. aureus biofilms with ALA-PDT. In the future, it could also be referenced in PDT studies utilizing other photosensitizers for different bacterial strains with minimal adjustments.

An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm

This manuscript describes a protocol to study the antimicrobial effect of 5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) on a Staphylococcus aureus biofilm. This protocol can be used to develop an in vitro model to study the treatment of bacterial biofilms with PDT in the future.

用体外模型研究 5-氨基乙酰丙酸介导光动力疗法对金黄色葡萄球菌生物膜的影响

Staphylococcus aureus (S. aureus) is a common human pathogen, which causes pyogenic and systemic infections. S. aureus infections are difficult to ...

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.

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 ...

A Plate Competition Assay As a Quick Preliminary Assessment of Disease Suppression

The goal was to develop and optimize a simple, affordable, and effective bioassay to detect disease suppressive ability of a specific compost against soilborne fungus Rhizoctonia solani. R. solani is a pathogen of a wide range of plant hosts worldwide. The fungus survives in soils as a saprophyte and grows rapidly on simple water agar media. The plate assay is a rapid method to compare composts for their ability to slow the growth of R. solani. The assay also correlates well with suppression of other soilborne fungal pathogens that survive as saprophytes in soils such as Alternaria early blights, Fusarium wilt, Phytophthora root rot, and Pythium root rot.

Presented is a protocol for a plate competition assay to identify whether a specific compost is likely to contain bacteria and fungi that suppress growth of Rhizoctonia solani.

一种快速的疾病抑制初步评估的板块竞争分析法

The goal was to develop and optimize a simple, affordable, and effective bioassay to detect disease suppressive ability of a specific compost against ...

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm cell to penetrate the zona pellucida and fertilize the egg. It is therefore of great interest to test compounds (e.g., environmental chemicals or drug candidates) for their effect on Ca2+-signaling and acrosome reaction in human sperm either to examine the potential adverse effects on human sperm cell function or to investigate a possible role as a contraceptive. Here, two medium-throughput assays are described: 1) a fluorescence-based assay for assessment of effects on Ca2+-signaling in human sperm, and 2) an image cytometric assay for assessment of acrosome reaction in human sperm. These assays can be used to screen a large number of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm. Furthermore, the assays can be used to generate highly specific dose-response curves of individual compounds, determine potential additivity/synergism for two or more compounds, and to study the pharmacological mode of action through competitive inhibition experiments with CatSper inhibitors.

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Here, two medium-throughput assays for assessment of effects on Ca2+-signaling and acrosome reaction in human sperm are described. These assays can be used to quickly and easily screen large amounts of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm.

中通量筛选法评估对精子中 ca2 +信号和反应的影响

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm ...