Name: aseptic laboratory techniques: plating methods
Uploaded: 2012-05-11T12:00:00
Duration: 18 min
Description: Watch this Scientific Journal Video about Aseptic Laboratory Techniques: Plating Methods at JoVE.com

<p class="jove_content">特别感谢IROC设计CORI桑德斯准备设立样品的文化和协助数字插图和克里斯Reddi在加州大学洛杉矶分校和Bhairav​​沙阿。霍华德休斯医学研究所（HHMI的批准号：52006944），为这个项目提供了资金。 </p>

<ol>
	<li>Barker, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> <em style='font-style: italic'>At the Bench: A Laboratory Navigator</em>. , (1998).</li><li>US Department of Health and Human Services (DHHS). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> <em style='font-style: italic'>Biosafety in Microbiological And Biomedical Laboratories (BMBL)</em>. , (2009).</li><li>Dulbecco, R., Vogt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+problems+of+animal+virology+as+studied+by+the+plaque+technique.">Some problems of animal virology as studied by the plaque technique.</a> <em style='font-style: italic'>Cold Spring Harbor Symp. Quant. Biol</em>. <b>18</b>, 273-279 (1953).</li><li>Fields, B. 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L., Huycke, M. M., Gilmore, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+agar+plate+method+for+quantifying+viable+bacteria.">Simplified agar plate method for quantifying viable bacteria.</a> <em style='font-style: italic'>BioTechniques</em>. <b>23</b>, 648-650 (1997).</li><li>Karam, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> <em style='font-style: italic'>Molecular Biology of Bacteriophage T4</em>. , (1994).</li><li>Knipe, D. M., Howley, P. M., Griffin, D. E., Lamb, R. A., Martin, M. A., Roizman, B., Straus, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> <em style='font-style: italic'>Fundamental Virology</em>. , (2001).</li><li>Lederberg, J., Lederberg, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replica+plating+and+indirect+selection+of+bacterial+mutants.">Replica plating and indirect selection of bacterial mutants.</a> <em style='font-style: italic'>J. Bacteriol</em>. <b>63</b>, 399-406 (1952).</li><li>Miller, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> <em style='font-style: italic'>A Short Course in Bacterial Genetics: A Laboratory Manual and Handbook for Escherichia coli and related bacteria</em>. , (1992).</li><li>Mills, K. V., Gareau, J. R., Garcia, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-cost+modification+to+the+Copacabana+method+for+spreading+transformation+mixtures.">Low-cost modification to the Copacabana method for spreading transformation mixtures.</a> <em style='font-style: italic'>BioTechniques</em>. <b>39</b>, 188 (2005).</li><li>Jordan, T., 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=.">.</a> <em style='font-style: italic'>RESEARCH: National Genomics Research Initiative Phage Resource Laboratory Manual</em>. , (2008).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> <em style='font-style: italic'>Molecular Cloning - A Laboratory Manual</em>. , (2001).</li><li>Sanders, E. R., Miller, J. H. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> <em style='font-style: italic'>Microbiologist: A Discovery-based Course in Microbial Ecology and Molecular Evolution</em>. , (2010).</li><li>Slonczewski, J. L., Foster, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> <em style='font-style: italic'>Microbiology - An Evolving Science</em>. , (2011).</li><li>Teather, R. M., Wood, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Congo+Red-Polysaccharide+Interactions+in+Enumeration+and+Characterization+of+Cellulolytic+Bacteria+from+the+Bovine+Rumen.">Use of Congo Red-Polysaccharide Interactions in Enumeration and Characterization of Cellulolytic Bacteria from the Bovine Rumen.</a> <em style='font-style: italic'>Applied and Environmental Microbiology</em>. <b>43</b>, 777-780 (1982).</li><li>Wise, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> <em style='font-style: italic'>Preparing Spread Plates Protocols</em>. , (2006).</li><li>Worthington, M., Luo, R. Q., Pelo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Copacabana+method+for+spreading+E.+coli+and+yeast+colonies.">Copacabana method for spreading E. coli and yeast colonies.</a> <em style='font-style: italic'>BioTechniques</em>. <b>30</b>, 738-742 (2001).</li></ol>

<p class="jove_content">我什么都没有透露。 </p>

<p class="jove_content">培养微生物涉及电镀方法，所有这些都需要保持无菌技术在整个细胞和媒体操纵。在这个协议，五个不同的程序进行了描述。虽然这些电镀技术通常用于操纵细菌和噬菌体，他们也可以应用于哺乳动物细胞培养中常用的分子遗传学，如酵母（即， <em>酿酒酵母，白色念珠菌，裂殖酵母</em> ），藻类和原生动物和真核微生物（即， <em>团藻，衣藻，变形虫，草履虫</em> ），线虫（即<em>线虫</em> ）。也有许多（更复杂）各电镀方法的变化，根据实验的目标或正在研究的有机体。因此，重要的是不仅要选择最合适的技术对于一个给定的实验或目标微生物，而且裁缝的方法，这种T帽子的实验结果，适当解决所研究的问题或问题。 </p><p class="jove_content">一些涉及本议定书中所讨论的电镀技术的最新应用技术的进步，产生高通量筛选和药物发现实验结果。例如，基因组测序中心，为扩电镀克隆库使用“科帕卡巴纳方法”，这是<em>E。</em>含有来自一种微生物的基因组DNA片段的质粒转化<em>大肠杆菌</em>细胞。因为几十大板（称为生物活性托盘）一次编写，自动化板振动筛是用于托盘的整批玻璃珠摇。此外，选择从这些板块的殖民地后，孵化时，机器人的殖民地选择器用来收集从LB培养基接种适当的殖民地，在384孔板的细胞。对于这种高通量筛选法，蔓延-P的程序原则申请后期的技术，但技术允许各种措施来实现自动化和扩展，允许大量的样品进行分析，同时在很短的时间框架。 </p><p class="jove_content">在发展高通量技术在微生物学和分子遗传学的最基本的技术，生物技术和制药公司投入大量资源。例如，有多渠道micropipettors，进行长达8个或12个样品一次量转让。即使是96通道移液器头的机器人工作站，机动！这些工作涉及多学科的科学家小组，配对生物学家，拥有工程师和计算机程序员可以开发执行与实验相关的机械操作所需的仪器与方法的专门知识。无论研究中的应用，开发这些技术的公司共同的目标是相同的 - 自动化laboratoRY流程，工具，系统和工具，使他们少劳力密集和更有效。 </p>

<p class="jove_step">1. Yeast Tryptone Agar (YTA)</p>
<table border="1">
	<tbody>
		<tr>
			<td>Yeast extract</td>
			<td>2.0 g</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>10.0 g</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>15.0 g</td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td>up to 1000.0 ml</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>pH 7.0</td>
		</tr>
	</tbody>
</table>
<p class="jove_content">Autoclave at 121&deg;C for 20 minutes to sterilize. Store at 4&deg;C.</p>
<p class="jove_content">If preparing tubes for the pour-plate procedure, allow the agar to cool to ~55&deg;C then add 2.0 ml of 50 mg/ml cycloheximide. Aseptically dispense 18.0 ml of the melted agar per 18 mm tube then store at 4&deg;C. The agar will solidify and will need to be melted in a steamer or microwave prior to use.</p>
<p class="jove_step">2. Minimal Salts Agar (MSA) + 0.1% (w/v) carbon source</p>
<table border="1">
	<tbody>
		<tr>
			<td>NH<sub>4</sub>Cl</td>
			<td>1.0 g</td>
		</tr>
		<tr>
			<td>NH<sub>2</sub>HPO<sub>4</sub>&bull;2H<sub>2</sub>O</td>
			<td>2.14 g</td>
		</tr>
		<tr>
			<td>KH<sub>2</sub>PO<sub>4</sub></td>
			<td>1.09 g</td>
		</tr>
		<tr>
			<td>MgSO<sub>4</sub>&bull;7H<sub>2</sub>O</td>
			<td>0.2 g</td>
		</tr>
		<tr>
			<td>Carbon source*</td>
			<td>1.0 g</td>
		</tr>
		<tr>
			<td>Trace salts solution**</td>
			<td>10.0 ml</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>15.0 g</td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td>up to 1000.0 ml</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>pH 7.0</td>
		</tr>
	</tbody>
</table>
<p class="jove_content">Autoclave at 121&deg;C for 20 minutes to sterilize. Store at 4&deg;C.</p>
<p class="jove_content">* Carbon sources used for experiments presented in <strong>Figure 13</strong> include acetamide, lactose, and glycine.</p>
<p class="jove_content">** Trace salts solution is prepared in 0.1 N HCl as follows. It is added to the base before sterilization (autoclave at 121&deg;C for 20 minutes).</p>
<table border="1">
	<tbody>
		<tr>
			<td>FeSO<sub>4</sub>&bull;7H<sub>2</sub>O</td>
			<td>300.0 mg</td>
		</tr>
		<tr>
			<td>MnCl<sub>2</sub>&bull;4H<sub>2</sub>O</td>
			<td>180.0 mg</td>
		</tr>
		<tr>
			<td>Co(NO<sub>3</sub>)<sub>2</sub>&bull;6H<sub>2</sub>O</td>
			<td>130.0 mg</td>
		</tr>
		<tr>
			<td>ZnSO<sub>4</sub>.7H<sub>2</sub>O</td>
			<td>40.0 mg</td>
		</tr>
		<tr>
			<td>H<sub>2</sub>MoO<sub>4</sub></td>
			<td>20.0 mg</td>
		</tr>
		<tr>
			<td>CuSO<sub>4</sub>&bull;5H<sub>2</sub>O</td>
			<td>1.0 mg</td>
		</tr>
		<tr>
			<td>CaCl<sub>2</sub></td>
			<td>1000.0 mg</td>
		</tr>
		<tr>
			<td>HCl (0.1 N)</td>
			<td>up to 1000.0 ml</td>
		</tr>
	</tbody>
</table>
<p class="jove_step">3. EHA soft agar (0.65 % w/v)</p>
<table border="1">
	<tbody>
		<tr>
			<td>Agar</td>
			<td>6.5 g</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Tryptone</td>
			<td>13.0 g</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>NaCl</td>
			<td>8.0 g</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Na Citrate&bull;2H<sub>2</sub>O</td>
			<td>2.0 g</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Glucose</td>
			<td>3.0 g</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Distilled water</td>
			<td>up to 1000.0 ml</td>
		</tr>
	</tbody>
</table>
<p class="jove_content">Autoclave at 121&deg;C for 20 minutes to sterilize. Store at 4&deg;C.</p>
<p class="jove_step">4. EHA hard agar (1.2% w/v)</p>
<table border="1">
	<tbody>
		<tr>
			<td>Agar</td>
			<td>12.0 g</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>13.0 g</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>8.0 g</td>
		</tr>
		<tr>
			<td>Na Citrate&bull;2H<sub>2</sub>O</td>
			<td>2.0 g</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>3.0 g</td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td>up to 1000.0 ml</td>
		</tr>
	</tbody>
</table>
<p class="jove_content">Autoclave at 121&deg;C for 20 minutes to sterilize. Store at 4&deg;C.</p>
<p class="jove_step">5. 1X Middlebrook Top Agar (MBTA soft agar, 0.5% w/v)</p>
<table border="1">
	<tbody>
		<tr>
			<td>100 mM CaCl<sub>2</sub> stock*</td>
			<td>1 ml</td>
		</tr>
		<tr>
			<td>7H9 liquid medium: <em>Neat </em>**</td>
			<td>50 ml</td>
		</tr>
		<tr>
			<td>2XTA ***</td>
			<td>50 ml</td>
		</tr>
	</tbody>
</table>
<p class="jove_content">Melt 50 ml of 2XTA and allow it to cool to ~55&deg;C. Using aseptic technique, add the CaCl<sub>2</sub> and 7H9 broth to the melted agar. Aseptically dispense 4.5 ml of the mixture per 13 mm tube and store in a 55&deg;C incubator &le;7 days. Cooling MBTA to room temperature or 4&deg;C will cause the CaCl<sub>2</sub> to precipitate out of solution.</p>
<p class="jove_content">* 100 mM CaCl<sub>2</sub>. stock must be stored at room temperature to prevent CaCl<sub>2</sub> from precipitating out of solution.</p>
<p class="jove_content">** 7H9 liquid medium: Neat</p>
<table border="1">
	<tbody>
		<tr>
			<td>7H9 broth base</td>
			<td>4.7 g</td>
		</tr>
		<tr>
			<td>40% glycerol stock</td>
			<td>5 ml</td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td>up to 900.0 ml</td>
		</tr>
	</tbody>
</table>
<p class="jove_content">Mix the base with water then add the glycerol while stirring. Autoclave at 121&deg;C for 20 minutes to sterilize. Store at 4&deg;C.</p>
<p class="jove_content">*** 2X Middlebrook Top Agar (2XTA, 1.0% w/v)</p>
<table border="1">
	<tbody>
		<tr>
			<td>7H9 broth base</td>
			<td>4.7 g</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>1.0 g</td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td>up to 1000.0 ml</td>
		</tr>
	</tbody>
</table>
<p class="jove_content">Autoclave at 121&deg;C for 20 minutes to sterilize. Dispense 50 ml aliquots into 100 ml bottles and store at 4&deg;C.</p>
<p class="jove_step">6. Middlebrook 7H10 Agar Plates (MHA hard agar, 1.9% w/v)</p>
<table border="1">
	<tbody>
		<tr>
			<td>7H10 agar base</td>
			<td>19.0 g</td>
		</tr>
		<tr>
			<td>40% glycerol stock</td>
			<td>12.5 ml</td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td>887.5 ml</td>
		</tr>
	</tbody>
</table>
<p class="jove_content">Mix the agar base with water then add the glycerol while stirring. Heat the solution to boiling then stir for one minute to completely dissolve the base powder. Autoclave at 121&deg;C for 20 minutes to sterilize. Allow the agar to cool to ~55&deg;C then aseptically add the following reagents:</p>
<table border="1">
	<tbody>
		<tr>
			<td>AD supplement (pre-warmed to 37&deg;C)*</td>
			<td>100 ml</td>
		</tr>
		<tr>
			<td>50 mg/ml Carbenicillin **</td>
			<td>1.0 ml</td>
		</tr>
		<tr>
			<td>10 mg/ml Cycloheximide **</td>
			<td>1.0 ml</td>
		</tr>
	</tbody>
</table>
<p class="jove_content">* AD supplement</p>
<table border="1">
	<tbody>
		<tr>
			<td>NaCl &gt;</td>
			<td>17 g &gt;</td>
		</tr>
		<tr>
			<td>Albumin (Fraction V)&gt;</td>
			<td>100 g&gt;</td>
		</tr>
		<tr>
			<td>Dextrose (<em>D</em>-Glucose)&gt;</td>
			<td>40 g&gt;</td>
		</tr>
		<tr>
			<td>Distilled water&gt;</td>
			<td>up to 2000.0 ml&gt;</td>
		</tr>
	</tbody>
</table>
<p class="jove_content">Filter-sterilize this solution; do not autoclave. Store at 4&deg;C.</p>
<p class="jove_content">** Filter-sterilize and store these solutions at 4&deg;C for &le;60 days.</p>
<p class="jove_step">7. LB agar (1.5% w/v) + X-Gal (60 &mu;g/ml)</p>
<table border="1">
	<tbody>
		<tr>
			<td>Tryptone</td>
			<td>10.0 g</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>5.0 g</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>10.0 g</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>15.0 g</td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td>up to 1000.0 ml</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>pH 7.5 at 25&deg;C</td>
		</tr>
	</tbody>
</table>
<p class="jove_content">Autoclave at 121&deg;C for 20 minutes to sterilize. Allow the agar to cool to ~55&deg;C then aseptically add 3.0 ml of 20 mg/ml X-Gal solution. Freshly prepare X-gal stock by dissolving 400 mg X-Gal in 20 ml dimethylformamide (DMF).</p>
<p class="jove_step">Table of specific reagents:</p>
<table border="1">
	<tbody>
		<tr>
			<td><strong>Name of the reagent</strong></td>
			<td><strong>Company</strong></td>
			<td><strong>Catalogue number</strong></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Becton Dickenson</td>
			<td>212750</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Becton Dickenson</td>
			<td>211705</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Becton Dickenson</td>
			<td>214030</td>
		</tr>
		<tr>
			<td>NH<sub>4</sub>Cl</td>
			<td>Acros Organics</td>
			<td>123340010</td>
		</tr>
		<tr>
			<td>NH<sub>2</sub>HPO<sub>4</sub>&bull;2H<sub>2</sub>O</td>
			<td>Sigma-Aldrich</td>
			<td>30435</td>
		</tr>
		<tr>
			<td>KH<sub>2</sub>PO<sub>4</sub></td>
			<td>Fisher Scientific</td>
			<td>BP 303-500</td>
		</tr>
		<tr>
			<td>MgSO<sub>4</sub>&bull;7H<sub>2</sub>O</td>
			<td>Sigma-Aldrich</td>
			<td>230391</td>
		</tr>
		<tr>
			<td>Acetamide</td>
			<td>Sigma-Aldrich</td>
			<td>A-0500</td>
		</tr>
		<tr>
			<td>Lactose</td>
			<td>Fisher Scientific</td>
			<td>L6-500</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>G-7126</td>
		</tr>
		<tr>
			<td>FeSO<sub>4</sub>&bull;7H<sub>2</sub>O</td>
			<td>Sigma-Aldrich</td>
			<td>F8048</td>
		</tr>
		<tr>
			<td>MnCl<sub>2</sub>&bull;4H<sub>2</sub>O</td>
			<td>Sigma-Aldrich</td>
			<td>M-3634</td>
		</tr>
		<tr>
			<td>Co(NO<sub>3</sub>)<sub>2</sub>&bull;6H<sub>2</sub>O</td>
			<td>Sigma-Aldrich</td>
			<td>230375</td>
		</tr>
		<tr>
			<td>ZnSO<sub>4</sub>.7H<sub>2</sub>O</td>
			<td>Sigma-Aldrich</td>
			<td>Z4750</td>
		</tr>
		<tr>
			<td>H<sub>2</sub>MoO<sub>4</sub></td>
			<td>Acros Organics</td>
			<td>213621000</td>
		</tr>
		<tr>
			<td>CuSO<sub>4</sub>&bull;5H<sub>2</sub>O</td>
			<td>Sigma-Aldrich</td>
			<td>209198</td>
		</tr>
		<tr>
			<td>CaCl<sub>2</sub></td>
			<td>Sigma-Aldrich</td>
			<td>C1016</td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Fisher Scientific</td>
			<td>A144-212</td>
		</tr>
		<tr>
			<td>Cycloheximide</td>
			<td>Sigma</td>
			<td>038K1561</td>
		</tr>
		<tr>
			<td>Carbenicillin</td>
			<td>Cellgro</td>
			<td>46-100-RG</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher</td>
			<td>S271-1</td>
		</tr>
		<tr>
			<td>Na Citrate&bull;2H<sub>2</sub>O</td>
			<td>Fisher</td>
			<td>S279-500</td>
		</tr>
		<tr>
			<td>Glucose (Dextrose)</td>
			<td>BD</td>
			<td>215530</td>
		</tr>
		<tr>
			<td>7H9 broth base</td>
			<td>BD</td>
			<td>271310</td>
		</tr>
		<tr>
			<td>7H10 agar base</td>
			<td>BD</td>
			<td>262710</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Shelton</td>
			<td>IB15760</td>
		</tr>
		<tr>
			<td>Albumin (Fraction V)</td>
			<td>Fisher</td>
			<td>S71907</td>
		</tr>
		<tr>
			<td>X-Gal (5-bromo-4-chloro-3-indolyl-&beta;-D-galactopyranoside)</td>
			<td>Teknova</td>
			<td>X1205</td>
		</tr>
		<tr>
			<td>Dimethylformamide (DMF)</td>
			<td>Sigma</td>
			<td>D4551</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fisher</td>
			<td>CDA19</td>
		</tr>
		<tr>
			<td>Chlorine Bleach</td>
			<td>Chlorox</td>
			<td>02490/06644884</td>
		</tr>
		<tr>
			<td>CiDecon (disinfectant)</td>
			<td>Decon Laboratories, Inc.</td>
			<td>8504</td>
		</tr>
	</tbody>
</table>
<p class="jove_step">Table of specific equipment:</p>
<table border="1">
	<tbody>
		<tr>
			<td><strong>Name of equipment</strong></td>
			<td><strong>Company</strong></td>
			<td><strong>Catalogue number</strong></td>
			<td><strong>Experiment</strong></td>
		</tr>
		<tr>
			<td>Metal loops</td>
			<td>American Educational Products</td>
			<td>S17352</td>
			<td> Streak plating</td>
		</tr>
		<tr>
			<td>Disposable plastic loops</td>
			<td>Fisher</td>
			<td>22-363-602</td>
			<td> Streak plating</td>
		</tr>
		<tr>
			<td>Wooden sticks</td>
			<td>Fisher</td>
			<td>23-400-104</td>
			<td> Streak plating</td>
		</tr>
		<tr>
			<td>Flat Toothpicks</td>
			<td>American Educational Products</td>
			<td>S67859</td>
			<td>Streak plating</td>
		</tr>
		<tr>
			<td>Turn tables</td>
			<td>Fisher</td>
			<td>08-758Q</td>
			<td>Spread plating</td>
		</tr>
		<tr>
			<td>Glass rods</td>
			<td>Bellco Glass</td>
			<td>NC9004380</td>
			<td>Spread plating</td>
		</tr>
		<tr>
			<td>4 mm glass beads</td>
			<td>Fisher</td>
			<td>11-312B</td>
			<td>Spread plating</td>
		</tr>
		<tr>
			<td>Velveteen cloth</td>
			<td>Bel-Art Products</td>
			<td>09-718-2</td>
			<td>Replica plating</td>
		</tr>
		<tr>
			<td>Cylindrical block</td>
			<td>Bel-Art Products</td>
			<td>09-718-1</td>
			<td>Replica plating</td>
		</tr>
	</tbody>
</table>

aseptic laboratory techniques: plating methods

<p class="jove_content">微生物是创造无处不在实验室中的可能污染来源，无生命表面上。实验的成功依赖于科学家消毒工作台面和设备，以及防止接触表面非无菌消毒仪器和解决方案的能力。在这里，我们提出几个电镀经常在实验室中用于隔离，传播，或枚举微生物，如细菌和噬菌体的方法步骤。所有五个方法采用无菌技术，或程序，维护实验材料的无菌。描述的过程包括：（1）条纹电镀细菌培养分离单菌落，（2）倒入电镀，（3）传播电镀枚举可行的细菌菌落，（4）软琼脂覆盖隔离噬菌体和枚举斑块，（ 5）副本电镀转移细胞从一个板块向另一个在相同的空间格局。这些程序可以在t他实验室长凳上，只要涉及非致病微生物菌种（生物安全等级1，BSL-1）。如果BSL-2有机体的工作，那么这些操作必须在生物安全柜。咨询<em>中</em>的<em>微生物和生物医学实验室</em>生物安全<em>的</em>最新版本（BMBL）以及感染性物质的<em>材料安全数据表</em> （MSDS），以确定生物危害的分类，以及问题中的微生物所需要的安全防范措施和防护设施。可从保持特定组织，如<em>美国菌种保藏中心</em> （ATCC）的研究调查，公司，集合菌株和噬菌体股票。它被推荐用于学习各种电镀方法时，非致病性菌株。在本协议中所述的程序之后，学生应该能够： </p><ul><li>执行无contaminati电镀程序吴媒体。 </li><li>连胜电镀法分离单菌落。 </li><li>使用倒电镀和扩散电镀方法，以确定细菌的浓度。 </li><li>噬菌体时，执行软琼脂覆盖。 </li><li>细菌细胞转移到另一个使用副本电镀程序从一个板块。 </li><li>由于实验任务，选择适当的电镀方法。 </li></ul></p>

Watch this Scientific Journal Video about Aseptic Laboratory Techniques: Plating Methods at JoVE.com

Aseptic Laboratory Techniques: Plating Methods

<p class="jove_content">媒体和试剂用于培养微生物的工作时，必须实行无菌技术，以确保污染降到最低。多种电镀方法通常用于隔离，传播，或列举细菌和噬菌体，所有这些都纳入程序，维护实验材料的无菌。</p>

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Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or ...