Name: aseptic laboratory techniques: plating methods
Uploaded: 2012-05-11T12:00:00.000+00:00
Duration: 18 min
Description: Watch this Scientific Journal Video about Aseptic Laboratory Techniques: Plating Methods at JoVE.com

샘플 문화권을 설정하고 그림과 함께 지원을 위해 그림을 준비하고 크리스 Reddi와 UCLA의 Bhairav​​ 샤에 대한 Iroc 설계에서 Cori 샌더스에 대한 특별 감사합니다. 이 프로젝트에 대한 자금 지원은 HHMI (HHMI 그랜트 번호 52006944)에 의해 제공되었다.

<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> At the Bench: A Laboratory Navigator. , (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> Biosafety in Microbiological And Biomedical Laboratories (BMBL). , (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> Cold Spring Harbor Symp. Quant. Biol. 18, 273-279 (1953).</li><li>Fields, B. N., Knipe, D. M., Chanock, R. M., Hirsch, M. S., Melnick, J. L., Monath, T. P., Roizman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fields Virology. 1, (1991).</li><li>Gilbert, R. A., Ouwerkerk, D., Zhang, L. H., Klieve, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooperative+Research+Center+for+Beef+Genetic+Technologies.+In+vitro+detection+and+primary+cultivation+of+bacteria+producing+materials+inhibitory+to+ruminal+methanogens.">Cooperative Research Center for Beef Genetic Technologies. In vitro detection and primary cultivation of bacteria producing materials inhibitory to ruminal methanogens.</a> Journal of Microbiological Methods. 80, 217-218 (2010).</li><li>Jett, B. D., Hatter, K. 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> BioTechniques. 23, 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> Molecular Biology of Bacteriophage T4. , (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> Fundamental Virology. , (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> J. Bacteriol. 63, 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> A Short Course in Bacterial Genetics: A Laboratory Manual and Handbook for Escherichia coli and related bacteria. , (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> BioTechniques. 39, 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> RESEARCH: National Genomics Research Initiative Phage Resource Laboratory Manual. , (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> Molecular Cloning - A Laboratory Manual. , (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> Microbiologist: A Discovery-based Course in Microbial Ecology and Molecular Evolution. , (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> Microbiology - An Evolving Science. , (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> Applied and Environmental Microbiology. 43, 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> Preparing Spread Plates Protocols. , (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> BioTechniques. 30, 738-742 (2001).</li></ol>

나는 공개 할게 없다.

Culturing의 미생물 무균 기술은 세포와 미디어의 조작에 걸쳐 유지하는 것이 필요한 모든 중 도금 방법의 수를 포함한다. 다섯 가지 절차는이 프로토콜에서 설명되었다. 이러한 도금 기법은 일상적으로 세균과 파지를 조작하는 데 사용하고 있지만, 그들은 또한 (일반적으로 같은 효모 (즉, Saccharomyces의 cerevisiae, 칸디다 albicans, Schizosaccharomyces의 pombe), 해조류 등 분자 유전학에서 사용하고 원생 동물문 포유 동물 세포 배양 및 진핵 미생물에 적용할 수 즉, Volvox, Chlamydomonas, 아메바, Paramecium) 및 nematodes (즉, Caenorhabditis elegans). 또한 연구 미만 실험 목표 또는 유기체에 따라 각각의 도금 방법의 다양한 (그리고 훨씬 더 정교한) 차이가 있습니다. 그러므로, 그것은 단지 방법론 같은 T를 주어진 실험 또는 대상 미생물에 가장 적합한 기술을 선택할뿐만 아니라 봉제에하지하는 것이 중요모자 실험 결과는 적절한 연구 질문이나 문제를 해결합니다. 이 프로토콜에서 언급한 도금 기술의 최신 애플 리케이션 중 일부는 선별 및 약물 발견 실험에 대한 높은 처리량 결과를 얻을 기술 진보를 포함. 예를 들어, 게놈 시퀀싱 센터 E.되는, 확산 도금 클론 라이브러리에 대해 &quot;Copacabana 방법&quot;을 사용 대장균 세포는 미생물의 게놈으로부터 파생된의 DNA 조각을 포함하는 plasmids으로 바꾸었다. 큰 접시 수십개이 (bioassay 트레이라고도 함) 한번에 준비가되어 있기 때문에, 자동 번호판 뿌리는 쟁반의 전체 배치를 위해 유리 구슬을 떨쳐낼하는 데 사용됩니다. 또한, 부화에 따라 다음과 접시에서 콜로니를 선택할 때, 로봇 식민지 따는 사람은 384 잘 microplates의 LB의 국물을위한 inoculum 따라 식민지에서 세포를 수집하는 데 사용됩니다. 이러한 높은 처리량 심사 분석, 확산-P의 절차적 원칙에 대한늦은 기법이 적용될 수 있지만 기술은 여러 단계가 자동화되어 있으며 동시에 짧은 시간 내에 분석해야 할 시료의 큰 숫자를 허용하도록 조정 수 있습니다. 생명 공학 및 제약 회사들은 미생물학과 분자 유전학의 가장 기본적인 기술에 대한 높은 처리량 기술 개발에 상당한 자원을 투자합니다. 예를 들어, 최대 한번에 8 또는 12 샘플에 대해 볼륨 전송을 수행하는 멀티 채널 micropipettors가 없습니다. 심지어 로봇 워크 스테이션 그 책략 96 채널 피펫 머리가있다! 이러한 노력은 실험과 관련된 기계적인 작업을 수행하기 위해 필요한 장비를 개발할 수 있습니다 엔지니어와 컴퓨터 프로그래머가 방법론적인 전문 지식을 가진 생물학 자 페어 링, 과학자의 멀티 징계 팀을 포함됩니다. 에 관계없이 연구에 응용 프로그램의 이러한 기술을 개발 회사에 의해 공동 목표는 동일 - laborato를 자동화공예 프로세스, 도구, 시스템 및 악기, 그들 집중 적은 노동과 더 효율적인.

<table><tbody><tr><td colspan="4">&lt;p class=&quot;jove_step&quot;&gt;1. Yeast Tryptone Agar (YTA)&lt;/p&gt;
&lt;table border=&quot;1&quot;&gt;
	&lt;tbody&gt;
		&lt;tr&gt;
			&lt;td&gt;Yeast extract&lt;/td&gt;
			&lt;td&gt;2.0 g&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;Tryptone&lt;/td&gt;
			&lt;td&gt;10.0 g&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;Agar&lt;/td&gt;
			&lt;td&gt;15.0 g&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;Distilled water&lt;/td&gt;
			&lt;td&gt;up to 1000.0 ml&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;&lt;/td&gt;
			&lt;td&gt;pH 7.0&lt;/td&gt;
		&lt;/tr&gt;
	&lt;/tbody&gt;
&lt;/table&gt;
&lt;p class=&quot;jove_content&quot;&gt;Autoclave at 121&amp;deg;C for 20 minutes to sterilize. Store at 4&amp;deg;C.&lt;/p&gt;
&lt;p class=&quot;jove_content&quot;&gt;If preparing tubes for the pour-plate procedure, allow the agar to cool to ~55&amp;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&amp;deg;C. The agar will solidify and will need to be melted in a steamer or microwave prior to use.&lt;/p&gt;
&lt;p class=&quot;jove_step&quot;&gt;2. Minimal Salts Agar (MSA) + 0.1% (w/v) carbon source&lt;/p&gt;
&lt;table border=&quot;1&quot;&gt;
	&lt;tbody&gt;
		&lt;tr&gt;
			&lt;td&gt;NH&lt;sub&gt;4&lt;/sub&gt;Cl&lt;/td&gt;
			&lt;td&gt;1.0 g&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;NH&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;2H&lt;sub&gt;2&lt;/sub&gt;O&lt;/td&gt;
			&lt;td&gt;2.14 g&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&lt;/td&gt;
			&lt;td&gt;1.09 g&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;7H&lt;sub&gt;2&lt;/sub&gt;O&lt;/td&gt;
			&lt;td&gt;0.2 g&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;Carbon source*&lt;/td&gt;
			&lt;td&gt;1.0 g&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;Trace salts solution**&lt;/td&gt;
			&lt;td&gt;10.0 ml&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;Agar&lt;/td&gt;
			&lt;td&gt;15.0 g&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;Distilled water&lt;/td&gt;
			&lt;td&gt;up to 1000.0 ml&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;&lt;/td&gt;
			&lt;td&gt;pH 7.0&lt;/td&gt;
		&lt;/tr&gt;
	&lt;/tbody&gt;
&lt;/table&gt;
&lt;p class=&quot;jove_content&quot;&gt;Autoclave at 121&amp;deg;C for 20 minutes to sterilize. Store at 4&amp;deg;C.&lt;/p&gt;
&lt;p class=&quot;jove_content&quot;&gt;* Carbon sources used for experiments presented in Figure 13 include acetamide, lactose, and glycine.&lt;/p&gt;
&lt;p class=&quot;jove_content&quot;&gt;** Trace salts solution is prepared in 0.1 N HCl as follows. It is added to the base before sterilization (autoclave at 121&amp;deg;C for 20 minutes).&lt;/p&gt;
&lt;table border=&quot;1&quot;&gt;
	&lt;tbody&gt;
		&lt;tr&gt;
			&lt;td&gt;FeSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;7H&lt;sub&gt;2&lt;/sub&gt;O&lt;/td&gt;
			&lt;td&gt;300.0 mg&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;MnCl&lt;sub&gt;2&lt;/sub&gt;&amp;bull;4H&lt;sub&gt;2&lt;/sub&gt;O&lt;/td&gt;
			&lt;td&gt;180.0 mg&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;Co(NO&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;&amp;bull;6H&lt;sub&gt;2&lt;/sub&gt;O&lt;/td&gt;
			&lt;td&gt;130.0 mg&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;ZnSO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;O&lt;/td&gt;
			&lt;td&gt;40.0 mg&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;H&lt;sub&gt;2&lt;/sub&gt;MoO&lt;sub&gt;4&lt;/sub&gt;&lt;/td&gt;
			&lt;td&gt;20.0 mg&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;CuSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;5H&lt;sub&gt;2&lt;/sub&gt;O&lt;/td&gt;
			&lt;td&gt;1.0 mg&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;CaCl&lt;sub&gt;2&lt;/sub&gt;&lt;/td&gt;
			&lt;td&gt;1000.0 mg&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;HCl (0.1 N)&lt;/td&gt;
			&lt;td&gt;up to 1000.0 ml&lt;/td&gt;
		&lt;/tr&gt;
	&lt;/tbody&gt;
&lt;/table&gt;
&lt;p class=&quot;jove_step&quot;&gt;3. EHA soft agar (0.65 % w/v)&lt;/p&gt;
&lt;table border=&quot;1&quot;&gt;
	&lt;tbody&gt;
		&lt;tr&gt;
			&lt;td&gt;Agar&lt;/td&gt;
			&lt;td&gt;6.5 g&lt;/td&gt;
			&lt;td&gt;&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;&lt;/td&gt;
			&lt;td&gt;Tryptone&lt;/td&gt;
			&lt;td&gt;13.0 g&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;&lt;/td&gt;
			&lt;td&gt;NaCl&lt;/td&gt;
			&lt;td&gt;8.0 g&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;&lt;/td&gt;
			&lt;td&gt;Na Citrate&amp;bull;2H&lt;sub&gt;2&lt;/sub&gt;O&lt;/td&gt;
			&lt;td&gt;2.0 g&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;&lt;/td&gt;
			&lt;td&gt;Glucose&lt;/td&gt;
			&lt;td&gt;3.0 g&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;&lt;/td&gt;
			&lt;td&gt;Distilled water&lt;/td&gt;
			&lt;td&gt;up to 1000.0 ml&lt;/td&gt;
		&lt;/tr&gt;
	&lt;/tbody&gt;
&lt;/table&gt;
&lt;p class=&quot;jove_content&quot;&gt;Autoclave at 121&amp;deg;C for 20 minutes to sterilize. Store at 4&amp;deg;C.&lt;/p&gt;
&lt;p class=&quot;jove_step&quot;&gt;4. EHA hard agar (1.2% w/v)&lt;/p&gt;
&lt;table border=&quot;1&quot;&gt;
	&lt;tbody&gt;
		&lt;tr&gt;
			&lt;td&gt;Agar&lt;/td&gt;
			&lt;td&gt;12.0 g&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;Tryptone&lt;/td&gt;
			&lt;td&gt;13.0 g&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;NaCl&lt;/td&gt;
			&lt;td&gt; 8.0 g&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;Na Citrate&amp;bull;2H&lt;sub&gt;2&lt;/sub&gt;O&lt;/td&gt;
			&lt;td&gt; 2.0 g&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;Glucose&lt;/td&gt;
			&lt;td&gt; 3.0 g&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;Distilled water&lt;/td&gt;
			&lt;td&gt;up to 1000.0 ml&lt;/td&gt;
		&lt;/tr&gt;
	&lt;/tbody&gt;
&lt;/table&gt;
&lt;p class=&quot;jove_content&quot;&gt;Autoclave at 121&amp;deg;C for 20 minutes to sterilize. Store at 4&amp;deg;C.&lt;/p&gt;
&lt;p class=&quot;jove_step&quot;&gt;5. 1X Middlebrook Top Agar (MBTA soft agar, 0.5% w/v)&lt;/p&gt;
&lt;table border=&quot;1&quot;&gt;
	&lt;tbody&gt;
		&lt;tr&gt;
			&lt;td&gt;100 mM CaCl&lt;sub&gt;2&lt;/sub&gt; stock*&lt;/td&gt;
			&lt;td&gt; 1 ml&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;7H9 liquid medium: &lt;em&gt;Neat &lt;/em&gt;**&lt;/td&gt;
			&lt;td&gt;50 ml&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;2XTA ***&lt;/td&gt;
			&lt;td&gt;50 ml&lt;/td&gt;
		&lt;/tr&gt;
	&lt;/tbody&gt;
&lt;/table&gt;
&lt;p class=&quot;jove_content&quot;&gt;Melt 50 ml of 2XTA and allow it to cool to ~55&amp;deg;C. Using aseptic technique, add the CaCl&lt;sub&gt;2&lt;/sub&gt; and 7H9 broth to the melted agar. Aseptically dispense 4.5 ml of the mixture per 13 mm tube and store in a 55&amp;deg;C incubator &amp;#8804;7 days. Cooling MBTA to room temperature or 4&amp;deg;C will cause the CaCl&lt;sub&gt;2&lt;/sub&gt; to precipitate out of solution.&lt;/p&gt;
&lt;p class=&quot;jove_content&quot;&gt;* 100 mM CaCl&lt;sub&gt;2&lt;/sub&gt;. stock must be stored at room temperature to prevent CaCl&lt;sub&gt;2&lt;/sub&gt; from precipitating out of solution.&lt;/p&gt;
&lt;p class=&quot;jove_content&quot;&gt;** 7H9 liquid medium: Neat&lt;/p&gt;
&lt;table border=&quot;1&quot;&gt;
	&lt;tbody&gt;
		&lt;tr&gt;
			&lt;td&gt;7H9 broth base&lt;/td&gt;
			&lt;td&gt;4.7 g&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;40% glycerol stock&lt;/td&gt;
			&lt;td&gt;5 ml&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;Distilled water&lt;/td&gt;
			&lt;td&gt;up to 900.0 ml&lt;/td&gt;
		&lt;/tr&gt;
	&lt;/tbody&gt;
&lt;/table&gt;
&lt;p class=&quot;jove_content&quot;&gt;Mix the base with water then add the glycerol while stirring. Autoclave at 121&amp;deg;C for 20 minutes to sterilize. Store at 4&amp;deg;C.&lt;/p&gt;
&lt;p class=&quot;jove_content&quot;&gt;*** 2X Middlebrook Top Agar (2XTA, 1.0% w/v)&lt;/p&gt;
&lt;table border=&quot;1&quot;&gt;
	&lt;tbody&gt;
		&lt;tr&gt;
			&lt;td&gt;7H9 broth base&lt;/td&gt;
			&lt;td&gt;4.7 g&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;Agar&lt;/td&gt;
			&lt;td&gt;1.0 g&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;Distilled water&lt;/td&gt;
			&lt;td&gt;up to 1000.0 ml &lt;/td&gt;
		&lt;/tr&gt;
	&lt;/tbody&gt;
&lt;/table&gt;
&lt;p class=&quot;jove_content&quot;&gt;Autoclave at 121&amp;deg;C for 20 minutes to sterilize. Dispense 50 ml aliquots into 100 ml bottles and store at 4&amp;deg;C. &lt;/p&gt;
&lt;p class=&quot;jove_step&quot;&gt;6. Middlebrook 7H10 Agar Plates (MHA hard agar, 1.9% w/v)&lt;/p&gt;
&lt;table border=&quot;1&quot;&gt;
	&lt;tbody&gt;
		&lt;tr&gt;
			&lt;td&gt;7H10 agar base&lt;/td&gt;
			&lt;td&gt;19.0 g&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;40% glycerol stock&lt;/td&gt;
			&lt;td&gt;12.5 ml&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;Distilled water&lt;/td&gt;
			&lt;td&gt;887.5 ml&lt;/td&gt;
		&lt;/tr&gt;
	&lt;/tbody&gt;
&lt;/table&gt;
&lt;p class=&quot;jove_content&quot;&gt;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&amp;deg;C for 20 minutes to sterilize. Allow the agar to cool to ~55&amp;deg;C then aseptically add the following reagents:&lt;/p&gt;
&lt;table border=&quot;1&quot;&gt;
	&lt;tbody&gt;
		&lt;tr&gt;
			&lt;td&gt;AD supplement (pre-warmed to 37&amp;deg;C)*&lt;/td&gt;
			&lt;td&gt;100 ml&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;50 mg/ml Carbenicillin **&lt;/td&gt;
			&lt;td&gt;1.0 ml&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;10 mg/ml Cycloheximide **&lt;/td&gt;
			&lt;td&gt;1.0 ml&lt;/td&gt;
		&lt;/tr&gt;
	&lt;/tbody&gt;
&lt;/table&gt;
&lt;p class=&quot;jove_content&quot;&gt;* AD supplement&lt;/p&gt;
&lt;table border=&quot;1&quot;&gt;
	&lt;tbody&gt;
		&lt;tr&gt;
			&lt;td&gt;NaCl &amp;gt;&lt;/td&gt;
			&lt;td&gt;17 g &amp;gt;&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;Albumin (Fraction V)&amp;gt;&lt;/td&gt;
			&lt;td&gt;100 g&amp;gt;&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;Dextrose (&lt;em&gt;D&lt;/em&gt;-Glucose)&amp;gt;&lt;/td&gt;
			&lt;td&gt;40 g&amp;gt;&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;Distilled water&amp;gt;&lt;/td&gt;
			&lt;td&gt;up to 2000.0 ml&amp;gt;&lt;/td&gt;
		&lt;/tr&gt;
	&lt;/tbody&gt;
&lt;/table&gt;
&lt;p class=&quot;jove_content&quot;&gt;Filter-sterilize this solution; do not autoclave. Store at 4&amp;deg;C.&lt;/p&gt;
&lt;p class=&quot;jove_content&quot;&gt;** Filter-sterilize and store these solutions at 4&amp;deg;C for &amp;le;60 days.&lt;/p&gt;
&lt;p class=&quot;jove_step&quot;&gt;7. LB agar (1.5% w/v) + X-Gal (60 &amp;mu;g/ml)&lt;/p&gt;
&lt;table border=&quot;1&quot;&gt;
	&lt;tbody&gt;
		&lt;tr&gt;
			&lt;td&gt;Tryptone&lt;/td&gt;
			&lt;td&gt;10.0 g&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;Yeast extract&lt;/td&gt;
			&lt;td&gt;5.0 g&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;NaCl&lt;/td&gt;
			&lt;td&gt;10.0 g&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;Agar&lt;/td&gt;
			&lt;td&gt;15.0 g&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;Distilled water&lt;/td&gt;
			&lt;td&gt;up to 1000.0 ml&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;&lt;/td&gt;
			&lt;td&gt;pH 7.5 at 25&amp;deg;C&lt;/td&gt;
		&lt;/tr&gt;
	&lt;/tbody&gt;
&lt;/table&gt;
&lt;p class=&quot;jove_content&quot;&gt;Autoclave at 121&amp;deg;C for 20 minutes to sterilize. Allow the agar to cool to ~55&amp;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). &lt;/p&gt;
&lt;p class=&quot;jove_step&quot;&gt;Table of specific reagents:&lt;/p&gt;
&lt;table border=&quot;1&quot;&gt;
	&lt;tbody&gt;
		&lt;tr&gt;
			&lt;td&gt;&lt;strong&gt;Name of the reagent&lt;/strong&gt;&lt;/td&gt;
			&lt;td&gt;&lt;strong&gt;Company&lt;/strong&gt;&lt;/td&gt;
			&lt;td&gt;&lt;strong&gt;Catalogue number&lt;/strong&gt;&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;Yeast extract&lt;/td&gt;
			&lt;td&gt;Becton Dickenson&lt;/td&gt;
			&lt;td&gt;212750&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;Tryptone&lt;/td&gt;
			&lt;td&gt;Becton Dickenson&lt;/td&gt;
			&lt;td&gt;211705&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;Agar&lt;/td&gt;
			&lt;td&gt;Becton Dickenson&lt;/td&gt;
			&lt;td&gt;214030&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;NH&lt;sub&gt;4&lt;/sub&gt;Cl&lt;/td&gt;
			&lt;td&gt;Acros Organics&lt;/td&gt;
			&lt;td&gt;123340010&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;NH&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;2H&lt;sub&gt;2&lt;/sub&gt;O&lt;/td&gt;
			&lt;td&gt;Sigma-Aldrich&lt;/td&gt;
			&lt;td&gt;30435&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&lt;/td&gt;
			&lt;td&gt;Fisher Scientific&lt;/td&gt;
			&lt;td&gt;BP 303-500&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;7H&lt;sub&gt;2&lt;/sub&gt;O&lt;/td&gt;
			&lt;td&gt;Sigma-Aldrich&lt;/td&gt;
			&lt;td&gt;230391 &lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;Acetamide&lt;/td&gt;
			&lt;td&gt;Sigma-Aldrich &lt;/td&gt;
			&lt;td&gt;A-0500&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;Lactose&lt;/td&gt;
			&lt;td&gt;Fisher Scientific &lt;/td&gt;
			&lt;td&gt;L6-500 &lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;Glycine&lt;/td&gt;
			&lt;td&gt;Sigma-Aldrich&lt;/td&gt;
			&lt;td&gt;G-7126&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;FeSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;7H&lt;sub&gt;2&lt;/sub&gt;O&lt;/td&gt;
			&lt;td&gt;Sigma-Aldrich&lt;/td&gt;
			&lt;td&gt;F8048&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;MnCl&lt;sub&gt;2&lt;/sub&gt;&amp;bull;4H&lt;sub&gt;2&lt;/sub&gt;O&lt;/td&gt;
			&lt;td&gt;Sigma-Aldrich&lt;/td&gt;
			&lt;td&gt;M-3634&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;Co(NO&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;&amp;bull;6H&lt;sub&gt;2&lt;/sub&gt;O&lt;/td&gt;
			&lt;td&gt;Sigma-Aldrich&lt;/td&gt;
			&lt;td&gt;230375&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;ZnSO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;O&lt;/td&gt;
			&lt;td&gt;Sigma-Aldrich&lt;/td&gt;
			&lt;td&gt;Z4750&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;H&lt;sub&gt;2&lt;/sub&gt;MoO&lt;sub&gt;4&lt;/sub&gt;&lt;/td&gt;
			&lt;td&gt;Acros Organics&lt;/td&gt;
			&lt;td&gt;213621000&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;CuSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;5H&lt;sub&gt;2&lt;/sub&gt;O&lt;/td&gt;
			&lt;td&gt;Sigma-Aldrich&lt;/td&gt;
			&lt;td&gt;209198 &lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;CaCl&lt;sub&gt;2&lt;/sub&gt;&lt;/td&gt;
			&lt;td&gt;Sigma-Aldrich&lt;/td&gt;
			&lt;td&gt;C1016 &lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;HCl&lt;/td&gt;
			&lt;td&gt;Fisher Scientific&lt;/td&gt;
			&lt;td&gt;A144-212&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;Cycloheximide&lt;/td&gt;
			&lt;td&gt;Sigma&lt;/td&gt;
			&lt;td&gt;038K1561&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;Carbenicillin&lt;/td&gt;
			&lt;td&gt;Cellgro&lt;/td&gt;
			&lt;td&gt;46-100-RG&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;NaCl&lt;/td&gt;
			&lt;td&gt;Fisher&lt;/td&gt;
			&lt;td&gt;S271-1&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;Na Citrate&amp;bull;2H&lt;sub&gt;2&lt;/sub&gt;O&lt;/td&gt;
			&lt;td&gt;Fisher&lt;/td&gt;
			&lt;td&gt;S279-500&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;Glucose (Dextrose)&lt;/td&gt;
			&lt;td&gt;BD&lt;/td&gt;
			&lt;td&gt;215530&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;7H9 broth base&lt;/td&gt;
			&lt;td&gt;BD&lt;/td&gt;
			&lt;td&gt;271310&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;7H10 agar base&lt;/td&gt;
			&lt;td&gt;BD&lt;/td&gt;
			&lt;td&gt;262710&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;Glycerol&lt;/td&gt;
			&lt;td&gt;Shelton&lt;/td&gt;
			&lt;td&gt;IB15760&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;Albumin (Fraction V)&lt;/td&gt;
			&lt;td&gt;Fisher&lt;/td&gt;
			&lt;td&gt;S71907&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;X-Gal (5-bromo-4-chloro-3-indolyl-&amp;#946;-D-galactopyranoside) &lt;/td&gt;
			&lt;td&gt;Teknova&lt;/td&gt;
			&lt;td&gt;X1205&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;Dimethylformamide (DMF)&lt;/td&gt;
			&lt;td&gt;Sigma&lt;/td&gt;
			&lt;td&gt;D4551&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;Ethanol&lt;/td&gt;
			&lt;td&gt;Fisher&lt;/td&gt;
			&lt;td&gt;CDA19&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;Chlorine Bleach&lt;/td&gt;
			&lt;td&gt;Chlorox&lt;/td&gt;
			&lt;td&gt;02490/06644884&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;CiDecon (disinfectant)&lt;/td&gt;
			&lt;td&gt;Decon Laboratories, Inc.&lt;/td&gt;
			&lt;td&gt;8504&lt;/td&gt;
		&lt;/tr&gt;
	&lt;/tbody&gt;
&lt;/table&gt;
&lt;p class=&quot;jove_step&quot;&gt;Table of specific equipment:&lt;/p&gt;
&lt;table border=&quot;1&quot;&gt;
	&lt;tbody&gt;
		&lt;tr&gt;
			&lt;td&gt;&lt;strong&gt;Name of equipment&lt;/strong&gt;&lt;/td&gt;
			&lt;td&gt;&lt;strong&gt;Company&lt;/strong&gt;&lt;/td&gt;
			&lt;td&gt;&lt;strong&gt;Catalogue number&lt;/strong&gt;&lt;/td&gt;
			&lt;td&gt;&lt;strong&gt;Experiment&lt;/strong&gt;&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;Metal loops&lt;/td&gt;
			&lt;td&gt;American Educational Products&lt;/td&gt;
			&lt;td&gt;S17352&lt;/td&gt;
			&lt;td&gt;Streak plating&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;Disposable plastic loops&lt;/td&gt;
			&lt;td&gt;Fisher&lt;/td&gt;
			&lt;td&gt;22-363-602&lt;/td&gt;
			&lt;td&gt;Streak plating&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;Wooden sticks&lt;/td&gt;
			&lt;td&gt;Fisher&lt;/td&gt;
			&lt;td&gt;23-400-104&lt;/td&gt;
			&lt;td&gt;Streak plating&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;Flat Toothpicks&lt;/td&gt;
			&lt;td&gt;American Educational Products&lt;/td&gt;
			&lt;td&gt;S67859&lt;/td&gt;
			&lt;td&gt;Streak plating&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;Turn tables&lt;/td&gt;
			&lt;td&gt;Fisher&lt;/td&gt;
			&lt;td&gt;08-758Q&lt;/td&gt;
			&lt;td&gt;Spread plating&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;Glass rods&lt;/td&gt;
			&lt;td&gt;Bellco Glass&lt;/td&gt;
			&lt;td&gt;NC9004380&lt;/td&gt;
			&lt;td&gt;Spread plating&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;4 mm glass beads&lt;/td&gt;
			&lt;td&gt;Fisher&lt;/td&gt;
			&lt;td&gt;11-312B&lt;/td&gt;
			&lt;td&gt;Spread plating&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;Velveteen cloth&lt;/td&gt;
			&lt;td&gt;Bel-Art Products&lt;/td&gt;
			&lt;td&gt;09-718-2&lt;/td&gt;
			&lt;td&gt;Replica plating&lt;/td&gt;
		&lt;/tr&gt;
		&lt;tr&gt;
			&lt;td&gt;Cylindrical block&lt;/td&gt;
			&lt;td&gt;Bel-Art Products&lt;/td&gt;
			&lt;td&gt;09-718-1&lt;/td&gt;
			&lt;td&gt;Replica plating&lt;/td&gt;
		&lt;/tr&gt;
	&lt;/tbody&gt;
&lt;/table&gt;</td></tr></tbody></table>

aseptic laboratory techniques: plating methods

미생물 실험실에서 가능한 오염의 유비 쿼터스 소스를 만들고 모든 생명이없는 표면에 존재한다. 실험 성공은 작업 표면 및 장비를 소독뿐만 아니라 비 멸균 표면 살균 장비와 솔루션의 접촉을 방지하기 위해 과학자의 능력에 의존하고 있습니다. 여기에서 우리는 정기적으로, 고립 전파, 또는 세균과 파지와 같은 미생물을 열거하기 위해 실험실에서 사용되는 여러 가지 도금 방법에 대한 단계를 제시한다. 다섯 가지 방법 무균 기술, 또는 실험 재료의 불임을 유지 절차를 포함. 설명된 절차는 단일 콜로니를 분리하기 (1) 탄력 - 도금 세균성 문화, (2) 붓다 - 도금 (3) 확산 도금 가능한 세균성 식민지를 열거하기 위해, 파지 및 열거 plaques를 분리하기 (4) 부드러운 한천 오버레이, 및 (포함 5) 복제 - 도금을 한 판에서 동일한 공간적 패턴에서 다른 세포를 전송할 수 있습니다. 이러한 절차는 t에서 수행할 수그는 실험실 벤치는 그들이 미생물의 비 병원성 변종을 (Biosafety 레벨 1, BSL-1) 참여 제공. BSL-2 생물과 협력한다면, 이러한 조작은 biosafety 캐비닛에서 개최한다. 생물 학적 분류는 물론 문제의 미생물에 필요한 안전 조치 및 억제 시설을 결정하는 감염성 물질에 대한 최신 미생물 및 바이오 메디컬 연구소의 Biosafety의 판 (BMBL)뿐만 아니라 재료 안전 데이터 시트 (MSDS)를 참조하십시오. 박테리아 변종과 파지 주식 같은 미국 유형 문화 수집 (ATCC)와 같은 특정 단체에 의해 관리 연구 조사관, 회사 및 컬렉션에서 얻을 수있다. 그것은 여러 가지 도금 방법을 배우고 비 병원성 종자를 사용하는 것이 좋습니다. 이 프로토콜에서 설명한 절차에 따라, 학생들은 수 있어야합니다 : <ul><li> contaminati없이 도금 절차를 수행NG 미디어. </li><li> 줄무늬-도금 방법으로 단일 세균성 식민지를 분리. </li><li> 박테리아의 농도를 결정 부어 - 도금 및 확산 도금 방법을 사용합니다. </li><li> 파지 작업시 부드러운 한천 오버레이를 수행합니다. </li><li> 한 접시의 복제 - 도금 절차를 사용하여 다른 세균 세포를 전송합니다. </li><li> 실험적인 작업을 감안할 때, 적절한 도금 방법을 선택합니다. </li></ul>

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

Aseptic Laboratory Techniques: Plating Methods

문화 미생물에 사용되는 미디어 및 시약과 함께 작업할 경우 무균 기술은 오염을 최소화하기 위해 실천해야합니다. 도금 방법의 다양한 정기적으로, 고립 전파, 또는 열거 세균 및 파지, 실험 재료의 불임을 유지 절차를 포함 모두의하는 데 사용됩니다.

무균 실험실 기술 : 도금 방법

Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies ...

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727.9K 조회수.  University of California, Los Angeles. 문화 미생물에 사용되는 미디어 및 시약과 함께 작업할 경우 무균 기술은 오염을 최소화하기 위해 실천해야합니다. 도금 방법의 다양한 정기적으로, 고립 전파, 또는 열거 세균 및 파지, 실험 재료의 불임을 유지 절차를 포함 모두의하는 데 사용됩니다.

비디오: Aseptic Laboratory Techniques: Plating Methods

Quantifying Bacterial Surface Swarming Motility on Inducer Gradient Plates

Bacterial swarming motility is a common microbiological phenotype that bacterial communities use to migrate over semisolid surfaces. In investigations of induced swarming motility, specific concentration&#160;of an inducer may not be able to report events occurring within the optimal concentration range to elicit the desired responses from a species. Semisolid plates containing multiple concentrations are commonly used to investigate the response within an inducer concentration range. However, separate semisolid plates increase variations in medium viscosity and moisture content within each plate due to nonuniform solidification time.
This paper describes a one-step method to simultaneously test surface swarming motility on a single gradient plate, where the isometrically arranged test wells allow the simultaneous acquisition of multiconcentration responses. In the present work, the surface swarming of Escherichia coli K12 and Pseudomonas aeruginosa PAO1 were evaluated in response to a concentration gradient of inducers such as resveratrol and arabinose. Periodically, the swarm morphologies were imaged using an imaging system to capture the entire surface swarming process.
The quantitative measurement of the swarm morphologies was acquired using ImageJ software, providing analyzable information of the swarm area. This paper presents a simple gradient swarm plate method that provides qualitative and quantitative information about the inducers&#39; effects on surface swarming, which can be extended to study the effects of other inducers on a broader range of motile bacterial species.

Here, we describe the use of inducer gradient plates to evaluate bacterial swarming motility while simultaneously obtaining multiple concentration responses.

유도기 그라디언트 플레이트의 박테리아 표면 떼 운동성 정량화

Bacterial swarming motility is a common microbiological phenotype that bacterial communities use to migrate over semisolid surfaces. In investigations of ...

연구

생화학

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global prevalence and severity of this disease, V. cholerae has been extensively studied in both environmental and laboratory settings, requiring proper maintenance and culturing techniques. Classical and El Tor are two main biotypes that compose the V. cholerae O1 serogroup, each displaying unique genotypic and phenotypic characteristics that provide reliable mechanisms for biotype characterization, and require distinct virulence inducing culturing conditions. Regardless of the biotype of the causative strain for any given infection or outbreak, the standard treatment for the disease involves rehydration therapy supplemented with a regimen of antibiotics. However, biotype classification may be necessary for laboratory studies and may have broader impacts in the biomedical field. In the early 2000&#39;s clinical isolates were identified which exhibit genotypic and phenotypic traits from both classical and El Tor biotypes. The newly identified hybrids, termed El Tor variants, have caused clinical and environmental isolate biotype identification to become more complex than previous traditional single assay identification protocols. In addition to describing V. cholerae general maintenance and culturing techniques, this manuscript describes a series of gene specific (ctxB and tcpA) PCR-based genetic screens and phenotypic assays (polymyxin B resistance, citrate metabolism, proteolytic activity, hemolytic activity, motility, and glucose metabolism via Voges-Proskauer assay) collectively used to characterize and/or distinguish between classical and El Tor biotypes. Together, these assays provide an efficient systematic approach to be used as an alternative, or in addition, to costly, labor-intensive experiments in the characterization of V. cholerae clinical (and environmental) isolates.

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

This manuscript describes proper Vibrio cholerae maintenance techniques in addition to a series of biochemical assays, collectively utilized for quick and reliable differentiation between clinical and environmental V. cholerae biotypes in a laboratory setting.

생물학적 유형을 유지하고 차별화하기 위해 사용되는 실험실 기술<em&gt; 콜레라 비브리오</em&gt; 임상 적 및 환경 적 분리 물

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global ...

면역학 및 감염병학

Aseptic Laboratory Techniques: Volume Transfers with Serological Pipettes and Micropipettors

Microorganisms are everywhere - in the air, soil, and human body as well as on inanimate surfaces like laboratory benches and computer keyboards. The ubiquity of microbes creates a copious supply of potential contaminants in a laboratory. To ensure experimental success, the number of contaminants on equipment and work surfaces must be minimized. Common among many experiments in microbiology are techniques involving the measurement and transfer of cultures containing bacterial cells or viral particles. To do so without contacting non-sterile surfaces or contaminating sterile media requires (1) preparing a sterile workspace, (2) precisely setting and accurately reading instruments for aseptic transfer of liquids, and (3) properly manipulating instruments, cultures flasks, bottles and tubes within a sterile field. Learning these procedures calls for training and practice. At first, actions should be slow, deliberate, and controlled with the goal being for aseptic technique to become second nature when working at the bench. Here we present the steps for measuring volumes using serological pipettes and micropipettors within a sterile field created by a Bunsen burner. Volumes range from microliters (&mu;l) to milliliters (ml) depending on the instrument used. Liquids commonly transferred include sterile broth or chemical solutions as well as bacterial cultures and phage stocks. By following these procedures, students should be able to: &#8226;Work within the sterile field created by the Bunsen burner flame. &#8226;Use serological pipettes without compromising instrument sterility.&#8226; Aspirate liquids with serological pipettes, precisely reading calibrated volumes by aligning the meniscus formed by the liquid to the graduation marks on the pipette. &#8226;Keep culture bottles, flasks, tubes and their respective caps sterile during liquid transfers. &#8226;Identify different applications for plastic versus glass serological pipettes. &#8226;State accuracy limitations for micropipettors. &#8226;Precisely and accurately set volumes on micropipettors. &#8226;Know how to properly use the first and second stop on a micropipettor to aspirate and transfer correct volumes.

When working in a laboratory, it is imperative to minimize sources of contamination. Aseptic technique refers to procedures that permit transfer of cultures and reagents while avoiding contact with non-sterile surfaces. Serological pipettes and micropipettors are used to measure precise volumes without compromising sterility of solutions used in experiments.

무균 실험실 기술 : Serological Pipettes 및 Micropipettors로 볼륨 이동

Microorganisms are everywhere - in the air, soil, and human body as well as on inanimate surfaces like laboratory benches and computer keyboards. The ...

생물학

Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, a significant stress response is induced during gastrointestinal transit and upon bile exposure, a normal component of human digestion. To overcome the bactericidal effects of bile, many enteric pathogens form a biofilm hypothesized to permit survival when transiting through the small intestine. Here we present methodologies to define biofilm formation through solid-phase adherence assays as well as extracellular polymeric substance (EPS) matrix detection and visualization. Furthermore, biofilm dispersion assessment is presented to mimic the analysis of events triggering release of bacteria during the infection process. Crystal violet staining is used to detect adherent bacteria in a high-throughput 96-well plate adherence assay. EPS production assessment is determined by two assays, namely microscopy staining of the EPS matrix and semi-quantitative analysis with a fluorescently-conjugated polysaccharide binding lectin. Finally, biofilm dispersion is measured through colony counts and plating. Positive data from multiple assays support the characterization of biofilms and can be utilized to identify bile salt-induced biofilm formation in other bacterial strains.

This protocol enables the reader to analyze bile salt-induced biofilm formation in enteric pathogens using a multifaceted approach to capture the dynamic nature of bacterial biofilms by assessing adherence, extracellular polymeric substance matrix formation, and dispersion.

담 즙 소금 유도 Biofilm 형성 장 병원 체에서: 식별 및 정량화 기술

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, ...

Phage-Mediated Genetic Manipulation of the Lyme Disease Spirochete Borrelia burgdorferi

Introducing foreign DNA into the spirochete Borrelia burgdorferi has been almost exclusively accomplished by transformation using electroporation. This process has notably lower efficiencies in the Lyme disease spirochete relative to other, better-characterized Gram-negative bacteria. The rate of success of transformation is highly dependent upon having concentrated amounts of high-quality DNA from specific backgrounds and is subject to significant strain-to-strain variability. Alternative means for introducing foreign DNA (i.e., shuttle vectors, fluorescent reporters, and antibiotic-resistance markers) into B. burgdorferi could be an important addition to the armamentarium of useful tools for the genetic manipulation of the Lyme disease spirochete. Bacteriophage have been well-recognized as natural mechanisms for the movement of DNA among bacteria in a process called transduction. In this study, a method has been developed for using the ubiquitous borrelial phage &#966;BB-1 to transduce DNA between B. burgdorferi cells of both the same and different genetic backgrounds. The transduced DNA includes both borrelial DNA and heterologous DNA in the form of small shuttle vectors. This demonstration suggests a potential use of phage-mediated transduction as a complement to electroporation for the genetic manipulation of the Lyme disease spirochete. This report describes methods for the induction and purification of phage &#966;BB-1 from B. burgdorferi, the use of this phage in transduction assays, and the selection and screening of potential transductants.

Phage-Mediated Genetic Manipulation of the Lyme Disease Spirochete Borrelia burgdorferi

The ability of bacteriophage to move DNA between bacterial cells makes them effective tools for the genetic manipulation of their bacterial hosts. Presented here is a methodology for inducing, recovering, and using &#966;BB-1, a bacteriophage of Borrelia burgdorferi, to transduce heterologous DNA between different strains of the Lyme disease spirochete.

라임병 스피로체테 보렐리아 부르그도르페리의 파지 매개 유전자 조작

Introducing foreign DNA into the spirochete Borrelia burgdorferi has been almost exclusively accomplished by transformation using electroporation. This ...

Serial Dilutions and Plating: Microbial Enumeration

Source: Jonathan F. Blaize1, Elizabeth Suter1, and Christopher P. Corbo1 
1 Department of Biological Sciences, Wagner College, 1 Campus Road, Staten Island NY, 10301
Quantitative assessment of prokaryotes can be onerous given their abundance, propensity for exponential proliferation, species diversity within a population, and specific physiological needs. Compounding this challenge, is the four-phase nature in which bacteria replicate (lag, log, stationary and death). The ability to accurately estimate microorganism concentration is necessary for successful identification, isolation, cultivation, and characterization (6). As such, microbiologists have employed serial dilution and various plating techniques for over a century to reliably quantify bacterial and viral load in clinical, industrial, pharmaceutical, and academic laboratory environments (2,4,6). Descriptions of this methodology first appeared in 1883 when the German scientist and physician Robert Koch published his work on infectious disease-causing agents (2). Often referred to as the father of modern bacteriology, Koch&#39;s forenamed techniques have become the gold standard for enumeration of microorganisms, culturable or otherwise, throughout the world.
Serial dilution is a systematic reduction of a known or unknown entity (a solute, organism, etc.) through successive re-suspension of an initial solution (solution0) into fixed volumes of a liquid diluent (blanks). These blanks usually consist of 0.45% saline, though the composition can be varied (7). While an experimenter can choose any volume for each diluent, it is most often a multiple of 10, facilitating logarithmic reduction of the sample. For example, solution0 contains a total of 100 E. coli cells suspended in 10 mL of nutrient broth. If 1 mL of solution0 is removed and added to 9 mL of saline (diluent1), the new solution (solution1) would contain 1/10th of the initial concentration of E. coli. In this example, the new solution (solution1) would contain 10 E. coli cells. Repeating this process by removing 1 mL of solution1 and adding it to another 9 mL of saline (diluent2) would yield solution2, containing only a single E. coli cell. Since each new solution (9 mL of diluent + 1 mL of solution) contains a total of 10mL, we can conclude that the dilution factor for this reduction is 10 or that this was a 10-fold serial dilution (Figure 1). Since we only began with 100 cells in this example and we are diluting by a factor of 10, only two steps are required to reach the absolute minimum concentration of 1 cell.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10507/10507fig1.jpg" /> 
Figure 1: Serial dilution of a stock solution. A 1 mL aliquot of the stock solution (solution0) is added to tube 1 which contains 9 mL of 0.45% saline (dilent1); the product of this mixture is solution1. Repeat by aliquoting 1 mL of the newly created solution1 and adding it to tube 2. Aliquoting and resuspension continues in this fashion until the final tube is reached, diluting the stock concentration by a factor of 10 each with each step. <a href="https://www.jove.com/files/ftp_upload/10507/10507fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Serial dilution is the simplest technique for obtaining manageable concentrations of a desired organism and it is complemented by petri dish streaking and spreading, just two of many plating techniques used by microbiologists. This benefit of this approach is that the experimenter can harvest pure strains of a single species or separate strains from a mixed population (7). Streaking is accomplished by introducing an organism to a solid medium (generally consisting of agarose) that it will grow upon if the appropriate nutrients are available. Gently sweeping a sterile inoculating loop across the medium (so that a subtle streak remains) in a rigid sinusoidal pattern will distribute the organism proportionally to the frequency of the experimenter&#39;s waveform. Dividing the Petri dish into thirds or fourths (quadrant streak) and decreasing the frequency of each streak as a new region of the dish is entered will gradually reduce the number of microorganisms that can occupy that region, producing single colonies instead of an unquantifiable bacterial lawn. Spread plating does not additionally dilute samples; a sterile glass spreader is used to distribute an aliquot of suspension media across an entire petri dish (Figure 2). The colonies that grow on the spread plate arise from a single cell and each colony on the dish can be counted to estimate the number of colony forming units per milliliter (CFU) in a given suspension, represented as CFU/mL (6) (Figure 3) Soft agar and replica plating are variations of the aforementioned techniques and allow for isolation of bacteriophage and mutant screening, respectively (1,7).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/10507/10507fig2.jpg" /> 
Figure 2: Plate streaking for bacterial enumeration and strain isolation. Label the bottom of a petri dish with identification information (bacteria name, date, media) and divide into thirds. After selecting an appropriate dilution of the stock sample, take a sterile (disposable or flamed) inoculating loop and submerge it the test tube (here, T3). Slightly raise the petri dish cover on one side so that only the inoculating loop can access the agar. Glide the inoculating loop across the top of the media in a zig-zag fashion being careful not to compromise the agar. Rotate the plate by roughly 1/3rd (~118&#176;) and reduce the frequency of zig-zag motion. Rotate a final time and reduce zig-zag frequency once more. <a href="https://www.jove.com/files/ftp_upload/10507/10507fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/10507/10507fig3.jpg" /> 
Figure 3: Spread plating. 1 g of the aerobic zone was resuspended in T1 and then serially diluted. A sterile glass or plastic disposable spreading rod is used to distribute inoculum throughout each dish. This was repeated with 1 g of the anaerobic zone. <a href="https://www.jove.com/files/ftp_upload/10507/10507fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
As with serial dilutions, a logarithmic scale is employed to express organismal concentration. The number of colonies grown in standard petri dishes measuring 100mm x15mm can be enumerated manually (or automated with the aid of computational processing) by identifying isolated clusters of growth. Counts that total fewer than 30 or greater than 300 should be defined as too few to count (TFTC) or too numerous to count (TNTC), respectively. In the case of the latter, a serial dilution should be performed to reduce concentration before restreaking a new petri dish. Averaging the number of self-contained colonies identified from three separate petri dishes and multiplying the mean by the dilution factor will yield CFU/mL; plotting the log10 of CFU/mL against time will reveal the mean generation time of the organism (7).

연속 희석 및 플레이팅: 미생물 나열

Source: Jonathan F. Blaize1, Elizabeth Suter1, and Christopher P. Corbo1
1 Department of Biological Sciences, Wagner College, 1 Campus Road, Staten Island ...

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심화생물학

미생물학

Aseptic Technique in Environmental Science

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - The University of Arizona 
Demonstrating Author: Luisa Ikner
Aseptic technique is a fundamental skill widely practiced in the field of environmental microbiology that requires a balance of mindfulness and practice in the laboratory. Proper use of this technique reduces the likelihood of bacterial or fungal contamination of reagents, culture media, and environmental samples. Aseptic technique is also vital to ensure data integrity and maintain the purity of culture libraries that may be comprised of very rare and difficult to culture isolates. Sources of contamination in the laboratory environment include airborne microorganisms (including those adhering to dust and lint particles), microbes present on the laboratory bench workspace or on unsterilized glassware or equipment, and microbes transferred from the body and hair of the researcher. The use of aseptic technique is also a safety measure that lowers the potential for the transmission of microorganisms to researchers, which is particularly important when working with pathogens.

환경 과학의 무균 기술

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - The University of Arizona
Demonstrating Author: Luisa Ikner
Aseptic technique is a ...

환경 과학

환경 미생물학

Enrichment Cultures: Culturing Aerobic and Anaerobic Microbes on Selective and Differential Medias

Source: Christopher P. Corbo1, Jonathan F. Blaize1, Elizabeth Suter1 
1 Department of Biological Sciences, Wagner College, 1 Campus Road, Staten Island NY, 10301
Prokaryotic cells are able to inhabit nearly every environment on this planet. As a kingdom, they possess a great metabolic diversity, allowing them to use a wide variety of molecules for energy generation (1). Therefore, when cultivating these organisms in the lab, all necessary and specific molecules required to make energy must be provided in the growth media. While some organisms are metabolically diverse, others are able to survive in extreme environments such as high or low temperatures, alkaline and acidic pH, reduced or oxygen absent environments, or environments containing high salt (2,3,4). Termed &#34;extremophiles&#34;, these organisms often require these intense environments to proliferate. When scientists look to grow such organisms, the media components as well as any specific environmental conditions all need to be taken into account in order to successfully cultivate the organisms of interest.
Scientists are able to grow culturable organisms in the lab because they understand the specific requirements that those species need to grow. However, culturable organisms account for less than 1% of species estimated to be on the planet (5). Organisms that we have detected by gene sequencing but are not able to grow in the lab are considered unculturable (6). At this time, we do not know enough about the metabolism and growth conditions of these organisms to replicate their environment in the lab.
Fastidious organisms lie somewhere in between the former two. These organisms are culturable, but they require very specific growth conditions, such as specific growth media components and/or specific growth conditions. Two examples of such genera are Neisseria sp. and Haemophilus sp., both of which require partially broken-down red blood cells (also known as chocolate agar), as well as specific growth factors and an environment rich in carbon dioxide (7). Without all of the required specific components, these organisms will not grow at all. Often, even with all of their requirements, these organisms grow poorly.
Unlike eukaryotic cells, which are only able to grow in an aerobic, or oxygen containing, environment, prokaryotic cells are able to grow anaerobically using several fermentation pathways to generate ample energy (8). Other prokaryotes prefer a microaerophilic, or reduced oxygen environment, or even a capnophilic, or high carbon dioxide environment (9). These organisms are more challenging to enrich for, since the atmosphere must be altered. Scientists that frequently work with organisms sensitive to an oxygenated environment would normally work in an anaerobic chamber and incubator, where a heavy, inert gas such as argon is pumped in to displace the oxygen (10). Others make use of conventionally available sealed gas packet systems that use water to generate hydrogen and carbon dioxide, along with a catalyst like palladium to remove all atmospheric oxygen. These commercially available kits can create any of the above-mentioned atmospheric conditions (10).
Whether cultivating a pathogen to determine potential infection or looking to identify a specific species of bacteria present in a natural environment, one problem exists. No one bacterial species inhabits one habitat. Bacteria live as multicellular communities everywhere from the skin of humans to the oceans of our planet (11). When attempting to isolate one species of bacteria, scientists must work to exclude the numerous other organisms that are also inhabiting the isolated area. For this reason, enriched growth media for bacteria often carry out two functions. The first is to make the media selective. A selective agent will prevent some species from growing, while not inhibiting and often even promoting others to grow (12). The second function of media ingredients may be to work as differential agents. Such agents allow for the identification of a particular biochemical feature of an isolated organism. By pairing several different selective and differential medias along with appropriate growth conditions, scientists and diagnosticians are able to identify the presence of specific bacterial species from a particular isolate.
One example of a selective and differential media aiding in to identification is in the case of the clinically significant organism Staphylococcus aureus. This organism is typically cultured on mannitol salt agar. This media not only selects for only organisms which can live in a high salt environment, which include some gram positives like Staphylococcus, but it also inhibits any organisms sensitive to salt. The mannitol sugar is the differential component of this medium. Of all the clinically significant Staphylococcus species, only S. aureus is able to ferment mannitol. This fermentation reaction produces acid as a by-product which causes the red methyl red indicator in the media to turn yellow. Other Staphylococcus species (such as Staphylococcus epidermidis) although able to grow, will leave the media red in color.
This lab exercise demonstrates proper aseptic technique, as well as proper inoculation of growth media from broth. It also introduces the growth of common contaminant organisms on enrichment media, the use of a gas package anaerobic culture system for anaerobic bacteria, and the use of different selective and differential medias for the presumptive identification of gram positive and gram-negative bacteria.

Enrichment Cultures, Selective and Differential Media

농축 배양: 선택 및 차동 매체에서 호기성 및 혐기성 미생물 배양

Source: Christopher P. Corbo1, Jonathan F. Blaize1, Elizabeth Suter1
1 Department of Biological Sciences, Wagner College, 1 Campus Road, Staten Island NY, ...

Pure Cultures and Streak Plating: Isolation of Single Bacterial Colonies from a Mixed Sample

Source: Tilde Andersson1, Rolf Lood1 
1 Department of Clinical Sciences Lund, Division of Infection Medicine, Biomedical Center, Lund University, 221 00 Lund, Sweden
Seemingly impossible to determine, microbial biodiversity is truly astounding with an estimated one trillion coexisting species (1,2). Although particularly harsh climates, like the acidic environment of the human stomach (3) or the subglacial lakes of Antarctica (4), may be dominated by a specific species, bacteria are typically found in mixed cultures. As each strain may influence the growth of another (5), the ability to separate and cultivate &#34;pure&#34; (consisting only of one type) colonies has become essential in clinical and academic settings alike. Pure cultures enable further genetic (6) and proteomic examinations (7), analysis of sample purity and, perhaps more noteworthy, the identification and characterization of infectious agents from clinical samples.
Bacteria have a wide range of growth requirements and there are numerous types of nutrient media designed to sustain both the un-demanding and the fastidious species (8). Growth media can be prepared either in liquid form (as a broth) or in a typically agar-based (a gelling agent derived from red algae) solid form. Whereas direct inoculation into broth carries the risk of generating a genetically diverse or even mixed bacterial population, plating and re-streaking creates a purer culture where each cell has a highly similar genetic makeup. The streak plate technique is based on progressive dilution of a sample (Figure 1), with the aim of separating individual cells from one another. Any viable cell (hereafter referred to as a colony forming unit, CFU) sustained by the media and designated environment can subsequently found an isolated colony of daughter-cells through binary fission. In spite of the rapid mutation rates within bacterial communities, this cell-group is generally regarded as clonal. Harvesting and re-streaking this population consequently ensures that subsequent work involves only a single bacterial type.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10509/10509fig1.jpg" /> 
Figure 1: A streak plate is based on progressive dilution of the original sample. I) The inoculum is initially dispersed using a zig-zag motion, creating an area with a relatively dense bacterial population. II-IV) Streaks are drawn from the preceding area, using a sterile inoculation loop each time, until the fourth quadrant is reached. V) A final zig-zag motion directed towards the middle of the plate forms a region where the inoculum has been markedly diluted, allowing colonies to appear separate from one another.
The streak plate technique can also be combined with the use of selective and/or differential media. A selective medium will inhibit the growth of certain organisms (e.g. through addition of antibiotics) whilst a differential medium will solely help distinguish one from another (e.g. through hemolysis on blood agar plates).
Underlying all work in microbiology is the use of aseptic (sterile) techniques. Every bacterial culture should be considered potentially pathogenic as there is a risk of unintended growth of treacherous strains, aerosol formation and contamination of equipment/personnel. To minimize these risks, all media, plastic-, metal- and glass-ware are typically sterilized through autoclaving before and after use, subjecting them to high-pressure saturated steam at around 121&#176;C that effectively wipes out any lingering cells. The work space is generally disinfected using ethanol both prior to, and after, use. Lab coat and gloves are always worn during work with infectious agents.

Pure Cultures and Streak Plating: Isolation of Bacterial Colonies

순수 배양 및 줄무늬 평판배양: 혼합 검체에서 단일 박테리아 군집 분리

Source: Tilde Andersson1, Rolf Lood1
1 Department of Clinical Sciences Lund, Division of Infection Medicine, Biomedical Center, Lund University, 221 00 ...

Plaque Assay: A Method to Determine Viral Titer as Plaque Forming Units (PFU)

Source: Tilde Andersson1, Rolf Lood1 
1 Department of Clinical Sciences Lund, Division of Infection Medicine, Biomedical Center, Lund University, 221 00 Lund, Sweden
Viruses that infect prokaryotic organisms, called bacteriophages or simply phages, were identified in the early 20th century by Twort (1) and d&#39;H&#233;relle (2) independently. Phages have since been widely recognized for their therapeutic value (3) and their influence on human (4), as well as global, ecosystems (5). Current concerns have fueled a renewed interest in the use of phages as an alternative to modern antibiotics in treatment of infectious disease (6). Essentially all phage research relies on the ability to purify and quantify viruses, also known as a viral titer. Initially described in 1952, this was the purpose of the plaque assay (7). Decades and multiple technological advancements later, the plaque assay remains one of the most reliable methods for determination of viral titer (8).
Bacteriophages subsist by injecting their genetic material into host cells, hijacking the machineries for production of new phage particles, and eventually causing the host to release numerous progeny virions through cell lysis. Because of their minute size, bacteriophages cannot be observed using solely light microscopy; therefore, scanning electron microscopy is required (Figure 1). Additionally, phages cannot be cultivated on nutritional agar plates like bacteria, as they need host cells to prey on.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10514/10514fig1.jpg" /> 
Figure 1: The morphology of a bacteriophage, here exemplified by an E. coli phage, can be studied using scanning electron microscopy. Most bacteriophages belong to Caudovirales (tailed bacteriophages). This particular phage has a very short tail structure and an icosahedral head, placing it in the family of Podoviruses.
The plaque assay (Figure 2) is based on incorporation of host cells, preferentially in log-phase growth, into the medium. This creates a dense, turbid layer of bacteria able to sustain viral growth. An isolated phage can subsequently infect, replicate within, and lyse one cell. With each lysed cell, multiple adjacent ones become immediately infected. Several cycles in, a clear zone (a plaque) can be observed in the otherwise turbid plate (Figure 2B/Figure 3A), indicating the presence of what was initially a single bacteriophage particle. The number of plaque forming units per volume (i.e. PFU/mL) of a sample, can thus be determined from the number of plaques generated.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/10514/10514fig2.jpg" /> 
Figure 2: Testing for plaque forming units (PFU) is a common method for determining the number of bacteriophages in a sample. (A) The base of a sterile Petri dish is covered with an appropriate solid nutrient medium, followed by a mixture of soft media, susceptible host cells and a dilution of the original bacteriophage sample. Note that the phage-suspension could, in some cases, also be evenly spread-out across the surface of already solidified soft agar. (B) Growth of the host bacteria forms a lawn of cells in the top agar layer. Bacteriophage replication generates clear zones, or plaques, caused by host cell lysis.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/10514/10514fig3.jpg" /> 
Figure 3: Results from PFU-testing show multiple plaques generated by bacteriophages. Due to lysis of susceptible host cells, plaques can be seen as clearing zones in the bacterial lawn, either with (A) full clearance, or (B) partial re-growth caused by generation of resistant bacteria (or possibly by temperate phages in the lysogenic cycle).
Certain temperate phages can adopt what is referred to as a lysogenic lifecycle, in addition to the formerly described lytic growth. In lysogeny, the virus assumes a latent state through incorporation of its genetic material into the genome of the host cell (9), often conferring resistance to further phage infections. This is sometimes revealed through a slight clouding of the plaque (Figure 3B). It is worth noting however, that plaques can also appear blurred due to re-growth of bacteria that have evolved resistance to the phage independent of previous phage infections.
Viruses can attach, or adsorb, to only a limited range of host bacteria (10). Host ranges are further limited by intracellular anti-viral strategies such as the CRISPR-Cas system (11). The resistance/sensitivity towards specific phages displayed by bacterial subgroups has historically been used to categorize bacterial strains into different phage types (Figure 4). Although the effectiveness of this method has now been surpassed by novel sequencing techniques, phage typing can still provide valuable information about bacteria-phage interactions, for instance, facilitating the design of a phage cocktail for clinical usage.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/10514/10514fig4.jpg" /> 
Figure 4: Phage sensitivity of different bacterial strains. Soft agar plates with Cutibacterium acnes strain (A) AD27 and (B) AD35, were spotted with 21 different C. acnes bacteriophages. Only phage 11 were able to infect and kill AD27 while strain AD35 showed sensitivity towards all phages. This technique, termed phage typing, can be used to divide bacterial species and strains into different subgroups based on phage susceptibility.

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