Un ringraziamento speciale a Cori a Sanders Designs IROC per la preparazione di illustrazioni e di Kris Reddi e Bhairav ​​Shah presso la UCLA per la creazione di culture campione e l&#39;assistenza con figure. Il finanziamento per questo progetto è stato fornito dal HHMI (HHMI Grant No. 52006944).

<ol><li>Barker, K. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aBarker%2C+K+%22At+the+Bench%3A+A+Laboratory+Navigator%22">At the Bench: A Laboratory Navigator</a>. , Cold Spring Harbor Laboratory Press. Cold Spring Harbor, New York. (1998).</li>
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<li>Jett, B. D., Hatter, K. L., Huycke, M. M., Gilmore, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Simplified+agar+plate+method+for+quantifying+viable+bacteria%5BTitle%5D)+AND+%22BioTechniques%22%5BJournal%5D)">Simplified agar plate method for quantifying viable bacteria.</a> BioTechniques. 23, 648-650 (1997).</li>
<li>Karam, J. D. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aKaram%2C+JD+%22Molecular+Biology+of+Bacteriophage+T4%22">Molecular Biology of Bacteriophage T4</a>. , ASM Press. Washington, DC. (1994).</li>
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<li>Lederberg, J., Lederberg, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Replica+plating+and+indirect+selection+of+bacterial+mutants%5BTitle%5D)+AND+%22J.+Bacteriol%22%5BJournal%5D)">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://scholar.google.com/scholar?hl=en&safe=off&q=author%3aMiller%2C+JH+%22A+Short+Course+in+Bacterial+Genetics%3A+A+Laboratory+Manual+and+Handbook+for+Escherichia+coli+and+related+bacteria%22">A Short Course in Bacterial Genetics: A Laboratory Manual and Handbook for Escherichia coli and related bacteria</a>. , Cold Spring Harbor Laboratory Press. Plainview, NY. (1992).</li>
<li>Mills, K. V., Gareau, J. R., Garcia, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Low-cost+modification+to+the+Copacabana+method+for+spreading+transformation+mixtures%5BTitle%5D)+AND+%22BioTechniques%22%5BJournal%5D)">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://scholar.google.com/scholar?hl=en&safe=off&q=author%3aJordan%2C+T+%22RESEARCH%3A+National+Genomics+Research+Initiative+Phage+Resource+Laboratory+Manual%22">RESEARCH: National Genomics Research Initiative Phage Resource Laboratory Manual</a>. , Howard Hughes Medical Institute. (2008).</li>
<li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSambrook%2C+J+%22Molecular+Cloning+-+A+Laboratory+Manual%22">Molecular Cloning - A Laboratory Manual</a>. , 3rd, Cold Spring Harbor Laboratory Press. Cold Spring Harbor, New York. (2001).</li>
<li>Sanders, E. R., Miller, J. H. I. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSanders%2C+ER+%22Microbiologist%3A+A+Discovery-based+Course+in+Microbial+Ecology+and+Molecular+Evolution%22">Microbiologist: A Discovery-based Course in Microbial Ecology and Molecular Evolution</a>. , ASM Press. Washington, DC. (2010).</li>
<li>Slonczewski, J. L., Foster, J. W. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSlonczewski%2C+JL+%22Microbiology+-+An+Evolving+Science%22">Microbiology - An Evolving Science</a>. , 2nd, W.W. Norton & Co., Inc. New York, NY. (2011).</li>
<li>Teather, R. M., Wood, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Use+of+Congo+Red-Polysaccharide+Interactions+in+Enumeration+and+Characterization+of+Cellulolytic+Bacteria+from+the+Bovine+Rumen%5BTitle%5D)+AND+%22Applied+and+Environmental+Microbiology%22%5BJournal%5D)">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>
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 <a target="_blank" href="http://www.microbelibrary.org/index.php/component/resource/laboratory-test/3085-preparing-spread-plates-protocols">http://www.microbelibrary.org/index.php/component/resource/laboratory-test/3085-preparing-spread-plates-protocols</a> (2006).</li>
<li>Worthington, M., Luo, R. Q., Pelo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Copacabana+method+for+spreading+E.+coli+and+yeast+colonies%5BTitle%5D)+AND+%22BioTechniques%22%5BJournal%5D)">Copacabana method for spreading E. coli and yeast colonies.</a> BioTechniques. 30, 738-742 (2001).</li>
<li>American Type Culture Collection (ATCC). , Available from: 
 <a target="_blank" href="http://www.atcc.org/">http://www.atcc.org/</a> (2012).</li>
<li>EPA Microbiology Home Page. , Available from: 
 <a target="_blank" href="http://www.epa.gov/nerlcwww/index.html">http://www.epa.gov/nerlcwww/index.html</a> (2012).</li>
<li>The GENE Project: Laboratory Methods and Materials. , Available from: 
 <a target="_blank" href="http://www.phys.ksu.edu/gene/g1.html">http://www.phys.ksu.edu/gene/g1.html</a> (2012).</li>
<li>Joint Genome Institute (JGI) Genome Sequencing Protocols: Library Plating Protocol. , Available from: 
 <a target="_blank" href="http://www.jgi.doe.gov/sequencing/protocols/prots_production.html">http://www.jgi.doe.gov/sequencing/protocols/prots_production.html</a> (2012).</li>
<li>Microbial Genetics (maintained by Stanley Maloy at UCSD). , Available from: 
 <a target="_blank" href="http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics">http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics</a> (2012).</li>
<li>Organization of American States (OAS) Department of Sustainable Development (DSD). , Available from: 
 <a target="_blank" href="http://www.oas.org/DSD/publications/Unit/oea59e/ch26.htm">http://www.oas.org/DSD/publications/Unit/oea59e/ch26.htm</a> (2012).</li>
<li>Public Health Agency of Canada: Material Safety Data Sheets (MSDS) for Infectious Substances. , Available from: 
 <a target="_blank" href="http://www.phac-aspc.gc.ca/msds-ftss">http://www.phac-aspc.gc.ca/msds-ftss</a> (2012).</li>
<li>United States Environmental Protection Agency (EPA) Office of Water. , Available from: 
 <a target="_blank" href="http://water.epa.gov">http://water.epa.gov</a> (2012).</li>
<li>American Society for Microbiology (ASM)'s Curriculum Recommendations Microbiology Majors Program. , Available from: 
 <a target="_blank" href="http://www.asm.org/asm/index.php/unknown/asm-s-curriculum-recommendations-microbiology-majors-program.html">http://www.asm.org/asm/index.php/unknown/asm-s-curriculum-recommendations-microbiology-majors-program.html</a> (2012).</li>
<li>Introductory Course in Microbiology. , Available from: 
 <a target="_blank" href="http://www.asm.org/asm/index.php/unknown/asm-s-curriculum-recommendations-introductory-course-in-microbiology.html">http://www.asm.org/asm/index.php/unknown/asm-s-curriculum-recommendations-introductory-course-in-microbiology.html</a> (2012).</li>
</ol>

Non ho nulla da rivelare.

Coltivare microrganismi comporta un certo numero di metodi di placcatura, le quali richiedono che asepsi essere mantenuta durante tutta la manipolazione di cellule e media. Cinque diverse procedure sono state descritte in questo protocollo. Sebbene queste tecniche di placcatura sono abitualmente utilizzati per manipolare i batteri e fago, possono anche essere applicato a colture cellulari di mammifero e microrganismi eucarioti comunemente usato in genetica molecolare come lievito (cioè, Saccharomyces cerevisiae, Candida albicans, Schizosaccharomyces pombe), alghe e protozoi ( cioè, Volvox, Chlamydomonas, Amoeba, Paramecium) e nematodi (cioè, Caenorhabditis elegans). Ci sono anche numerosi (e ancor più sofisticate) variazioni di ogni metodo di placcatura a seconda dell&#39;obiettivo sperimentale o dell&#39;organismo in fase di studio. Pertanto, è importante non solo selezionare la tecnica più appropriato per un dato microrganismo esperimento o bersaglio, ma anche per adattare il t tale metodologiacappello dei risultati sperimentali adeguatamente affrontare la questione o il problema di ricerca. Alcune delle applicazioni più attuali delle tecniche di placcatura discusse in questo protocollo comporta progressi tecnologici che producono risultati di high-throughput per gli esperimenti di screening e di scoperta della droga. Ad esempio, centri di sequenziamento del genoma utilizzare il &quot;metodo Copacabana&quot; per la diffusione-placcatura librerie di cloni, che sono E. coli trasformate con i plasmidi contenenti i frammenti di DNA derivate dal genoma di un microrganismo. Perché decine di piatti di grandi dimensioni (chiamato vassoi saggi biologici) sono preparati in una sola volta, un sistema automatico agitatore viene utilizzata per agitare le perline in vetro per l&#39;intera partita di vassoi. Inoltre, quando si selezionano le colonie di queste piastre dopo l&#39;incubazione, una raccoglitrice di colonia robotica viene utilizzata per raccogliere le cellule delle colonie appropriate come l&#39;inoculo per brodo LB a 384 e micropiastre. Per questo saggio ad alta prestazione di screening, i principi procedurali di diffusione-ptecnica di ritardo ma la tecnologia consente di applicare vari passi da automatizzare e scalata per consentire un gran numero di campioni da analizzare contemporaneamente e in un breve arco di tempo. Aziende biotecnologiche e farmaceutiche investono considerevoli risorse nello sviluppo di high-throughput tecnologia per le tecniche più basilari di microbiologia e genetica molecolare. Ad esempio, ci sono multi-canale Micropipettatrici per effettuare trasferimenti di volume fino a 8 o 12 campioni alla volta. Ci sono anche stazioni di lavoro robotizzate che manovra a 96 canali testa pipetta! Questi sforzi coinvolgono team multi-disciplinare di scienziati, biologi associazione che possiedono le competenze metodologiche con ingegneri e programmatori di computer che possano sviluppare la strumentazione necessaria per eseguire le operazioni meccaniche associate con gli esperimenti. Qualunque sia l&#39;applicazione di ricerca, l&#39;obiettivo condiviso da aziende che sviluppano queste tecnologie è lo stesso - per automatizzare laboratoriry processi, strumenti, sistemi e strumenti, rendendoli meno manodopera e più efficienti.

<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

I microrganismi sono presenti su tutte le superfici inanimate creazione di fonti di possibile contaminazione onnipresenti in laboratorio. Successo sperimentale si basa sulla capacità di uno scienziato per sterilizzare le superfici di lavoro e attrezzature, nonché prevenire il contatto degli strumenti sterili e soluzioni con superfici non sterili. Qui vi presentiamo i passaggi per la placcatura di diversi metodi comunemente impiegati in laboratorio per isolare, propagare, o elencare i microrganismi come batteri e fagi. Tutti e cinque i metodi di incorporare una tecnica asettica, o procedure che consentono di mantenere la sterilità dei materiali sperimentali. Procedure descritte includono (1) streak-placcatura colture batteriche per isolare singole colonie, (2) pour-placcatura e (3) spread-plating per enumerare vitali colonie batteriche, (4) overlay agar morbide per isolare fago e le placche enumerare, e ( 5) replica-placcatura per trasferire le cellule da una piastra all&#39;altra in un identico modello spaziale. Queste procedure possono essere eseguite a tegli banco di laboratorio, a condizione che coinvolgono non-ceppi di microrganismi patogeni (Biosafety Level 1, BSL-1). Se si lavora con BSL-2 organismi, allora queste manipolazioni devono avvenire in un armadio biosicurezza. Consulta l&#39;edizione più recente del biosicurezza nei laboratori microbiologici e biomedici (BMBL), così come il materiale Schede di Sicurezza (MSDS) per le sostanze infettive di determinare la classificazione di rischio biologico, nonché le precauzioni di sicurezza e le strutture di contenimento necessari per il microrganismo in questione. Ceppi batterici e le scorte di fagi possono essere ottenute da investigatori di ricerca, aziende e collezioni particolari tenuti da organizzazioni come l&#39;American Type Culture Collection (ATCC). Si raccomanda di non utilizzare i ceppi patogeni per imparare i metodi di placcatura vari. Seguendo le procedure descritte in questo protocollo, gli studenti dovrebbero essere in grado di: <ul><li> Eseguire le procedure di placcatura senza contaminating supporti. </li><li> Isolare singole colonie batteriche dalla striscia-plating metodo. </li><li> Utilizzare pour-placcatura e la diffusione-plating metodi per determinare la concentrazione di batteri. </li><li> Effettuare sovrapposizioni agar morbidi quando si lavora con fago. </li><li> Trasferire le cellule batteriche da un piatto all&#39;altro utilizzando la replica-plating procedura. </li><li> Dato un compito sperimentale, selezionare il metodo appropriato di placcatura. </li></ul>

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Aseptic Laboratory Techniques: Plating Methods

Quando si lavora con i media e dei reagenti utilizzati per i microrganismi cultura, tecnica asettica deve essere praticato per garantire la contaminazione è ridotto al minimo. Una varietà di metodi di placcatura sono abitualmente utilizzati per isolare, propagare o batteri enumerare e fago, tutte che incorporano procedure che consentono di mantenere la sterilità dei materiali sperimentali.

Tecniche di laboratorio asettico: Metodi Placcatura

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

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JoVE Journal

Biology

729.4K Views.  University of California, Los Angeles. Quando si lavora con i media e dei reagenti utilizzati per i microrganismi cultura, tecnica asettica deve essere praticato per garantire la contaminazione è ridotto al minimo. Una varietà di metodi di placcatura sono abitualmente utilizzati per isolare, propagare o batteri enumerare e fago, tutte che incorporano procedure che consentono di mantenere la sterilità dei materiali sperimentali.

Video: Aseptic Laboratory Techniques: Plating Methods

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium. Because resistance to ampicillin and vancomycin in E. faecalis is still uncommon compared to resistance in E. faecium, the development of rapid tests allowing differentiation between enterococcal species is important for appropriate therapy and resistance surveillance. The E. faecalis OE PNA FISH assay (AdvanDx, Woburn, MA) uses species-specific peptide nucleic acid (PNA) probes in a fluorescence in situ hybridization format and offers a time to results of 1.5 hours and the potential of providing important information for species-specific treatment. Multicenter studies were performed to assess the performance of the 1.5 hour E. faecalis/OE PNA FISH procedure compared to the original 2.5 hour assay procedure and to standard bacteriology methods for the identification of enterococci directly from a positive blood culture bottle.

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

A rapid protocol for the direct identification of Enterococcus faecalis and other Enterococcus species from a positive blood culture using a Peptide Nucleic Acid fluorescent in situ hybridization assay (PNA FISH).

Una procedura 1,5 ore per l&#39;identificazione di Enterococcus Specie Direttamente da emocolture

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium.  Because resistance to ampicillin and ...

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Immunologia e infezioni

Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.

A rapid bacterial pellet preparation from a positive blood culture can be used as a sample for applications such as identification by MALDI-TOF, Gram staining, antibiotic susceptibility testing and PCR-based test. The results can be rapidly communicated to clinicians to improve the outcome of patients suffering from bloodstream infections.

Preparazione di un Sangue cultura pellet per il Rapid batterica identificazione e test di sensibilità agli antibiotici

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a ...

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.

Tecniche di laboratorio utilizzate per mantenere e differenziare i biotipi di<em&gt; Vibrio cholerae</em&gt; Isolati clinici ed ambientali

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

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.

Tecniche asettiche di laboratorio: Trasferimenti Volume con pipette sierologiche e Micropipettatrici

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

Biologia

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.

Formazione di Biofilm indotta da sali biliari in patogeni enterici: tecniche di identificazione e quantificazione

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

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).

Diluizioni seriali e piastratura: la conta microbica

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

Didattica

JoVE Science Education

Biologia avanzata

Microbiologia

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.

Tecnica asettica nelle scienze ambientali

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

Scienze ambientali

Microbiologia ambientale

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

Culture di arricchimento: coltura di microbi aerobici e anaerobici su terreni selettivi e differenziali

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

Colture pure e piastratura per striscio: isolamento di singole colonie batteriche da un campione misto

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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