תודה מיוחדת קורי סנדרס על עיצובים Iroc להכנת איורים וכדי קריס Reddi ו Bhairav ​​השאה ב-UCLA להקמת תרבויות מדגם וסיוע עם דמויות. המימון לפרויקט זה סופק על ידי HHMI (HHMI גרנט מס &#39;52006944).

<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>

אין לי מה לחשוף.

מיקרואורגניזמים culturing כוללת מספר שיטות ציפוי, כל אלה דורשים כי טכניקה אספטי להישמר לאורך כל מניפולציה של תאים מדיה. חמישה הליכים שונים תוארו בפרוטוקול זה. למרות הטכניקות האלה ציפוי משמשים באופן שגרתי כדי לתפעל חיידקים הפאג, הם גם יכולים להיות מיושמים על תרבית תאים יונקים מיקרואורגניזמים האיקריוטים נפוץ גנטיקה מולקולרית כמו שמרים (כלומר, cerevisiae Saccharomyces, קנדידה אלביקנס, pombe Schizosaccharomyces), אצות פרוטוזואה ( כלומר, Volvox, Chlamydomonas, אמבה, הסנדלית), ו נמטודות (כלומר, Caenorhabditis elegans). יש גם רבים (ועוד יותר מתוחכמת) וריאציות של כל שיטה ציפוי בהתאם המטרה האורגניזם הניסוי או על פי המחקר. לכן, חשוב לא רק כדי לבחור את הטכניקה המתאימה ביותר עבור מיקרואורגניזם הניסוי או יעד נתון, אלא גם כדי להתאים את המתודולוגיה לא כזהכובע תוצאות הניסוי כראוי להתמודד עם שאלת המחקר או בעיה. חלק מהיישומים העדכניים ביותר של טכניקות ציפוי שנדונו בפרוטוקול זה כרוך כי ההתקדמות הטכנולוגית להניב תפוקה גבוהה תוצאות ניסויים ההקרנה ו גילוי תרופות. לדוגמה, רצף הגנום מרכזים להשתמש &quot;שיטת קופקבנה&quot; להתפשטות-ציפוי ספריות לשכפל, אשר א תאים קולי הפך עם פלסמידים המכילים קטעי דנ&quot;א שמקורם הגנום של החיידק. בגלל עשרות צלחות גדולות (המכונה מגשים המבדק) מוכנים בבת אחת, אוטומטית צלחת שאכר משמש לנער את חרוזי זכוכית עבור אצווה שלמה של מגשים. יתר על כן, כאשר אתם בוחרים מושבות של הצלחות אלה לאחר הדגירה, בורר המושבה רובוטית משמש לאיסוף תאים ממושבות בהתאם הבידוד של מרק LB ב 384 גם microplates. עבור assay תפוקה גבוהה זה ההקרנה, עקרונות פרוצדורליים של p-התפשטותהטכניקה מאוחר להחיל אבל הטכנולוגיה מאפשרת צעדים שונים כדי להיות אוטומטי ו scaled כדי לאפשר מספר גדול של דגימות כדי לנתח בו זמנית תוך פרק זמן קצר. חברות ביוטכנולוגיה ותרופות משקיעות משאבים רבים בפיתוח טכנולוגיה תפוקה גבוהה עבור הטכניקות הבסיסיות ביותר במיקרוביולוגיה וגנטיקה מולקולרית. למשל, יש הרב ערוצית micropipettors לבצע העברות בנפח של עד 8 או 12 דגימות בו זמנית. יש אפילו הם תחנות עבודה רובוטיות תמרון הראש 96 ערוצים פיפטה! מאמצים אלה כוללים רב תחומית צוותים של מדענים, זיווג ביולוגים שיש להם מומחיות מתודולוגית עם מהנדסים, מתכנתי מחשבים, אשר יכולה לפתח את המכשור הדרוש כדי לבצע את הפעולות המכניות הקשורות הניסויים. ללא תלות ביישום המחקר, המטרה המשותפת חברות המפתחות טכנולוגיות אלה היא אחת - כדי להפוך laboratoר&quot;י תהליכים, כלים, מערכות ומכשירים, מה שהופך אותם פחות עבודה אינטנסיבית ויעילה יותר.

1. Yeast Tryptone Agar (YTA)
<table border="1">
	<tbody>
		<tr>
			<td>Yeast extract</td>
			<td>2.0 g</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>10.0 g</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>15.0 g</td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td>up to 1000.0 ml</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>pH 7.0</td>
		</tr>
	</tbody>
</table>
Autoclave at 121&deg;C for 20 minutes to sterilize. Store at 4&deg;C.
If preparing tubes for the pour-plate procedure, allow the agar to cool to ~55&deg;C then add 2.0 ml of 50 mg/ml cycloheximide. Aseptically dispense 18.0 ml of the melted agar per 18 mm tube then store at 4&deg;C. The agar will solidify and will need to be melted in a steamer or microwave prior to use.
2. Minimal Salts Agar (MSA) + 0.1% (w/v) carbon source
<table border="1">
	<tbody>
		<tr>
			<td>NH4Cl</td>
			<td>1.0 g</td>
		</tr>
		<tr>
			<td>NH2HPO4&bull;2H2O</td>
			<td>2.14 g</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>1.09 g</td>
		</tr>
		<tr>
			<td>MgSO4&bull;7H2O</td>
			<td>0.2 g</td>
		</tr>
		<tr>
			<td>Carbon source*</td>
			<td>1.0 g</td>
		</tr>
		<tr>
			<td>Trace salts solution**</td>
			<td>10.0 ml</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>15.0 g</td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td>up to 1000.0 ml</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>pH 7.0</td>
		</tr>
	</tbody>
</table>
Autoclave at 121&deg;C for 20 minutes to sterilize. Store at 4&deg;C.
* Carbon sources used for experiments presented in Figure 13 include acetamide, lactose, and glycine.
** Trace salts solution is prepared in 0.1 N HCl as follows. It is added to the base before sterilization (autoclave at 121&deg;C for 20 minutes).
<table border="1">
	<tbody>
		<tr>
			<td>FeSO4&bull;7H2O</td>
			<td>300.0 mg</td>
		</tr>
		<tr>
			<td>MnCl2&bull;4H2O</td>
			<td>180.0 mg</td>
		</tr>
		<tr>
			<td>Co(NO3)2&bull;6H2O</td>
			<td>130.0 mg</td>
		</tr>
		<tr>
			<td>ZnSO4.7H2O</td>
			<td>40.0 mg</td>
		</tr>
		<tr>
			<td>H2MoO4</td>
			<td>20.0 mg</td>
		</tr>
		<tr>
			<td>CuSO4&bull;5H2O</td>
			<td>1.0 mg</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>1000.0 mg</td>
		</tr>
		<tr>
			<td>HCl (0.1 N)</td>
			<td>up to 1000.0 ml</td>
		</tr>
	</tbody>
</table>
3. EHA soft agar (0.65 % w/v)
<table border="1">
	<tbody>
		<tr>
			<td>Agar</td>
			<td>6.5 g</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Tryptone</td>
			<td>13.0 g</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>NaCl</td>
			<td>8.0 g</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Na Citrate&bull;2H2O</td>
			<td>2.0 g</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Glucose</td>
			<td>3.0 g</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Distilled water</td>
			<td>up to 1000.0 ml</td>
		</tr>
	</tbody>
</table>
Autoclave at 121&deg;C for 20 minutes to sterilize. Store at 4&deg;C.
4. EHA hard agar (1.2% w/v)
<table border="1">
	<tbody>
		<tr>
			<td>Agar</td>
			<td>12.0 g</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>13.0 g</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>8.0 g</td>
		</tr>
		<tr>
			<td>Na Citrate&bull;2H2O</td>
			<td>2.0 g</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>3.0 g</td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td>up to 1000.0 ml</td>
		</tr>
	</tbody>
</table>
Autoclave at 121&deg;C for 20 minutes to sterilize. Store at 4&deg;C.
5. 1X Middlebrook Top Agar (MBTA soft agar, 0.5% w/v)
<table border="1">
	<tbody>
		<tr>
			<td>100 mM CaCl2 stock*</td>
			<td>1 ml</td>
		</tr>
		<tr>
			<td>7H9 liquid medium: Neat **</td>
			<td>50 ml</td>
		</tr>
		<tr>
			<td>2XTA ***</td>
			<td>50 ml</td>
		</tr>
	</tbody>
</table>
Melt 50 ml of 2XTA and allow it to cool to ~55&deg;C. Using aseptic technique, add the CaCl2 and 7H9 broth to the melted agar. Aseptically dispense 4.5 ml of the mixture per 13 mm tube and store in a 55&deg;C incubator &le;7 days. Cooling MBTA to room temperature or 4&deg;C will cause the CaCl2 to precipitate out of solution.
* 100 mM CaCl2. stock must be stored at room temperature to prevent CaCl2 from precipitating out of solution.
** 7H9 liquid medium: Neat
<table border="1">
	<tbody>
		<tr>
			<td>7H9 broth base</td>
			<td>4.7 g</td>
		</tr>
		<tr>
			<td>40% glycerol stock</td>
			<td>5 ml</td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td>up to 900.0 ml</td>
		</tr>
	</tbody>
</table>
Mix the base with water then add the glycerol while stirring. Autoclave at 121&deg;C for 20 minutes to sterilize. Store at 4&deg;C.
*** 2X Middlebrook Top Agar (2XTA, 1.0% w/v)
<table border="1">
	<tbody>
		<tr>
			<td>7H9 broth base</td>
			<td>4.7 g</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>1.0 g</td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td>up to 1000.0 ml</td>
		</tr>
	</tbody>
</table>
Autoclave at 121&deg;C for 20 minutes to sterilize. Dispense 50 ml aliquots into 100 ml bottles and store at 4&deg;C.
6. Middlebrook 7H10 Agar Plates (MHA hard agar, 1.9% w/v)
<table border="1">
	<tbody>
		<tr>
			<td>7H10 agar base</td>
			<td>19.0 g</td>
		</tr>
		<tr>
			<td>40% glycerol stock</td>
			<td>12.5 ml</td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td>887.5 ml</td>
		</tr>
	</tbody>
</table>
Mix the agar base with water then add the glycerol while stirring. Heat the solution to boiling then stir for one minute to completely dissolve the base powder. Autoclave at 121&deg;C for 20 minutes to sterilize. Allow the agar to cool to ~55&deg;C then aseptically add the following reagents:
<table border="1">
	<tbody>
		<tr>
			<td>AD supplement (pre-warmed to 37&deg;C)*</td>
			<td>100 ml</td>
		</tr>
		<tr>
			<td>50 mg/ml Carbenicillin **</td>
			<td>1.0 ml</td>
		</tr>
		<tr>
			<td>10 mg/ml Cycloheximide **</td>
			<td>1.0 ml</td>
		</tr>
	</tbody>
</table>
* AD supplement
<table border="1">
	<tbody>
		<tr>
			<td>NaCl &gt;</td>
			<td>17 g &gt;</td>
		</tr>
		<tr>
			<td>Albumin (Fraction V)&gt;</td>
			<td>100 g&gt;</td>
		</tr>
		<tr>
			<td>Dextrose (D-Glucose)&gt;</td>
			<td>40 g&gt;</td>
		</tr>
		<tr>
			<td>Distilled water&gt;</td>
			<td>up to 2000.0 ml&gt;</td>
		</tr>
	</tbody>
</table>
Filter-sterilize this solution; do not autoclave. Store at 4&deg;C.
** Filter-sterilize and store these solutions at 4&deg;C for &le;60 days.
7. LB agar (1.5% w/v) + X-Gal (60 &mu;g/ml)
<table border="1">
	<tbody>
		<tr>
			<td>Tryptone</td>
			<td>10.0 g</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>5.0 g</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>10.0 g</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>15.0 g</td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td>up to 1000.0 ml</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>pH 7.5 at 25&deg;C</td>
		</tr>
	</tbody>
</table>
Autoclave at 121&deg;C for 20 minutes to sterilize. Allow the agar to cool to ~55&deg;C then aseptically add 3.0 ml of 20 mg/ml X-Gal solution. Freshly prepare X-gal stock by dissolving 400 mg X-Gal in 20 ml dimethylformamide (DMF).
Table of specific reagents:
<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Becton Dickenson</td>
			<td>212750</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Becton Dickenson</td>
			<td>211705</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Becton Dickenson</td>
			<td>214030</td>
		</tr>
		<tr>
			<td>NH4Cl</td>
			<td>Acros Organics</td>
			<td>123340010</td>
		</tr>
		<tr>
			<td>NH2HPO4&bull;2H2O</td>
			<td>Sigma-Aldrich</td>
			<td>30435</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Fisher Scientific</td>
			<td>BP 303-500</td>
		</tr>
		<tr>
			<td>MgSO4&bull;7H2O</td>
			<td>Sigma-Aldrich</td>
			<td>230391</td>
		</tr>
		<tr>
			<td>Acetamide</td>
			<td>Sigma-Aldrich</td>
			<td>A-0500</td>
		</tr>
		<tr>
			<td>Lactose</td>
			<td>Fisher Scientific</td>
			<td>L6-500</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>G-7126</td>
		</tr>
		<tr>
			<td>FeSO4&bull;7H2O</td>
			<td>Sigma-Aldrich</td>
			<td>F8048</td>
		</tr>
		<tr>
			<td>MnCl2&bull;4H2O</td>
			<td>Sigma-Aldrich</td>
			<td>M-3634</td>
		</tr>
		<tr>
			<td>Co(NO3)2&bull;6H2O</td>
			<td>Sigma-Aldrich</td>
			<td>230375</td>
		</tr>
		<tr>
			<td>ZnSO4.7H2O</td>
			<td>Sigma-Aldrich</td>
			<td>Z4750</td>
		</tr>
		<tr>
			<td>H2MoO4</td>
			<td>Acros Organics</td>
			<td>213621000</td>
		</tr>
		<tr>
			<td>CuSO4&bull;5H2O</td>
			<td>Sigma-Aldrich</td>
			<td>209198</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C1016</td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Fisher Scientific</td>
			<td>A144-212</td>
		</tr>
		<tr>
			<td>Cycloheximide</td>
			<td>Sigma</td>
			<td>038K1561</td>
		</tr>
		<tr>
			<td>Carbenicillin</td>
			<td>Cellgro</td>
			<td>46-100-RG</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher</td>
			<td>S271-1</td>
		</tr>
		<tr>
			<td>Na Citrate&bull;2H2O</td>
			<td>Fisher</td>
			<td>S279-500</td>
		</tr>
		<tr>
			<td>Glucose (Dextrose)</td>
			<td>BD</td>
			<td>215530</td>
		</tr>
		<tr>
			<td>7H9 broth base</td>
			<td>BD</td>
			<td>271310</td>
		</tr>
		<tr>
			<td>7H10 agar base</td>
			<td>BD</td>
			<td>262710</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Shelton</td>
			<td>IB15760</td>
		</tr>
		<tr>
			<td>Albumin (Fraction V)</td>
			<td>Fisher</td>
			<td>S71907</td>
		</tr>
		<tr>
			<td>X-Gal (5-bromo-4-chloro-3-indolyl-&beta;-D-galactopyranoside)</td>
			<td>Teknova</td>
			<td>X1205</td>
		</tr>
		<tr>
			<td>Dimethylformamide (DMF)</td>
			<td>Sigma</td>
			<td>D4551</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fisher</td>
			<td>CDA19</td>
		</tr>
		<tr>
			<td>Chlorine Bleach</td>
			<td>Chlorox</td>
			<td>02490/06644884</td>
		</tr>
		<tr>
			<td>CiDecon (disinfectant)</td>
			<td>Decon Laboratories, Inc.</td>
			<td>8504</td>
		</tr>
	</tbody>
</table>
Table of specific equipment:
<table border="1">
	<tbody>
		<tr>
			<td>Name of equipment</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Experiment</td>
		</tr>
		<tr>
			<td>Metal loops</td>
			<td>American Educational Products</td>
			<td>S17352</td>
			<td> Streak plating</td>
		</tr>
		<tr>
			<td>Disposable plastic loops</td>
			<td>Fisher</td>
			<td>22-363-602</td>
			<td> Streak plating</td>
		</tr>
		<tr>
			<td>Wooden sticks</td>
			<td>Fisher</td>
			<td>23-400-104</td>
			<td> Streak plating</td>
		</tr>
		<tr>
			<td>Flat Toothpicks</td>
			<td>American Educational Products</td>
			<td>S67859</td>
			<td>Streak plating</td>
		</tr>
		<tr>
			<td>Turn tables</td>
			<td>Fisher</td>
			<td>08-758Q</td>
			<td>Spread plating</td>
		</tr>
		<tr>
			<td>Glass rods</td>
			<td>Bellco Glass</td>
			<td>NC9004380</td>
			<td>Spread plating</td>
		</tr>
		<tr>
			<td>4 mm glass beads</td>
			<td>Fisher</td>
			<td>11-312B</td>
			<td>Spread plating</td>
		</tr>
		<tr>
			<td>Velveteen cloth</td>
			<td>Bel-Art Products</td>
			<td>09-718-2</td>
			<td>Replica plating</td>
		</tr>
		<tr>
			<td>Cylindrical block</td>
			<td>Bel-Art Products</td>
			<td>09-718-1</td>
			<td>Replica plating</td>
		</tr>
	</tbody>
</table>

aseptic laboratory techniques: plating methods

מיקרואורגניזמים נמצאים בכל משטחים דוממים יצירת מקורות בכל מקום אפשרי של זיהום במעבדה. הצלחת הניסוי מסתמך על היכולת של המדען לעקר משטחי עבודה וציוד, כמו גם למנוע מגע של כלים ופתרונות סטריליים עם לא סטריליים משטחים. כאן אנו מציגים את השלבים כמה שיטות ציפוי המשמשים באופן שגרתי במעבדה לבודד, להפיץ, או למנות מיקרואורגניזמים כגון חיידקים הפאג. כל חמש שיטות לשלב טכניקה מזוהם, או נהלים, כי לשמור על סטריליות של חומרים ניסיוניים. הנהלים שתוארו כוללים (1) רצף, ציפוי תרבויות לבודד מושבות חיידקים בודדים, (2) לשפוך, ציפוי ו (3) התפשטות, ציפוי על מנת לספור מושבות חיידקים קיימא (4) שכבות אגר רך לבודד הפאג ושלטים למנות, ו ( 5) העתק, ציפוי להעביר תאים מהצלחת אחד למשנהו בתבנית מרחבית זהה. נהלים אלה ניתן לבצע tהוא ספסל במעבדה, בתנאי שהם כרוכים פתוגניים שאינם זנים של מיקרואורגניזמים (רמת בטיחות ביולוגית 1, BSL-1). אם עובדים עם BSL-2 אורגניזמים, אז המניפולציות האלה חייב להתבצע בתוך ארון בטיחות ביולוגית. להתייעץ המהדורה העדכנית ביותר של בטיחות ביולוגית במעבדות מיקרוביולוגיות ביו (BMBL), כמו גם בטיחות חומרים גיליונות נתונים (MSDS) של חומרים זיהומיות על מנת לקבוע את סיווג Biohazard, כמו גם את אמצעי בטיחות ומתקנים הבלימה הנדרש מיקרואורגניזם הנדון. זני חיידקים ומניות הפאג ניתן לקבל החוקרים מחקר, חברות, ואוספים מתוחזק על ידי ארגונים מסוימים כגון אוסף התרבות האמריקאית סוג (ATCC). מומלץ לא להשתמש זנים פתוגניים כאשר לומדים את שיטות ציפוי שונים. על ידי ביצוע הליכים המתוארים בפרוטוקול זה, התלמידים צריכים להיות מסוגלים: <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> נהלי ציפוי ללא contaminatiנג התקשורת. </li><li style=";text-align:right;direction:rtl"> לבודד מושבות חיידקים בודדים בשיטת פס-ציפוי. </li><li style=";text-align:right;direction:rtl"> להשתמש בשיטות לשפוך, ציפוי, פרוסים ציפוי כדי לקבוע את ריכוז החיידקים. </li><li style=";text-align:right;direction:rtl"> לבצע שכבות אגר רך בעבודה עם הפאג. </li><li style=";text-align:right;direction:rtl"> העברת תאים חיידקיים מהצלחת אחד לאחר באמצעות ההליך העתק-ציפוי. </li><li style=";text-align:right;direction:rtl"> לאור ניסיוני המשימה, בחר את שיטת ציפוי מתאים. </li></ul>

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

Aseptic Laboratory Techniques: Plating Methods

כאשר עובדים עם התקשורת ריאגנטים המשמשים מיקרואורגניזמים תרבות, הטכניקה aseptic חייב להיות מתורגל כדי להבטיח זיהום ממוזער. מגוון שיטות ציפוי משמשים באופן שגרתי כדי לבודד, להפיץ, או חיידקים למנות ו הפאג, אשר כולם לשלב נהלים השומרים על עקרות של חומרים ניסיוניים.

טכניקות מעבדה אספטיים: שיטות ציפוי

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

aseptic-laboratory-techniques-plating-methods

Research

JoVE Journal

Biology

722.1K Views.  University of California, Los Angeles. כאשר עובדים עם התקשורת ריאגנטים המשמשים מיקרואורגניזמים תרבות, הטכניקה aseptic חייב להיות מתורגל כדי להבטיח זיהום ממוזער. מגוון שיטות ציפוי משמשים באופן שגרתי כדי לבודד, להפיץ, או חיידקים למנות ו הפאג, אשר כולם לשלב נהלים השומרים על עקרות של חומרים ניסיוניים.

Video: Aseptic Laboratory Techniques: Plating Methods

Microbial Communities in Nature and Laboratory - Interview

הקהילות מיקרוביאלית בטבע מעבדה - ראיון

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.

טכניקות מעבדה אספטיים: העברות נפח עם טפטפות ו Micropipettors סרולוגיות

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

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

אופטימיזציה שיטות דוגמנות מכתימה והפצת נשק לניטור חלוקת תא באמצעות צבעי מעקב סלולרי

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal&#8211;to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes.

Many different methods exist for the measurement of extracellular vesicles (EVs) using flow cytometry (FCM). Several aspects should be considered when determining the most appropriate method to use. Two protocols for measuring EVs are presented, using either individual detection or a bead-based approach.

טכניקות לניתוח של תאית שלפוחית ​​שימוש cytometry הזרימה

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we know about C. elegans is limited to laboratory cultivation of the nematodes that may not necessarily reflect the environments they normally inhabit in nature. Cultivation of C. elegans in a 3D habitat that is more similar to the 3D matrix that worms encounter in rotten fruits and vegetative compost in nature could reveal novel phenotypes and behaviors not observed in 2D. In addition, experiments in 3D can address how phenotypes we observe in 2D are relevant for the worm in nature. Here, a new method in which C. elegans grows and reproduces normally in three dimensions is presented. Cultivation of C. elegans in Nematode Growth Tube-3D (NGT-3D) can allow us to measure the reproductive fitness of C. elegans strains or different conditions in a 3D environment. We also present a novel method, termed Nematode Growth Bottle-3D (NGB-3D), to cultivate C. elegans in 3D for microscopic analysis. These methods allow scientists to study C. elegans biology in conditions that are more reflective of the environments they encounter in nature. These can help us to understand the overlying evolutionary relevance of the physiology and behavior of C. elegans we observe in the laboratory.

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

We present a simple method to construct 3D nematode cultivation systems called NGT-3D and NGB-3D. These can be used to study nematode fitness and behaviors in habitats that are more similar to natural Caenorhabditis elegans habitats than the standard 2D laboratory C. elegans culture plates.

טיפוח<em&gt; Elegans Caenorhabditis</em&gt; ב בשלושה ממדים במעבדה

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we ...

Techniques to Induce and Quantify Cellular Senescence

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

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

טכניקות כדי להשרות ולכמת סלולר הזדקנות

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

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to study the functions and evolution of bacterial symbionts in their host. As most symbionts cannot be cultured in vitro, methods to isolate an adequate quantity of bacteria for genome sequencing are very important. In the whitefly Bemisia tabaci, a number of endosymbionts have been identified and are predicted to be of importance in the development and reproduction of the pests through multiple approaches. However, the mechanism underpinning the associations remains largely unknown. The obstacle partially comes from the fact that the endosymbionts in whitefly, mostly restrained in bacteriocytes, are hard to separate from the host cells. Here we report a step-by-step protocol for the identification, extraction and purification of endosymbionts from the whitefly B. tabaci mainly by dissection and filtration. Endosymbiont samples prepared by this method, although still a mixture of different endosymbiont species, are suitable for subsequent genome sequencing and analysis of the possible roles of endosymbionts in B. tabaci. This method may also be used to isolate endosymbionts from other insects.

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Here, we present a protocol to isolate endosymbionts from the whitefly Bemisia tabaci through dissection and filtration. After amplification, the DNA samples are suitable for subsequent sequencing and study of the mutualism between endosymbionts and whitefly.

שיטות להפקת endosymbionts מן Whitefly<em&gt; Bemisia Tabaci</em

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to ...

Simple and Effective Administration and Visualization of Microparticles in the Circulatory System of Small Fishes Using Kidney Injection

The systemic administration of micro-size particles into a living organism can be applied for vasculature visualization, drug and vaccine delivery, implantation of transgenic cells and tiny optical sensors. However, intravenous microinjections into small animals, which are mostly used in biological and veterinary laboratories, are very difficult and require trained personnel. Herein, we demonstrate a robust and efficient method for the introduction of microparticles into the circulatory system of adult zebrafish (Danio rerio) by injection into the fish kidney. To visualize the introduced microparticles in the vasculature, we propose a simple intravital imaging technique in fish gills. In vivo monitoring of the zebrafish blood pH was accomplished using an injected microencapsulated fluorescent probe, SNARF-1, to demonstrate one of the possible applications of the described technique. This article provides a detailed description of the encapsulation of pH-sensitive dye and demonstrates the principles of the quick injection and visualization of the obtained microcapsules for in vivo recording of the fluorescent signal. The proposed method of injection is characterized by a low mortality rate (0-20%) and high efficiency (70-90% success), and it is easy to institute using commonly available equipment. All described procedures can be performed on other small fish species, such as guppies and medaka.

This article demonstrates the principles of a quick, minimally invasive injection of fluorescent microparticles into the circulatory system of small fishes and the in vivo visualization of the microparticles in fish blood.

ניהול פשוטה ויעילה והדמיה של Microparticles במערכת הדם של דגים קטנים באמצעות הזרקת כליות

The systemic administration of micro-size particles into a living organism can be applied for vasculature visualization, drug and vaccine delivery, ...

A Versatile Model of Hard Tick Infestation on Laboratory Rabbits

The use of live animals in tick research is crucial for a variety of experimental purposes including the maintenance of hard tick colonies in the laboratory. In ticks, all developmental stages (except egg) are hematophagous, and acquiring a blood-meal when attached to their vertebrate hosts is essential for the successful completion of their life cycle. Here we demonstrate a simple method that uses easily openable capsules for feeding of hard ticks on rabbits. The advantages of the proposed method include its simplicity, short duration and most importantly versatile adjustment to the needs of specific experimental requirements. The method makes possible the use of multiple chambers (of various sizes) on the same animal, which permits feeding of multiple stages or different experimental groups while reducing the overall animal requirement. The non-irritating and easily accessible materials used minimizes discomfort to the animals, which can be easily recovered from an experiment and offered for adoption or reused if the ethical protocol allows it.

We have developed a simple and versatile system to feed hard ticks on laboratory rabbits. Our non-laborious protocol uses easily accessible materials and can be adjusted depending on the requirements of the various experimental settings. The method allows comfortable monitoring and/or sampling of ticks during the entire feeding period.

מודל רב תכליתי של שנתות קשה שרץ על ארנבים מעבדה

The use of live animals in tick research is crucial for a variety of experimental purposes including the maintenance of hard tick colonies in the ...

Bovine Mammary Gland Biopsy Techniques

Bovine mammary gland biopsies allow researchers to collect tissue samples to study cell biology including gene expression, histological analysis, signaling pathways, and protein translation. This article describes two techniques for biopsy of the bovine mammary gland (MG). Three healthy Holstein dairy cows were the subjects. Before biopsies, cows were milked and subsequently restrained in a cattle chute. An analgesic (flunixin meglumine, 1.1 to 2.2 mg/kg of body weight) was administered via jugular intravenous [IV] injection 15-20 min prior to biopsy. For standing sedation, xylazine hydrochloride (0.01-0.05 mg/kg of body weight) was injected via the coccygeal vessels 5-10 min before the procedure. Once adequately sedated, the biopsy site was aseptically prepared and locally anaesthetized with 6 mL of 2% lidocaine hydrochloride via subcutaneous injection. Using aseptic technique, a 2 to 3 cm vertical incision was made using a number 10 scalpel. Core and needle biopsy tools were used. The core biopsy tool was attached to a cordless drill and inserted into the MG tissue through the incision using a clock-wise drill action. The needle biopsy tool was manually inserted into the incision site. Immediately after the procedure, an assistant applied pressure on the incision site for 20 to 25 min using a sterile towel to achieve hemostasis. Stainless steel surgical staples were used to oppose the skin incision. The staples were removed 10 days post-procedure. The main advantages of core and needle biopsies is that both approaches are minimally invasive procedures that can be safely performed in healthy cows. Milk yield following the biopsy was unaffected. These procedures require a short recovery time and result in fewer risks of complications. Specific limitations may include bleeding after the biopsy and infection on the biopsy site. Applications of these techniques include tissue collection for clinical diagnosis and research purposes, such as primary cell culture.

This article presents a bovine mammary gland biopsy using core and needle biopsy tools. Harvested tissue can be used for cell culture or to assess mammary physiology and metabolism including gene expression, protein expression, protein modifications, immunohistochemistry, and metabolite concentrations.