Name: aseptic laboratory techniques: volume transfers with serological pipettes and micropipettors
Uploaded: 2012-05-31T12:00:00
Duration: 11 min 43 s
Description: Watch this Scientific Journal Video about Aseptic Laboratory Techniques: Volume Transfers with Serological Pipettes and Micropipettors at JoVE.com

Special thanks to Cori Sanders at Iroc Designs for preparing illustrations and to Kris Reddi at UCLA for setting up sample cultures for figures. Funding for this project was provided by HHMI (HHMI Grant No. 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>. <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>Bykowski, T., Stevenson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aseptic+Technique.">Aseptic Technique.</a> Current Protocols in Microbiology. Appendix 4, Appendix 4D (2008).</li><li>Cot&#233;, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aseptic+Technique+for+Cell+Culture.">Aseptic Technique for Cell Culture.</a> Current Protocols in Cell Biology. Chapter 1, Unit 1.3 (2001).</li><li>Grimes, 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+basic+laboratory+manual+for+the+small-scale+production+and+testing+of+I-2+Newcastle+disease+vaccine.">A basic laboratory manual for the small-scale production and testing of I-2 Newcastle disease vaccine.</a> RAP Publication. AC802/E, 12-13 (2002).</li><li>Guzman, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pipetting:+A+Practical+Guide.">Pipetting: A Practical Guide.</a> The American Biology Teacher. 63 (2), 128-131 (2001).</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. , 978-087969576 (2001).</li><li>Seidman, L. A., Moore, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Basic Laboratory Methods for Biotechnology: Textbook and Laboratory Reference. , (2000).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> PIPETMAN P User's Guide. , (2012).</li></ol>

I have nothing to disclose.

Aseptic technique refers to a set of routine procedures done to prevent sterile solutions and cultures from becoming contaminated by unwanted microorganisms in the laboratory. Such techniques are essential for experiments that require growing cells. Although a work setting that is completely sterile cannot be achieved, procedures such as disinfecting laboratory surfaces, creating a sterile field using a Bunsen burner, limiting exposure of uncapped cultures and media to the air, sterilizing materials such as bottles, tubes and glass pipettes, and avoiding contact of sterile instruments with non-sterile surfaces reduces the possibility of contaminating solutions and cultures in an experiment. The goal is for these precautionary procedures to become second nature; this comes with training and practice while working in a laboratory.
Volume transfers with sterile solutions and cultures using instruments such as serological pipettes and micropipettors are one of many types of routine techniques done in a laboratory. Different experimental applications call for instruments capable of transferring distinct, yet precise and accurate, volumes. Serological pipettes are used in microbiology laboratories to prepare cell cultures requiring media preparations involving milliliter volumes, while micropipettors are essential to molecular biology experiments that need only microliter amounts of solutions. When aseptic technique is practiced with these instruments, contamination is minimized during volume transfers regardless of amount of liquid or type of experiment.
Although not discussed in this protocol, one other means commonly used to prevent contamination is to work within a laminar flow hood (Figure 10). This equipment is critical for tissue culture and for experiments performed with microorganisms classified as BLS-2 or higher. A laminar flow hood contains a HEPA (high-efficiency particulate air) filter that removes airborne contaminants from the air flowing into the hood while preventing unfiltered air from the room from permeating the workspace. Of note, a Bunsen burner cannot be used inside a laminar flow hood because the heat from the flame disrupts air flow essential to the functionality of the hood.
It is often helpful to check the quality of your aseptic technique when performing experiments. To confirm solutions and culture media do not get contaminated during experimental manipulations, always prepare a negative control. For example, if preparing tubes of broth for growth of bacterial cultures, do not inoculate one tube leaving only sterile media. Incubate the medium alongside the inoculated tubes then inspect uninoculated control tube for signs of contamination such as turbidity from growth of unwanted cells unintentionally introduced into the tube. If the control tube is contaminated, the experimental tubes likely are contaminated as well, and the experiment will have to be repeated. These precautionary measures should be done with every experiment.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments </td>
		</tr>
		<tr>
			<td>LB Broth</td>
			<td>Difco</td>
			<td>244620</td>
			<td>Recipe also available in reference 6</td>
		</tr>
		<tr>
			<td colspan="4">TE Buffer:</td>
		</tr>
		<tr>
			<td>EDTA disodium salt dihydrate</td>
			<td>Sigma</td>
			<td>E5134</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trizma-HCl</td>
			<td>Sigma</td>
			<td>T-3253</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CiDecon</td>
			<td>Decon Laboratories, Inc.</td>
			<td>8504</td>
			<td>Disinfectant</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fisher Scientific</td>
			<td>A406</td>
			<td>For use as disinfectant, prepare 70%(v/v) with distilled water</td>
		</tr>
	</tbody>
</table>

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.

Watch this Scientific Journal Video about Aseptic Laboratory Techniques: Volume Transfers with Serological Pipettes and Micropipettors at JoVE.com

Aseptic Laboratory Techniques: Volume Transfers with Serological Pipettes and Micropipettors

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.

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