Name: cell culture techniques and practices to avoid contamination by fungi and bacteria in the research cell culture laboratory
Uploaded: 2023-07-07T13:40:00
Description: Watch this Scientific Journal Video about Cell Culture Techniques and Practices to Avoid Contamination by Fungi and Bacteria in the Research Cell Culture Laboratory at JoVE.com

This work has been made possible thanks to funding from the Howard Hughes Medical Institute (HHMI). We wish to thank our head of lab, Jue Chen, for reading the manuscript and for her continued support, Donna Tallent for her helpful edits and comments, and Jeff Hennefeld from the Information Technology Department at The Rockefeller University for his help with the video component of this manuscript.

1. Preparations 
<ol>
	<li>General
	<ol>
		<li>Wear a clean lab coat designated to be worn only in the cell culture room and no other parts of the laboratory. 
		NOTE: The lab coat does not need to be sterile.</li>
		<li>Wear new gloves that have not touched any other surfaces. Make sure the gloves fit tightly. Nitrile, powder-free gloves are best. 
		NOTE: The gloves do not need to be sterile.</li>
		<li>To prepare for work, spray the gloves, lab coat sleeves, and interior of the biological ...

<ol>
	<li>Johnson, Z. L., Chen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+of+substrate+recognition+by+the+multidrug+resistance+protein+MRP1.">Structural basis of substrate recognition by the multidrug resistance protein MRP1.</a> Cell. 168 (6), 1075-1085 (2017).</li><li>Capes-Davis, A., 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=Cell+lines+as+biological+models:+practical+steps+for+more+reliable+research.">Cell lines as biological models: practical steps for more reliable research.</a> Chemical Research in Toxicology. 32 (9), 1733-1736 (2019).</li><li>Segeritz, C. P., Vallier, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+culture:+growing+cells+as+model+systems+in+vitro.">Cell culture: growing cells as model systems in vitro.</a> Basic Science Methods for Clinical Researchers. , 151-172 (2017).</li><li>Drexler, H. G., Uphoff, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycoplasma+contamination+of+cell+cultures:+Incidence,+sources,+effects,+detection,+elimination,+prevention.">Mycoplasma contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention.</a> Cytotechnology. 39 (2), 75-90 (2002).</li><li>Lincoln, C. K., Gabridge, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+culture+contamination:+sources,+consequences,+prevention,+and+elimination.">Cell culture contamination: sources, consequences, prevention, and elimination.</a> Methods in Cell Biology. 57, 49-65 (1998).</li><li>Nikfarjam, L., Farzaneh, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+and+detection+of+mycoplasma+contamination+in+cell+culture.">Prevention and detection of mycoplasma contamination in cell culture.</a> Cell Journal. 13 (4), 203-212 (2012).</li><li>Stacey, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+culture+contamination.">Cell culture contamination.</a> Methods in Molecular Biology. 731, 79-91 (2011).</li><li>Young, L., Sung, J., Stacey, G., Masters, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+Mycoplasma+in+cell+cultures.">Detection of Mycoplasma in cell cultures.</a> Nature Protocols. 5 (5), 929-934 (2010).</li><li>Barth, O. M., Majerowicz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+detection+by+transmission+electron+microscopy+of+mycoplasma+contamination+in+sera+and+cell+cultures.">Rapid detection by transmission electron microscopy of mycoplasma contamination in sera and cell cultures.</a> Memorias do Instituto Oswaldo Cruz. 83 (1), 63-66 (1988).</li><li>Mirabelli, P., Coppola, L., Salvatore, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+cell+lines+are+useful+model+systems+for+medical+research.">Cancer cell lines are useful model systems for medical research.</a> Cancers. 11 (8), 1098 (2019).</li><li>A cell culture master class: What your cells wish they could tell you. Science Available from: <a target="_blank" href="https://www.science.org/content/webinar/cell-culture-master-class-your-cells-wish-they-could-tell-you">https://www.science.org/content/webinar/cell-culture-master-class-your-cells-wish-they-could-tell-you</a> (2020)</li><li>Langdon, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+culture+contamination:+an+overview.">Cell culture contamination: an overview.</a> Methods in Molecular Medicine. 88, 309-317 (2004).</li><li>Babic, Z., 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=Meta-research:+Incidences+of+problematic+cell+lines+are+lower+in+papers+that+use+RRIDs+to+identify+cell+lines.">Meta-research: Incidences of problematic cell lines are lower in papers that use RRIDs to identify cell lines.</a> eLife. 8, e41676 (2019).</li><li>Visconti, P., 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=Short+tandem+repeat+profiling+for+the+authentication+of+cancer+stem-like+cells.">Short tandem repeat profiling for the authentication of cancer stem-like cells.</a> International Journal of Cancer. 148 (6), 1489-1498 (2021).</li><li>Horbach, S. P. J. M., Halffman, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ghosts+of+HeLa:+How+cell+line+misidentification+contaminates+the+scientific+literature.">The ghosts of HeLa: How cell line misidentification contaminates the scientific literature.</a> PLoS One. 12 (10), 0186281 (2017).</li><li>Why 70% isopropyl alcohol is a better disinfectant than 99% isopropyl alcohol when it comes to Covid-19. MunGlobal Available from: <a target="_blank" href="https://munglobal.com.au/resources/knowledge-base/pathogens/why-70-isopropyl-alcohol-is-a-better-disinfectant-than-99-isopropyl-alcohol/#:~:text=Due%20to%20the%20concentration%20difference">https://munglobal.com.au/resources/knowledge-base/pathogens/why-70-isopropyl-alcohol-is-a-better-disinfectant-than-99-isopropyl-alcohol/#:~:text=Due%20to%20the%20concentration%20difference</a> (2023)</li><li>United States Department of Agriculture. Processing and Safety. Food Safety Publications Available from: <a target="_blank" href="https://www.ars.usda.gov/ARSUserFiles/60701000/FoodSafetyPublications/p328.pdf">https://www.ars.usda.gov/ARSUserFiles/60701000/FoodSafetyPublications/p328.pdf</a> (2004)</li><li>Sterilizing Practices. Centers for Disease Control and Prevention Available from: <a target="_blank" href="https://www.cdc.gov/infectioncontrol/guidelines/disinfection/sterilization/sterilizing-practices.html">https://www.cdc.gov/infectioncontrol/guidelines/disinfection/sterilization/sterilizing-practices.html</a> (2023)</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. , (2001).</li><li>Uphoff, C. C., Drexler, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eradication+of+Mycoplasma+contamination+from+cell+cultures.">Eradication of Mycoplasma contamination from cell cultures.</a> Current Protocols in Molecular Biology. 106, 1-12 (2014).</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. 56 (1), e98 (2020).</li><li>How a Class II, Type A2 Biosafety Cabinet Works. Nuaire Available from: <a target="_blank" href="https://www.nuaire.com/resources/class-ii-type-a2-biosafety-cabinet-how-it-works-article">https://www.nuaire.com/resources/class-ii-type-a2-biosafety-cabinet-how-it-works-article</a> (2020)</li><li>Phelan, K., May, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+cell+tissue+culture+techniques.">Mammalian cell tissue culture techniques.</a> Current Protocols in Pharmacology. 73, 1-23 (2016).</li></ol>

While contamination is one of the primary concerns when performing cell culture work, the practices and techniques outlined in this manuscript will help mitigate the risks. The critical steps include wearing a clean lab coat, which is only used in the cell culture room, using clean, powder-free gloves that are sprayed with 70% EtOH often and which are changed when switching between cell lines, encouraging each individual to not share media bottles, cleaning the cabinet thoroughly prior to and after finishing work, neatly...

Cell culture has many uses in the research laboratory. Since the origins of cell culture in the early 20th century, cell lines have helped advance science. Cell lines have several advantages; various cell lines can help researchers study cell biology, produce baculovirus for further studies, or produce large quantities of a protein of interest, to name a few1. Some additional uses include studying tissue growth, helping to advance vaccine development, toxicology research, studying the role of genes in healthy organisms and diseased models, and the production of hybrid cell lines2,3. Cell lines can also enable drug production3. Proper aseptic techniques are necessary when working with cell lines; the practices and techniques outlined in this manuscript are applicable to research laboratories where cell culture work is performed. Other laboratory settings are not discussed.
Contamination is often the primary concern when performing cell culture work. In the context of this paper, contamination generally refers to fungi and bacteria. The overall goal of the method outlined in this paper is to thoroughly describe the best practices for avoiding contamination. All lab members should adhere to these practices when working in a research laboratory&#8217;s cell culture room. Laboratories should ensure all workers are active participants in using these best practices to prevent contamination. The knowledge of the correct practices and techniques will help ensure cell cultures remain viable, healthy, and free from contamination. The development of this technique is based on research of the literature, seven years of experience working with cell cultures, and the need for a method that both novices and experienced cell culture workers can refer to on an annual basis.
There is a need for a clear, standardized technique that all research cell culture laboratories should follow. Much of the literature on cell culture contamination discusses the detection of mycoplasma, aseptic techniques, sources of contamination, elimination of contaminants, and prevention by use of antibiotics and regular testing4,5,6,7,8. While this information is helpful, there are no videos present in the literature that demonstrate the proper cell culture techniques one should follow. The advantage of the practices presented over alternative techniques is a focus on preventing contamination before it happens, rather than detecting and correcting mistakes later. Moreover, a thorough demonstration of aseptic techniques, a discussion about preventing fungi and bacterial growth, and information regarding biosafety cabinet airflow are valuable for both novice and experienced cell culture workers alike.
Bacteria and fungi are the two most common types of contaminants. Within the bacteria category, mycoplasma is a major concern due to its small size and ability to proliferate while remaining unnoticed. They are self-replicating organisms with no rigid cell wall that rely on eukaryotic cells to grow. They have reduced metabolic capabilities and can multiply greatly while remaining unrecognized in the routine visual inspection of cell cultures and regular microscopic analysis, although transmission electron microscopy can detect mycoplasma9,10. Moreover, they can pass through microbiological filters10. Cell culture medium provides mycoplasma with nutrients, although unfortunately supplementing media with antibiotics does not affect mycoplasma10. One should note that, in general, it is not necessary to supplement media with antibiotics; proper techniques should suffice in keeping contamination at bay. Infection with mycoplasma does not lead to immediate cell death, but it is concerning to researchers as it affects data reproducibility and quality.&#160;
All lab personnel should strictly adhere to good cell culture practices. Cultures should be tested for mycoplasma after they&#8217;ve been newly purchased, while they are currently being grown, before cryopreservation, and when thawed from liquid nitrogen2,10. Different tests are available on the market using polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), or immunostaining.3 The literature indicates that &#8220;human isolates represent a large percentage of the mycoplasma contaminants found in cell culture&#8221;5. Although more than 200 mycoplasma species have been described, about six of these account for most infections. These six species are M. arginini, M. fermentans, M. hominis, M. hyorhinis, M. orale, and Acholeplasma laidlawii10. As with other types of contamination, air and aerosols bring these into cell cultures5. This is echoed in other papers since the &#8220;human operator is potentially the greatest hazard in the laboratory&#8221;7. Although this is done through human error, the risk can be eliminated if a standard procedure is followed. Shedding from personnel is not restricted to only mycoplasma contamination; cell cultures in one lab are usually infected with the same mycoplasma species, indicating that contamination spreads from one flask to another due to improper cell culture techniques10.
The prevention of cross-contamination is also another reason why proper cell culture techniques should be followed. It is noted that at least 15%&#8211;18% of cell lines worldwide may be cross-contaminated or misidentified11,12. In addition to testing cell lines for mycoplasma contamination, they should also be tested for cross-contamination10. For human cell lines, cell line authentication by an inexpensive DNA-based technique called short tandem repeat (STR) profiling is the current international reference standard, as it&#8217;s an easy way to confirm cell line identity2,10,13,14. STR can identify mislabeled or cross-contaminated cell lines, but it cannot detect incorrect tissue origin10,13,14. The validity of research data can be compromised if cell lines are mislabeled, wrongly identified, or contaminated13.&#160;Similar to other types of contamination, cross-contamination can occur due to poor technique causing aerosols to spread, mistaken contact leading to the wrong cell type entering a flask, or using the same media bottle and reagents with different cell lines10. No sharing of media bottles should occur; sharing one bottle of media between two different cell lines can allow those cell populations to be mixed, leading the faster growing cell type to completely take over the flask. This replacement is not noticeable and leads to mislabeling and misidentification2. A cell line can also be mistaken for another if cultures are confused during handling or labeling10. Careful attention should be paid to keeping reagents, media, and flasks separate from one another. Each lab member should have their own media bottles; no sharing should occur between lab members. Cell lines themselves should be purchased from a qualified cell bank and provider. Laboratories should not share cells. Studies show that, although STR and mycoplasma tests are regularly used, many research papers in the literature have already used misidentified or contaminated cell lines15. Sifting through the research to find these problematic papers and retroactively inform readers about this matter is cumbersome. Prevention is the best way to ensure this problem does not occur in the first place.
The simple action of spraying items with 70% EtOH can kill organisms; 70% EtOH works by denaturing proteins and dissolving lipids in the most commonly contaminating organisms, including bacteria and fungi16. Studies have shown that 70% is the most effective concentration; surface proteins do not coagulate rapidly with 70% EtOH so they can enter the cell, while the water it contains is necessary for the denaturing process of proteins. Due to the concentration difference of water and alcohol on either side of the cell wall, 70% EtOH enters the cell to denature both enzymatic and structural proteins. If mold growth is observed in flasks, the entire incubator must be decontaminated by first spraying it with 70% EtOH and wiping it dry, followed by a 16 h overnight incubation at 60 &#176;C17. This kills most mold and any bacteria.
The main advantage of prevention practices over alternative techniques of eliminating contamination after it occurs is that by preventing contamination early on, laboratory workers can be sure their cell cultures are healthy and there will be no high costs associated with the decontamination of incubators or discarding of cell cultures. The elimination of mycoplasma contaminants after, for instance, is not efficient7. Taking the time early on to ensure laboratory personnel are properly trained, the cell culture room is self-contained, and a standard procedure is used will save time and money.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14-190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM F-12 Media</td>
			<td>ATCC</td>
			<td>30-2006</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Baffled Flask</td>
			<td>Pyrex&#160;</td>
			<td>09-552-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Pipettes</td>
			<td>Fisher&#160;</td>
			<td>13-678-6B</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Aid</td>
			<td>Drummond</td>
			<td>13-681-15A&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological Pipette</td>
			<td>Corning</td>
			<td>07-200-573</td>
			<td></td>
		</tr>
		<tr>
			<td>T75 flask</td>
			<td>Corning</td>
			<td>07-202-004</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco</td>
			<td>25-300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>*Items may vary because this video is about general cell culture techniques</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

cell culture techniques and practices to avoid contamination by fungi and bacteria in the research cell culture laboratory

Cell culture is a delicate skill necessary for growing human, animal, and insect cells, or other tissues, in a controlled environment. The goal of the protocol is to emphasize the correct techniques used in a research laboratory to prevent contamination from fungi and bacteria. Special emphasis is placed on avoiding mycoplasma contamination, a major concern in the cell culture room due to its small size and resistance to most antibiotics used for cell culture. These same techniques ensure continuous growth and maintain healthy cells. For new and experienced cell culture users alike, it&#8217;s important to consistently adhere to these best practices to mitigate the risk of contamination. Once a year, laboratories should review cell culture best practices and follow-up with a discussion or additional training if needed. Taking early action to prevent contamination in the first place will save time and money, as compared to cleaning up after contamination occurs. Universal best practices keep cell cultures healthy, thereby reducing the need to constantly thaw new cells, purchase expensive cell culture media, and reducing the amount of incubator decontamination and downtime.&#160;

If the proper cell culture techniques and practices outlined in this paper are not followed, contamination by fungi and bacteria may occur in the research cell culture laboratory. Figure 2 shows flasks containing contamination in both the suspension and adherent cultures.
When not following aseptic techniques, mold contamination may occur 2&#8211;3 days later. Round fuzzy balls floating in the media are noticeable in suspension cells, while mold growth in attached...

Watch this Scientific Journal Video about Cell Culture Techniques and Practices to Avoid Contamination by Fungi and Bacteria in the Research Cell Culture Laboratory at JoVE.com

Cell Culture Techniques and Practices to Avoid Contamination by Fungi and Bacteria in the Research Cell Culture Laboratory

This protocol presents essential cell culture techniques and practices to be used in the research cell culture laboratory to avoid contamination by fungi and bacteria. Within the category of bacteria, special emphasis will be placed on preventing mycoplasma contamination.&#160;&#160;

Cell culture is a delicate skill necessary for growing human, animal, and insect cells, or other tissues, in a controlled environment. The goal of the ...

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