Biotech Basics: Aseptic Techniques

What are they?

Aseptic techniques are a large set of practices that prevent contamination by eliminating the unintentional transfer of microorganisms and cells. In general, these practices involve a combination of environmental control, personal hygiene, and sterilizing equipment and media.

Scientists in fields like microbiology, medical research, cell based agriculture, and molecular biology use these techniques to guarantee experiment success and consistency. In particular, scientists whose experiments involve cells must practice aseptic techniques to reduce the risk of contamination by microorganisms and/or different types of cells.

Healthcare workers also use aseptic techniques to minimize the spread of infections. To find out more about medical aseptic techniques read this article or this more detailed report.

Why practice it?

In many research labs cells are grown – or “cultured” – in growth media. This is a chemically complex solution that provides the nutrients necessary for cells to grow. Media typically contains essential amino acids, buffers, salts, and a carbon source like glucose. Many also contain additional hormones and growth factors. These media are a great environment for cells to flourish but also serve as a place for other forms of life to live and thrive.

At the same time, microorganisms are ubiquitous. Thousands of species can be found on most surfaces – including human skins – as well as in the air. Consequently, everything that comes in contact with the culture (e.g. the cells in their growth media) must be as sterile as possible. 

Contamination can stop an experiment in its tracks or lead to incorrect conclusions. In addition, many labs have “lines” of cells that are maintained for many years through a process called cell passaging. In these cases, contamination can destroy an essential part of the living lab equipment. While some cell cultures are readily available and can be purchased for a few hundred dollars others are far more expensive or completely irreplaceable. 

Know your enemy

Knowing and practicing the key steps of aseptic technique is important (many of these are listed in the next section). Understanding what can contaminate a culture and how these contaminants behave is equally essential. Different places, media, and cell types have specific contamination threats. However, the usual suspects fall into the following categories:  

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• Bacteria: These are a prolific and highly diverse domain of life consisting of single-celled prokaryotic organisms. Media contaminated with bacteria will appear cloudy or may have a white/cream/pink/yellow film on the surface. Under the microscope small, granular cells will be visible as black dots.

• Fungi: These are eukaryotes like molds and mushrooms that are heterotrophs and that have cell walls with chitin. Like bacteria, this kingdom also boasts tremendous diversity. Contaminated cell cultures often have thin threadlike strands called mycelia that form a fuzzy white or black growth easily seen by the naked eye.

• Yeasts: Yeasts are also members of the fungal kingdom. However, in cell culture, they are considered separately because of their unique appearance. Contaminated media will have small round particles that are often in chains of two or more. 

• Other cells: Often labs have multiple cell lines. In these labs, contamination with another cell line is a large risk. Contaminating cells can also come from the individuals in the lab. Detecting this contamination can be easy or incredibly hard depending on the cell types involved. 

• Viruses: These are small infectious agents that replicate only inside living cells. Viruses alter the growth, behavior, and survival of cells. Because they are much smaller than cells they cannot be detected by most standard magnification microscopes. 

The Golden Dos and Don’ts of Cell Culture Aseptic Technique

DO choose a designated area for your experiment. This area should be in a low traffic area and far away from any windows or air vents. Often labs use specialized hoods that are easy to sterilize and help control airflow. Top of the line cell hoods can also prevent viral infections and provide some level of user protection.

DO have a plan for the day. Make a list and then gather everything you’ll need. Also, schedule your experiments so that the day starts will cell lines that are expensive or difficult to replace and with simple procedures that do not involve additional contaminants. 

DO sterilize the hood before beginning using 70% Ethanol or Methanol. Do the same for any pieces of equipment or bottles that you plan to bring into the work area. 

DO wear protective gear like lab coats, face masks, and gloves. These serve as barriers that protect both you (the user) and the cell culture. During the experiment change gloves or disinfect them by spraying them with 70% ethanol. 

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DON’T clutter your work area with unneeded items. If you have too many things close to you, you will inevitably brush the tip of a sterile pipette against a non-sterile surface. 

DON’T keep open bottles and flask vertical. Lay them flat or if they are full of solution keep them at a shallow angle. 

DON’T pour from one sterile container to another unless the bottle you are pouring will be used only once and will deliver all its contents in one transfer. 

DO close open bottles and cover plates/trays whenever possible. 

DO wash hands before and after handling any cell culture material, even if you are wearing gloves.

DO inspect cultures regularly. If contamination happens stay calm. While prevention is the best strategy, many situations can be addressed by adding antibiotics or antifungal medicines.

How to practice it a home

In some situations, a sterile lab with easy access to advanced equipment like an autoclave and tissue culture hood isn’t available. However, that’s no reason to throw up your hands and call it day. Many of the early discoveries in microbiology and cell studies were made under much less sterile conditions than your average classroom or home lab. Just remember to follow the above steps to the best of your ability, stay adaptable, and prioritize safety. 

Some additional ideas:

• Carry out your work in a box. An empty box that’s been wiped with 70% ethanol makes an excellent makeshift incubator and/or hood. 
• Location still matters. Choose somewhere free of heavy traffic, clean, and away from windows or air vents.   
• Use blue flames to sterilize metal equipment like transfer loops.  
• Wash your hands before and after. Keep your hair back or covered. Reserve a set of long-sleeved clothing for your cell work. And when possible still use gloves that go over the sleeves.

If you’re ready to get your gloved hands “dirty” and practice these skills check out our cell culture kits: Eukaryotic Cell Biology (Kit #1001) or Cell Culture Toxicity Screening (Kit #1002). Or try growing a plant not from a seed but from leaf, root, or bud cutting with our kit Introduction to Plant Cell Culture (Kit #908).