What is Sterile? Find Your Way around a Sterile Tissue Culture Hood

You’ve been told that maintaining a sterile environment in a tissue culture hood is vital to preventing contamination of cell cultures. But what exactly is meant by sterile? The definition of sterile is ‘completely clean, sanitized, and free of all forms of life’. Obviously you still want your cells and/or any other organisms you are studying to live, but any reagents or equipment that are used for tissue culture should be sterile. A better word for how you want to work in the hood is asepsis, or the state of being free from biological contaminants. Aseptic technique will ensure that only the things you want to grow in your culture will. If you are mindful of sources of contamination and work carefully, then you will be able to work effectively in a tissue culture hood. Below are several tips for good aseptic technique. Plan your experiments.  Plan your experiments ahead of time so you can make sure that you have time to sterilize all reagents and equipment. Make a list of everything you will need in the hood to minimize leaving the hood during your experiment. Set up your hood with everything needed for the experiment prior to starting your work. Be good go your hood.   A clean, functioning tissue culture hood is critical for aseptic work.  Make sure you know how to use the hood properly. If you have questions you can start by reading Bitesize Bio’s Quick Protocol and Ways to Abuse Biological Safety Cabinets. A qualified technician should certify the hood annually, and a sticker on the hood should indicate certification. Take apart and deep clean the hood about twice a year. Take out all removable parts, clean with disinfectant and sterilize in the autoclave. Every time you turn the hood on, make sure the fan is working properly by reading the magnahelic gauge (Figure 1). Clean the hood with disinfectant before and after every experiment. If you spill something, clean it up as soon as you can without compromising your experiment. This includes checking and cleaning the drip pans if large amounts of liquid are spilled. Do not store unnecessary equipment or reagents in the hood: this will make it harder to keep the hood clean.

Start clean.  Make sure everything you bring into the hood is clean. Sterilize or sterile filter (with a 0.22micron filter) all media and reagents. Designate store bought reagents for tissue culture use only. Sterilize or disinfectant all equipment used in the hood. Remember to spray the outside of all bottles with disinfectant prior to placing them in the hood. Prior to starting your work, wash your hands well, put on clean gloves and make sure your clothing does not bring in unwanted organisms. Many labs designate lab coats for tissue culture use. If you use a lab coat, make sure it is cleaned on a regular basis. Work from clean to dirty.  This concept should be used throughout your day as you move from experiment to experiment, and as you work on each experiment within the hood. Plan your day so that you do your clean experiments first and your dirty experiments later on. For example, I usually split cells for maintaining cell lines in the morning then do any work infecting tissue culture cells with viruses in the afternoon. Within the hood I usually designate the right side of the hood my “clean” side and the left side my “dirty” side. As much as possible I work from the clean side over to the dirty side. Don’t trap things with your trap.  If you use a vacuum trap for removing large amounts of liquid, make sure you take care of the trap. On a regular basis inspect all hoses and lines to make sure nothing is growing in them. Clean the outside of all hoses and lines within the hood with disinfectant before and after every experiment. If the vacuum hose is stored in the hood, make sure liquid is not dripping out of the hose into the hood when the vacuum is turned off. Do not allow the trap to overfill. Empty and clean the collection flask on a regular basis, preferably at the end of your experiment. If the vacuum hoses are connected to a filter, check the filter when you empty the flask to make sure it is not wet; replace the filter as needed. Knowing how to find your way around a tissue culture hood using aseptic technique will prevent contamination of your experiments with unwanted organisms. Follow these tips and you will have a good start!