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 various types of cells and tissues in a controlled laboratory setting. The primary concern when performing cell culture work is contamination. This contamination can come in the form of bacteria, mold or mycoplasma.

The techniques and practices all outlined in this video will ensure that your cells remain viable, healthy and contamination free. These practices should be strictly adhered to, and all lab personnel should receive training at least once a year. Before beginning work, wear a clean lab coat designated for use in the cell culture room only.

Wear new gloves that have not touched any other surfaces. Make sure the gloves fit tightly. Nitrile powder-free gloves are best.

The lab coat and gloves do not need to be sterile. To prepare for work, spray gloves, lab coat sleeves and the interior of the biological safety cabinet with 70%ethanol. Spray the working surface, the sides, the back, any items in the cabinet and the front glass panel, both from the inside and outside.

Wipe the working surface and glass panel dry with a lint-free paper towel. Take some time to arrange the workspace inside the cabinet. Keep water baths inside the cell culture room and only use these for cell culture purposes, such as warming media or thawing cells.

The water baths should be drained and washed once a week. Limit the amount of items brought into the cabinet and do not interrupt the air airflow inside the biological safety cabinet by blocking the front or back grills. Spray all items placed inside the cabinet with 70%ethanol and wipe them dry.

Begin by spraying the top of the media bottle and work your way down. With a clean paper towel, start from the top of the media bottle to wipe it dry, progressively working your way to the bottom. Do not go back up towards the cap.

The way these cabinets work is that they pull air from the room and the cabinet itself through the front and back grills. The air goes towards the back of the cabinet and up to the top. There, contaminated air goes through heap of filters, and part of the filtered clean air gets pushed down upon the working area in the cabinet.

If the cabinet is large enough to accommodate serological pipettes, they can be placed inside, otherwise, they can be stored in a receptacle mounted on the outside of the cabinet. It's good practice to check either side of the encased pipette for holes, tears or punctures in the packaging before use. Do not rip off the wrapping.

Instead, gently peel the ends of the wrapping. Insert the serological pipette into a pipette aid and remove the wrapping in one fluid motion. Do not hover over any open bottles or flasks.

Reaching over an open bottle or flask, or working above open flasks is not recommended. The airflow inside the cabinets push down upon anything inside. Reaching over will allow your sleeve or glove to be on top of items which could potentially push down any contamination present on your sleeve for instance, into your cell culture.

Whenever possible, do not pour liquids. Instead, add them using a serological pipette. Mix contents thoroughly and initial the bottle.

Write down what the media is supplemented with. If you're working with adherent cultures and need a plastic flask, then spray the entire package and get it inside. Anytime your gloves have dried up, spray them with the ethanol again.

When working with an adherent cell line, use autoclaved glass pipettes or disposable sterile plastic pipettes to aspirate media or washing solutions. Carefully remove the metal cap from the storage container. Isolate one glass pipette by gently shaking the container at an angle.

When reaching into the container, avoid touching any other pipettes. Handle the chosen pipette from one end only. Quickly replace caps on bottles and flasks as soon as possible.

Place caps on the work surface upside down so that the rim does not touch the work surface. Do not grab the cap from the top or bottom. Instead, touch caps from the sides.

When aspirating liquids, use a vacuum trap flask located outside of the cabinet in a secondary container on the floor. Waste will be created while working inside the cabinet. Because moving hands in and out of the cabinet too often will interrupt the airflow, it's fine to temporarily leave any waste inside the cabinet.

Place it off to the side so it will not interrupt your work. Generously spray gloves with 70%ethanol anytime they become dry. Rub your hands together so that they're not dripping wet.

A quick note about serological pipettes. If they accidentally touch something else in the hood, do not hesitate to throw them out. It's best to start fresh with a clean serological pipette instead of using one that may be contaminated.

Before placing cells in the incubator, check to see how they look under the microscope. Single cells should be observed. This indicates the cells have been resuspended thoroughly.

Do not speak, sneeze, cough or breathe heavily into the incubators. Quickly open and close incubator doors. Leaving them open for longer than necessary may allow contaminants present in the air to enter the incubators.

Ensure the caps on all bottles are thoroughly closed before removing them out of the cabinet. Media is light sensitive. It should be stored in the dark at four degrees Celsius when not in use.

Spray the interior of the cabinet with 70%ethanol again after cell culture work is completed and wipe the surface dry with a paper towel. Empty the biohazard waste bag. Repeat this process and replace gloves when switching to a different cell line.

When working with suspension cultures grown in glass flasks, ensure gloves are wet. Touch the aluminum foil with your wet gloves and then only spray the bottom of the flask before placing it inside the biological safety cabinet. When taking a sample for cell counting, remove only one 1.5 ml tube from its container.

Do not touch any of the other tubes. Place the cap upside down on the working surface. Do not touch the inside rim.

Handle it with care from the sides and replace it once you're done. When handling suspension cells and glass flasks, carefully remove the double folded piece of aluminum foil that covers the entire neck of the flask. Gently pull on all four corners and place the foil upside down on the working surface.

Handle the glass flask from the bottom only. Do not touch it from the neck once the foil is off. Use a 1 ml serological pipette to take a sample for cell counting.

Do not let media drip down the side of flasks. If it does, spray paper towel with 70%ethanol and clean it up right away. Ensure the caps or aluminum foil are tightened before removing bottles or flasks from the biological safety cabinets.

Use separate incubators for different cell types. This will prevent cross-contamination of different cell types. Placing the vacuum trap flask for collecting liquid waste outside of the cabinet on the floor in a secondary container labeled waste is recommended.

The hose is connected to a filter which is replaced on a monthly basis. The hose goes in through the side of the cabinet. Remove the biohazard trash once work is completed.

Keep a glass washing protocol on the wall by the sink. First, soak the discarded cells with 10%bleach for about five minutes. Then discard the bleach mixture and rinse the flask thoroughly with water.

Add soap with water, scrub it with a brush very well, rinse with more water. And then the final step is to rinse the glassware with the DI water on the tap on the right. Wash glass flasks as soon as possible, scrub the glassware well.

Sf9 cells will leave this rim of dead cells if it's not scrubbed properly. Use these flasks only for cell culture, autoclave cell culture glassware instead of sending it to a glass washing facility. Use the vacuum cycle on the autoclave for 40 minutes sterilized time and 40 minutes dry time.

Keep it separate from glassware in the main area of the lab. Organize the cell culture room so that all supplies are located in one area, thereby minimizing the need for lab members to exit the cell culture room in search of supplies. Label any plastic bottles used to harvest cells and reused in cell culture afterwards, for cell culture use only.

Use the designated cell culture bottles for one specific cell type. Store these bottles in the cell culture room for easy access. For cultures that are grown in suspension, mold will be recognized as floating balls in the media.

As for bacterial contamination, it will turn the media white and cloudy. Similarly, for adherent cells, mold contamination can be easily recognized as fuzzy patches. Bacterial contamination will turn normally clear media, white and cloudy.

Many species of mycoplasma can be reliably identified using a PCR based assay. The band on the left shows the molecular weight standards for DNA. The four bands on the right are positive controls.

No bands appear under tested cell types because mycoplasma was not detected. Cell culture media will change color from red to yellow if pH indicators are present. The yellow color indicates the pH is low and the media should be replaced.

If other growths or cell shapes are observed under the light microscope while performing cell counts, then this may be an indicator of contamination. Note, that a thorough cleansing of the hemocytometer should be done prior to cell counting as this type of debris may be present only on the hemocytometer and not in the cell culture itself. While contamination is one of the primary concerns when performing cell culture work, these risks can be mitigated by following the practices and techniques outlined in this video.

If a monthly mycoplasma test is performed every month, lab personnel are trained on a yearly basis and these practices are strictly adhered to, then your cells will remain healthy and contamination free. This will also help reduce costs associated with purchasing expensive cell culture media, purchasing new cells, and it will reduce incubator decontamination downtime. Remember, the key to successful cell culture is early action in order to prevent contamination in the first place.