1. Preparations
2. Working with adherent cell lines
4. Checking and storing cells
5. Working with suspension cell lines
6. Cell incubation
7. Liquid waste collection
8. Cleanup
9. Organization
10. Identifying bacteria, fungi, and mycoplasma contamination
NOTE: Not following the workflow above can lead to bacterial, fungi, and mycoplasma contamination.
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 2–3 days later. Round fuzzy balls floating in the media are noticeable in suspension cells, while mold growth in attached cells can be observed as large, irregular, white, or green patches.
For bacteria, contamination is observed the following day. The media is turbulent, white, and cloudy. The white color is typical of bacterial cells, which multiply much more rapidly than cell lines. An experienced eye is able to tell the difference between non-contaminated media and contaminated media. For attached cells, one can compare a bottle of unopened media with a flask to check if any turbulence is seen in the flask.
Inside the biological safety cabinet, the number of items should be kept at a minimum. Avoid placing items on the back and front grills (see Figure 3). In this cabinet, a set of pipettes, tips, autoclaved glass pipettes, a pipette aid, and markers are inside. The working area in the middle is clear. Keeping cabinets organized in this way is a good idea. In addition, the operator should wear a clean lab coat and gloves before beginning work. A spray bottle with 70% EtOH should be kept nearby so the operator can spray their gloves often. The skin is covered by gloves or a lab coat. The operator should adjust their chair so their arms are at a 90° angle when working inside the cabinet, and items should be within easy reach inside the cabinet (see Figure 4).
Many species of mycoplasma can be reliably identified using a PCR-based assay. Figure 5 shows the results of a negative mycoplasma test. 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 the tested cell types because mycoplasma was not detected.
If the media contains pH indicators, it will be red for the optimal pH value of cells at 7.4. Once the cells grow, the media will change color from red to yellow3. This color change can also occur if bacteria take over the flask and overgrow (Figure 6). The yellow color indicates the pH is low. Observing a new, unopened bottle of media next to a flask is an objective way to observe contamination in attached cells. For suspension cultures, the user can closely observe the flask for any growths floating in the media or whether a thick ring of overgrown bacterial cells is present around the inside of the glass flask. For both cell types, a small sample can be taken and observed under the microscope. If other growths or cell shapes are observed, especially if the cells are moving, then this is an indicator of contamination (Figure 7). A thorough cleansing of the hemocytometer should be performed prior to cell counting, as this type of debris may be present only on the hemocytometer and not in the cell cultures themselves.
The smell may be another indicator of contamination in an incubator. Bacterial overgrowth has a typical smell that an experienced cell culture user will notice. The smell always coincides with a contaminated flask, although one infected flask may not always cause the entire incubator to smell.
Mold contamination tends to be more prevalent in HEK 293 S cells grown in suspension. Bacterial contamination is more common in SF-9 cells. This may be because RIC cells are grown with humidity, thereby leading to moisture accumulation in the incubators. SF-9 cells are grown without humidity, so the environment is drier. The rate of contamination in adherent cultures is less than the rate of contamination in suspension cultures. This may be due to the smaller flask size, the non-reusable nature of the flask, or the vented cap instead of the use of aluminum foil.
Mycoplasma cannot be observed with the naked eye nor with a regular light microscope, although specialized transmission electron microscopy can detect mycoplasma. A mycoplasma identification kit should be used to test the cell cultures monthly. Many species of mycoplasma can be reliably identified using a PCR-based assay. A brief description about how this PCR test is performed can be found in the protocol section, and more information about mycoplasma can be found in the discussion section.
Figure 1: How air flows in a biosafety cabinet. Biological safety cabinets pull contaminated air from the room itself and from the cabinet through the front and back grills. This air goes under the metal working surface, toward the back of the cabinet, and up to the top of the unit where a HEPA filter is located. There, the air passes through the filter and gets filtered. This clean air pushes down on the work surface. Due to how the flow of filtered air is pushed down inside the cabinet, it is good practice to not hover. For example, it is undesirable to have a sleeve on top of an open bottle and risk having any potential contaminants be pushed into the media. The number of items brought inside the cabinet should be kept at a minimum, and items should not be placed on the front or back grills in order not to interrupt the air flow. Moving arms in and out of the cabinet too quickly can also disturb the airflow. Created for NuAire, Inc., by Jeff Kaphingst, Jeff the Designer, LLC. Used with permission. Please click here to view a larger version of this figure.
Figure 2: Non-contaminated suspension and adherent cells and cells contaminated with mold or bacteria. The first image on the left contains insect cells (SF-9 cells) that are not contaminated. The second image shows another flask of these cells contaminated with mold. The third flask was contaminated by bacteria, as can be noted by the thick, white, cloudy appearance. The second and third flasks were contaminated because none of the proper cell culture techniques and practices were followed. All flasks were prepared on the same day. Growth was observed the next day for bacterial contamination and 2 days later for mold contamination. Non-contaminated adherent cells are shown (Hek293 cells) along with mold and bacterial contamination in adherent cultures. Mold contamination is shown in the second line in a round Petri dish. The photo is taken from the top of the dish. Please click here to view a larger version of this figure.
Figure 3: Cell culture cabinet organization. Inside the biological safety cabinet, the amount of items should be kept at a minimum. Placing items on the back and front grills should be avoided. In this cabinet, a set of pipettes, tips, autoclaved glass pipettes, a pipette aid, and markers are inside. The working area in the middle is clear. Please click here to view a larger version of this figure.
Figure 4: The correct way for an operator to work under the flow hood. An operator should wear a clean lab coat and gloves before beginning work. A spray bottle with 70% EtOH should be kept nearby so the operator can spray their gloves often. The skin is covered by the gloves or lab coat. The operator should adjust their chair so their arms are at a 90° angle when working inside the cabinet. Items should be within easy reach inside the cabinet. Please click here to view a larger version of this figure.
Figure 5: A negative mycoplasma test result. 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 the tested cell types because mycoplasma was not detected. Please click here to view a larger version of this figure.
Figure 6: Normal media color change from red to yellow. Cell culture media changes the color from red to yellow if pH indicators are present. The yellow color indicates the pH is low and the media should be replaced. Please click here to view a larger version of this figure.
Figure 7: Contaminants observed under the light microscope. If other growths or cell shapes are observed under the light microscope while performing cell counts, then this may be an indicator of contamination. It should be noted that a thorough cleansing of the hemocytometer should be performed prior to cell counting as this type of debris may be present only on the hemocytometer and not in the cell cultures themselves. Please click here to view a larger version of this figure.
Supplementary File 1: Appendix Please click here to download this File.
DPBS | Gibco | 14-190-144 | |
DMEM F-12 Media | ATCC | 30-2006 | |
Glass Baffled Flask | Pyrex | 09-552-40 | |
Glass Pipettes | Fisher | 13-678-6B | |
Pipette Aid | Drummond | 13-681-15A | |
Serological Pipette | Corning | 07-200-573 | |
T75 flask | Corning | 07-202-004 | |
Trypsin | Gibco | 25-300-054 | |
*Items may vary because this video is about general cell culture techniques |
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’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.
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’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.
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’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.