Gibco™ Leibovitz's L-15 Medium

Very often mycoplasma contamination cannot be removed from the culture so it should be discarded. You may have a unique culture that you prefer not to discard and would like to try to clean it. Ciprofloxacin and Plasmocin have reportedly been used for this application. If interested in a protocol or directions for use, check with the antibiotic supplier or published literature. Note that mycoplasma are very difficult to remove from culture and spread easily so the treated cultures should be quarantined until clear of mycoplasma, and your laboratory should be thoroughly cleaned.

Try changing the medium or serum. Compare media formulations for differences in glucose, amino acids, and other components. Compare an old lot of serum with a new lot. Increase initial cell inoculums. Lastly, adapt cells sequentially to new medium.

This can occur if cells are overly trypsinized. Trypsinize for a shorter time or use less trypsin. Mycoplasma contamination could also cause this problem. Segregate your culture and test for mycoplasma infection. Lastly, check for attachment factors in the medium.

This is most likely a sign of bacterial or fungal contamination. We would recommend discarding the media, and trying to decontaminate the culture.

The precipitate could be due to residual phosphate left over from detergent washing, which may precipitate powered medium components. Rinse glassware in deionized, distilled water several times, then sterilize. If the medium is frozen, try warming media to 37 degrees C and swirl to dissolve. If precipitate remains, discard medium.

Please see the possible causes for this rapid pH shift, and suggested solutions:

- Incorrect carbon dioxide tension: Increase or decrease percentage of carbon dioxide in the incubator based on concentration of sodium bicarbonate in the medium. For sodium bicarbonate concentrations of 2.0 to 3.7 g/L, use carbon dioxide amounts of 5-10%, respectively. Switch to carbon dioxide-independent medium.
- Overly tight caps on tissue culture flasks: Loosen caps one-quarter turn.
- Insufficient bicarbonate buffering: Add HEPES buffer to a final concentration of 10-25 mM.
- Incorrect salts in medium: Use an Earle's salts-based medium in a CO² environment and a Hanks' salts-based medium in atmospheric conditions.
- Bacterial, yeast, or fungal contamination: Discard culture and medium. Try to decontaminate culture.

Please see some common reasons below, and solutions to the issue:

- Growth medium is not correct: Use pre-warmed growth medium as recommended by the supplier.
- Serum in the growth medium is of poor quality: Use serum from a different lot.
- Cells have been passaged too many times: Use healthy, low-passage number cells.
- Cells were allowed to grow beyond confluency: Passage mammalian cells when they are in the log-phase before they reach confluency.
- Culture is contaminated with mycoplasma: Discard cells, media, and reagents. Obtain new stocks of cells, and use them with fresh media and reagents.

Please see the possible causes and solutions we recommend below:

- Cells were stored incorrectly: Obtain a new stock and store in liquid nitrogen. Keep cells in liquid nitrogen until thawing.
- Homemade freezer stock is not viable: Freeze cells at a density recommended by the supplier. Use low-passage cells to make your own freezer stocks. Follow procedures for freezing cells exactly as recommended by the supplier. Obtain a new stock.
- Cells were thawed incorrectly: Follow procedures for thawing cells exactly as recommended by the supplier. Make sure that you thaw the frozen cells quickly but dilute them slowly using pre-warmed growth medium before plating.
- Thawing medium is not correct: Use the medium recommended by the supplier. Make sure the medium is pre-warmed.
- Cells are too dilute: Plate thawed cells at the highest density recommended by the supplier to optimize recovery.
- Cells not handled gently: Freezing and thawing procedures are stressful to most cells. Do not vortex, bang the flasks to dislodge the cells (except when culturing insect cells), or centrifuge cells at high speeds.
- Glycerol used in the freezing medium was stored in light (if applicable): If stored in light, glycerol gets converted into acrolein, which is toxic to cells. Obtain a new stock.

There are a number of events that can contribute to this:

1. Incorrect CO² levels-monitor the level of CO² manually with a Fyrite kit, available from Bacharach (http://www.bacharach-inc.com/fyrite_analyzers.htm). Check if the manual readings concur with the readings displayed on the incubator. If the incubator has a trace readout, check the printout for fluctuations in CO² level. Check the settings to insure that CO² levels are set at appropriate levels for your cell line (usually between 5 and 10%). Check line connections frequently for leaks. Avoid frequent opening and closing of incubator doors.
2. Temperature fluctuations in the incubator-Monitor the temperature of incubator with a good thermometer inside the incubator.
3. Fungizone or other preventive antibiotics/antimycotics are present at toxic concentrations-use at recommended levels.
4. Humidity is incorrect-check the water level in the water pan. Humidity is vital to appropriate gas exchange for many types of cells and media (i.e., appropriate CO² levels are largely irrelevant for most cultures if the humidity level is not high enough).
5. Incorrect osmotic pressure in medium-check osmolality of complete medium. Most mammalian cells can tolerate an osmolality of 260 to 350 mOsm/kg. Additions of reagents such as HEPES and drugs may affect osmolality.
6. Contamination by microorganisms-bacterial and fungal contaminations are usually easily visible; symptoms of mycoplasma contamination are more subtle, and careful monitoring of culture morphology and regular testing are necessary to detect this type of contamination.
7. Inappropriate medium is being used-double-check that the medium used is appropriate for your cell type and culture application. For example, ensure that the medium being used for serum-free culture is actually designed for serum-free culture; make sure that appropriate selective drugs are used at appropriate levels; check the expiration dates for the reagents being used; and store medium at appropriate temperatures in the dark.

If the media formulation contains:
- NaHCO³ (g/L) <1.5, it needs CO² at 4%
- NaHCO³ (g/L) 1.5-2.2, it needs CO² at 5%
- NaHCO³ (g/L) 2.2-3.4, it needs CO² at 7%
- NaHCO³ (g/L) >3.5, it needs CO² at 10%

However, there are some exceptions. Gibco DMEM has always been made according to Dulbecco's original published formulation, with 3.7 g/L sodium bicarbonate. Customers have been using this medium in CO² incubators ranging from 5-10% CO² for decades, usually with no trouble maintaining physiological pH; this also depends on the cell type. Once cells are growing, the pH will drop (due to metabolic accumulation of lactic acid).

The serum concentration will vary with the cell line and basal medium used. Please go here to see our recommended sera supplementations for tested cell lines (https://www.thermofisher.com/us/en/home/life-science/cell-culture/mammalian-cell-culture/classical-media/advanced-d-mem-and-mem/recommended-sera-supplementations.html).

Yes. Our Gibco Custom Media Specialists and R&D staff can provide advice related to the addition or elimination of media components. For more comprehensive services, our PD-Direct Bioprocess Services team specializes in media development and optimization.

We have a dedicated team of Gibco Custom Media Specialists who can partner with you during customization, manufacturing, and delivery. Please email custommedia@lifetech.com or call the local phone number listed on www.thermofisher.com/mediaconfigurator with inquiries about your submission. For questions related to website effectiveness, please contact System Support through the toll-free or local phone number.

Using the Gibco Media Configurator, you can view specific customization options for your cell culture product, online, at any time. Also, this tool provides faster pricing for custom media, generally within 48 business hours.

No, the Gibco Media Configurator cannot be used to request pricing on non-Gibco cell culture products. However, we are very experienced at manufacturing customer-owned media formulations. Please visit www.thermofisher.com/custommedia or contact us at custommedia@lifetech.com for more information.

The Gibco Media Configurator allows you to add, remove, and adjust the concentration of components. In addition, you can select from a range of packaging, QC tests, and either cGMP or non-cGMP manufacturing.

Almost all Gibco cell culture basal media products can be customized using the Gibco Media Configurator. To check on a particular product, use the Media Configurator page (https://www.thermofisher.com/us/en/home/life-science/bioproduction/custom-cell-culture-media-and-services/gibco-custom-media-configurator.html to search by Gibco catalog number, or browse our offering by description. For the customization of non-media Gibco products, such as buffers and growth factors, contact us at custommedia@lifetech.com.

No, you do not need to adjust the pH of the liquid media when you receive it. It should be in the correct pH range for use. After you add your supplements (if needed), you can check the pH again and adjust if necessary. However, usually it does not change significantly. It would be necessary to pH media that you make up yourself from a powder, with the exception of AGT media where pH and osmolality are auto-adjusted.

Gibco cell culture media is formulated using water meeting all USP monograph requirements for Water For Injection.

You can find a protocol on our web site. In the main search box type “Media Preparation from Powder and Concentrates” and hit Enter or click Search. This should take you to a detailed protocol page.

Aseptic technique, designed to provide a barrier between the microrganisms in the environment and the sterile cell culture, depends upon a set of procedures to reduce the probability of contamination from these sources. The elements of aseptic technique are an aseptic work area, good personal hygiene, sterile (or sterile filtered) reagents and media, and aseptic handling. Go here to read more (https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/aseptic-technique.html) about aseptic technique or view our checklist (https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/aseptic-technique/aseptic-techniques-checklist.html).

Most normal mammalian cell lines grow well at pH 7.4, and there is very little variability among different cell strains. While most researchers usually use 5-7% CO² in air, 4-10% CO² is common for most cell culture experiments. Read more about buffering conditions here (https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/cell-culture-environment/ph-CO²-levels.html).

Please see the comparison chart here (https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/cell-culture-useful-numbers.html).

Yes. Store animal sera at -5 to -20 degrees C. Store media at 2 to 8 degrees C; use within recommended shelf life period. Store complete media (supplemented) at 2 to 8 degrees C, and for complete medium the recommended shelf life is 2 to 4 weeks. Additionally, minimize exposure of sera and media to light.

Please use our media formulation search tool (https://www.thermofisher.com/search/results?docTypes=MediaFormulation&persona=DocSupport&linkIn=true&query=).