Understand what goes into cell culture medium and the incubator conditions cells need, and keep a culture log.
Why it matters
Cultured cells are grown outside the body for research, drug testing, vaccine production and biologics such as monoclonal antibodies. They only behave consistently if their environment is consistent: the same medium, the same supplements, the same temperature, gas and humidity, handled the same way. Many "irreproducible" experiments trace back to a changed serum lot or an incubator that drifted. As a technician you prepare media, keep cultures fed and record every condition.
What is in culture medium
A basal medium (such as DMEM, RPMI 1640 or MEM) supplies:
- Salts that set osmolality (roughly 260 to 340 mOsm/kg for most mammalian cells).
- Glucose (energy) and amino acids, including L-glutamine, which breaks down in solution, so it is often added fresh or replaced by a stable dipeptide form.
- Vitamins and trace elements.
- A buffer, usually sodium bicarbonate, which works with the incubator's CO2.
- Often phenol red, a pH indicator: red-pink near pH 7.4, yellow when acidic (cells overgrown or bacteria), purple when too alkaline (for example a flask left out of the incubator).
Common supplements:
| Supplement | Purpose |
|---|---|
| Fetal bovine serum (FBS), often 10 percent | Growth factors, hormones, attachment factors; varies lot to lot |
| L-glutamine (if not in the basal medium) | Amino acid that degrades over weeks |
| Penicillin-streptomycin (optional) | Antibiotics; many labs avoid routine use because it can hide poor technique |
| Specific growth factors | For defined, serum-free media used in biomanufacturing |
Biomanufacturing increasingly uses chemically defined, serum-free media, because serum is variable and is a possible source of contaminants.
Worked example: making complete medium
You need 500 mL of DMEM with 10 percent FBS, 1 percent penicillin-streptomycin (supplied at 100x) and 1 percent L-glutamine (supplied at 100x, 200 mM).
FBS: 10% of 500 mL = 50 mL
Pen-strep: 500 mL / 100 = 5 mL
L-glutamine: 500 mL / 100 = 5 mL
Basal DMEM: 500 - 50 - 5 - 5 = 440 mL
Many labs simply add supplements to a 500 mL bottle and accept a slightly larger volume; your SOP says which. Either way, label the bottle with the contents, date, your initials and lot numbers, and the expiry your lab assigns (often a few weeks at 2 to 8 °C).
Growth conditions
Most mammalian cell lines grow at:
- 37 °C.
- 5 percent CO2 (some media use 10 percent). The CO2 dissolves into the medium and, with bicarbonate, holds the pH near 7.2 to 7.4. Medium matched to the wrong CO2 level drifts in pH.
- High humidity (a water pan in the incubator) to stop evaporation, which would concentrate the medium.
Insect cells, many microbes and some specialised lines need different temperatures and gases. Adherent cells grow attached to treated plastic; suspension cells (such as CHO cells in bioreactors) grow free in shaken or stirred flasks.
Aseptic technique and the biosafety cabinet
Cell culture work happens in a Class II biological safety cabinet, which protects the worker, the culture and the environment with HEPA-filtered airflow. Core habits: disinfect the work surface and items going in (commonly with 70 percent ethanol), work in the middle of the cabinet without blocking the grilles, never pass hands over open vessels, use each sterile pipette once and keep bottles capped. Human cell lines are usually handled at biosafety level 2, because they can carry unknown viruses. The CDC's BMBL describes biosafety levels: https://www.cdc.gov/labs/bmbl/index.html (Checked on 2026-10-23.) You learn this hands-on in a teaching or employer lab with your instructor, after institution training.
Keeping a culture log
For each culture, record: cell line and source, passage number, date, medium and lot numbers (especially serum), split ratio or seeding density, confluence or cell count, appearance, and the incubator used. Incubator CO2, temperature and water level are logged too, and the incubator's CO2 reading is checked periodically with an independent meter.
Common mistakes
- Using medium made up for 10 percent CO2 in a 5 percent incubator.
- Changing the serum lot without testing or recording it.
- Letting the incubator water pan run dry.
- Relying on antibiotics instead of aseptic technique.
- Unlabelled bottles of "complete medium" of unknown age.
Quick recap
- Medium = basal medium (salts, glucose, amino acids, vitamins, bicarbonate buffer) plus supplements.
- Phenol red shows pH: yellow acidic, purple alkaline.
- Typical conditions: 37 °C, 5 percent CO2, humidified.
- Work aseptically in a biosafety cabinet, and log lots, passage numbers and conditions.
Try it
Open the Do tab to plan a batch of complete medium and design a culture log, on paper.