Media & supplements
The two workhorse basal media. DMEM is a high-glucose, high-bicarbonate enrichment of Eagle's medium used for most adherent lines; RPMI 1640 was developed for lymphoid cells and is the default for suspension blood-cell lines.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
DMEM / RPMI
The two workhorse basal media. DMEM is a high-glucose, high-bicarbonate enrichment of Eagle's medium used for most adherent lines; RPMI 1640 was developed for lymphoid cells and is the default for suspension blood-cell lines.
How it differs from the standard method
- DMEM: 25 mM glucose (high-glucose version), 4 mM glutamine, 3.7 g/L (44 mM) bicarbonate; nominally for 10% CO₂ but widely used at 5%.
- RPMI 1640: 11 mM glucose, 2 mM glutamine, 2 g/L (24 mM) bicarbonate, for 5% CO₂; contains glutathione and more vitamins.
- Low-glucose DMEM, DMEM/F12 and others exist; match what the line's supplier recommends.
1What a medium has to do
A culture medium stands in for blood and tissue fluid. The basal medium is a buffered salt solution with glucose, amino acids, vitamins and trace elements in roughly the proportions a cell type uses them.
High glucose, high bicarbonate
25 mM glucose, 4 mM glutamine, 44 mM HCO₃⁻. For many adherent lines: HEK293, HeLa, fibroblasts.
Developed for lymphocytes
11 mM glucose, 2 mM glutamine, 24 mM HCO₃⁻, glutathione. For Jurkat, many leukaemia and lymphoma lines, and hybridomas.
Eagle's minimal recipe
5.5 mM glucose and fewer components. Fine for some robust lines; thin for demanding ones.
2Serum, glutamine and antibiotics
Fetal bovine serum is undefined and variable: lots differ in growth factors, hormones and endotoxin, so labs test a new lot on their own lines before switching and then reserve a supply. It is thawed slowly, aliquoted, and not refrozen repeatedly.
3How it goes wrong
- CO₂ supply runs out or the incubator drifts: medium turns purple, cells stop dividing.
- Bicarbonate medium titrated to 7.4 on the bench: the buffer is destroyed and the medium is acid once it equilibrates.
- A line grown in the wrong basal medium or with too little serum: slower growth and drifting phenotype.
- Old medium: glutamine gone, ammonia high, cells grow slowly with no obvious cause.
- An untested serum lot changes growth or an assay result overnight.
- Supplements added outside the cabinet contaminate every flask fed from that bottle.
Key terms
Practise these as flashcards →The defined mixture of salts, glucose, amino acids and vitamins before supplements (DMEM, RPMI 1640, MEM, DMEM/F12).
Basal medium plus the supplements a line needs, typically serum and glutamine, ready to use.
The physiological buffer system of most media; its pH is set by the bicarbonate concentration and the CO₂ in the atmosphere.
pH indicator in most media: yellow below ~6.8, red near 7.4, purple above ~7.8.
Holding serum at 56 °C for 30 min to destroy complement activity.
Comparing growth (and assay behaviour) on a new serum lot against the current one before switching.
A medium in which every component and its concentration is known: no serum, hydrolysates or tissue extracts.
Stepwise reduction of serum so that cells adapt to a serum-free medium over several passages.
When to use it
- Setting up a new line: matching the supplier's medium, serum and CO₂.
- Making up and storing complete medium so that it behaves the same from week to week.
- Moving a line to serum-free or chemically defined medium for protein production, reproducibility or regulatory reasons.
- Troubleshooting slow growth, odd colours or drifting pH.
Compared with alternatives
The general skills of weighing, dissolving, adjusting pH and sterilising solutions; culture media add the CO₂ buffer and labile supplements.
How a medium or supplement you have made up yourself is made sterile without heat.
The routine the medium supports; feeding schedules and split ratios depend on how fast the medium is used up.
What to do instead of relying on antibiotics: routine sterility and mycoplasma testing.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
The two workhorse basal media. DMEM is a high-glucose, high-bicarbonate enrichment of Eagle's medium used for most adherent lines; RPMI 1640 was developed for lymphoid cells and is the default for suspension blood-cell lines.
- DMEM: 25 mM glucose (high-glucose version), 4 mM glutamine, 3.7 g/L (44 mM) bicarbonate; nominally for 10% CO₂ but widely used at 5%.
- RPMI 1640: 11 mM glucose, 2 mM glutamine, 2 g/L (24 mM) bicarbonate, for 5% CO₂; contains glutathione and more vitamins.
- Low-glucose DMEM, DMEM/F12 and others exist; match what the line's supplier recommends.