Penicillin-Streptomycin and Antibiotic-Antimycotic in Cell Culture

Pen-strep is sold as a 100X concentrate, which is the only format you will normally encounter. The standard composition is fixed across suppliers:

Penicillin-Streptomycin, 100X

Antibiotic-Antimycotic, 100X

Both are typically formulated in 0.85-0.9% saline and sterile-filtered. To use, add 10 mL of 100X solution per litre of medium, or 5 mL per 500 mL bottle.

Note that penicillin is quoted in units, not mass, which is a historical convention from the days when penicillin preparations were of variable purity. For penicillin G sodium, 1 unit is approximately 0.6 ug, so 100 U/mL corresponds to roughly 60 ug/mL. Streptomycin is quoted by mass. Do not attempt to convert one to the other or assume the two components are present at equal potency.

A lower option. ATCC recommends a final concentration of 50-100 IU/mL penicillin and 50-100 ug/mL streptomycin where antibiotics are used at all. Working at the bottom of that range -- effectively 0.5X -- reduces the metabolic and off-target burden while retaining most of the antibacterial effect, and is worth considering for sensitive cells.

Penicillin G is a beta-lactam produced by Penicillium moulds. It binds penicillin-binding proteins and blocks the transpeptidase-catalysed cross-linking step of peptidoglycan synthesis, so bacteria cannot complete a functional cell wall and lyse as they attempt to divide. Because it acts on cell wall assembly, it is bactericidal only against actively growing organisms. Its spectrum in a cell culture context is predominantly Gram-positive.

Streptomycin is an aminoglycoside from Streptomyces griseus. It binds the 30S ribosomal subunit, causing misreading of mRNA and blocking translation initiation, which is bactericidal. Its coverage is predominantly Gram-negative, with useful activity against some Gram-positives. The complementary spectra are exactly why the two are combined: between them they cover the bacterial contaminants that most often reach a culture flask from skin, air and water.

Amphotericin B, the antimycotic component, is a polyene macrolide from Streptomyces nodosus and is often listed under the trade name Fungizone. It binds ergosterol in fungal membranes and assembles into pores that leak ions and small molecules, killing yeasts and moulds.

Amphotericin B carries a specific caution. Its selectivity comes from a preference for ergosterol over cholesterol, but that preference is a matter of degree rather than absolute. At concentrations above roughly 2.5 ug/mL it becomes measurably toxic to mammalian cells, which is why the working concentration is held at 0.25 ug/mL -- a tenfold margin, which is narrower than most people assume. It is also unstable, light-sensitive, and prone to precipitating out of solution. If a culture supplemented with anti-anti grows more slowly than the same cells in pen-strep alone, amphotericin B is the first thing to suspect.

Gentamicin, supplied separately, is a broader-spectrum aminoglycoside used at 10-50 ug/mL. It is considerably more stable in medium than penicillin and is often preferred for primary tissue work and for long culture periods where penicillin activity would decay.

The two components of pen-strep do not degrade at the same rate, and this matters more than it is usually given credit for.

Penicillin G is chemically fragile. The beta-lactam ring hydrolyses in aqueous solution, and the process accelerates at 37 C and at alkaline pH. In complete medium at incubator temperature, penicillin loses a substantial fraction of its activity within about three days and is largely spent within roughly five. A flask fed on Monday has meaningfully less penicillin coverage by Thursday.

Streptomycin is considerably more stable and retains useful activity across a normal medium-change interval.

The practical consequence is that pen-strep in a culture that is fed weekly is, for much of that week, effectively streptomycin alone with residual penicillin. This partly explains the common observation that Gram-positive contamination breaks through in cultures that are nominally protected. If antibiotic coverage genuinely matters for a particular experiment, it argues for more frequent medium changes or for a more stable agent such as gentamicin, rather than for a higher pen-strep dose.

Storage of the concentrate. Store 100X solutions frozen at -5 to -20 C, protected from light. Thaw at 2-8 C or at room temperature rather than at 37 C, mix gently, and avoid repeated freeze-thaw. Aliquot a large bottle into single-use volumes on first thaw. Once diluted into medium, treat the medium as having a limited antibiotic life as described above.

Do not autoclave or heat these solutions, and do not add the concentrate to medium that is being warmed at 37 C for extended periods before use.

This is the question most people are actually asking when they search for pen-strep, and it deserves a direct answer rather than a product description.

The consensus among cell banks and experienced culture labs is that routine, continuous antibiotic use in healthy established cultures is not good practice. ATCC states plainly that it does not use antibiotics or antimycotics for routine cell culture, and that routine use is not recommended unless specifically required -- for example, a selection agent such as G418 maintaining pressure on a transfected line. That position is not squeamishness; it rests on several concrete problems.

Antibiotics mask contamination rather than preventing it. This is the central argument. Pen-strep does not sterilise a culture; it suppresses bacterial growth to below the level at which you would notice it. A low-level contaminant that would have turned the medium cloudy in two days and been discovered immediately can instead persist quietly for months, altering pH, consuming nutrients, releasing endotoxin and shifting your cells' behaviour, while the flask looks normal. The contamination is then discovered late, after it has spread to other cultures and after it has silently influenced results.

They do nothing about mycoplasma. Mycoplasma has no cell wall, so penicillin has no target at all. Streptomycin at 100 ug/mL is not reliably effective either. Mycoplasma is by a wide margin the most consequential culture contaminant -- it is invisible, it is present in a substantial fraction of cultures in circulation, and it changes metabolism, growth rate and gene expression. Pen-strep provides no protection whatsoever against it, and by suppressing the visible bacterial contamination that would have prompted you to discard a suspect flask, it can extend the life of a mycoplasma-positive culture.

They are not biologically inert to your cells. Published work has shown that antibiotics at standard culture concentrations alter gene expression in mammalian cells, and can affect functional readouts -- one study found antibiotics inhibited sphere-forming ability in suspension culture. Amphotericin B in particular has a narrow margin between its antifungal and cytotoxic concentrations. If your experiment measures anything subtle, antibiotics are an uncontrolled variable in it.

They select for resistance. Continuous sub-lethal exposure in a warm, nutrient-rich environment is close to ideal conditions for selecting resistant organisms. Labs that use antibiotics continuously tend, over time, to acquire contaminants that antibiotics no longer touch.

They substitute for technique. The most damaging effect is cultural rather than biological. A lab that relies on antibiotics stops discovering its own aseptic failures, because the feedback signal -- a cloudy flask the day after a sloppy hood session -- has been removed.

Where antibiotics are genuinely justified:

• Primary tissue isolation, where the starting material is not sterile and the tissue cannot simply be replaced.
• Cell sorting and flow cytometry, where the instrument fluidics are not a sterile environment.
• Irreplaceable material -- a unique patient sample or a difficult primary line -- where losing the culture is worse than the drawbacks, and usually for a limited period rather than indefinitely.
• Short-term rescue of a valuable stock, followed by expansion of a clean working bank without antibiotics.
• Large-scale bioprocess operations where a defined risk assessment concludes the trade-off favours their use.

A workable compromise used by many labs: maintain master and working stocks antibiotic-free, so that any contamination declares itself immediately, and add antibiotics only to specific experiments where the risk profile justifies them. If you are currently using pen-strep in everything, the useful experiment is to take one well-behaved line antibiotic-free for a few passages and see whether anything actually happens. In a lab with sound technique, usually nothing does.

Adding or increasing antibiotics is rarely the right response to visible contamination.

Bacterial or fungal contamination in a replaceable culture: discard it. Autoclave or otherwise decontaminate the flask, discard the medium bottle and any reagent aliquots that were open at the same time, decontaminate the incubator and hood, and restart from a frozen vial. Attempting to treat a visibly contaminated culture usually produces a suppressed but persistent contaminant and risks spreading it across the incubator.

Find the source rather than treating the symptom. Recurrent contamination has a cause: a water bath, an incubator water tray, a cracked hood filter, an unclean pipettor, a shared medium bottle, or a specific technique failure. Antibiotics hide the evidence you need to find it.

Test for mycoplasma independently and regularly. PCR-based and luminescence-based mycoplasma tests are inexpensive and quick. Test new lines on arrival, test before banking, and test periodically thereafter. No antibiotic in routine use substitutes for testing. Dedicated anti-mycoplasma reagents exist and can clear an infected culture, but they are a rescue tool for irreplaceable material, not a maintenance strategy, and cleared cultures should be re-tested and re-banked.

Quarantine incoming cultures. Cells arriving from a collaborator are the single most common route by which mycoplasma enters a lab. Handle them separately until tested.

Check the reagents. Serum, trypsin and medium are potential sources. Reputable suppliers test for bioburden and mycoplasma, and certificates of analysis should be reviewed rather than filed unread.

Adding to medium. Add 100X concentrate at 10 mL per litre after the medium has been prepared and, if applicable, filtered. Mix by gentle inversion. There is no need to filter again -- the concentrate is supplied sterile-filtered.

Do not compensate for degradation by overdosing. Doubling the pen-strep concentration does not double the protection, and increases the off-target effects on your cells. If coverage duration is the problem, change medium more often or use a more stable agent.

Serum-free and chemically defined media. Cells in serum-free medium are generally more sensitive to chemical stress than the same cells in 10% serum, since serum proteins buffer and bind many compounds. If you use antibiotics in a serum-free process, watch growth rate and viability carefully after the switch, and consider the lower end of the concentration range.

Selection antibiotics are a separate matter. Puromycin, G418, hygromycin, blasticidin and zeocin are used to maintain selective pressure on engineered cells, not to prevent contamination. The arguments against routine antibacterial use do not apply to them, though the point about verifying that your transgene is still expressed does.

Documentation. Record whether a given experiment was run with or without antibiotics. It is a variable, it does affect some readouts, and it is worth being able to check retrospectively when results are inconsistent between labs.