Penicillin-Streptomycin 100X Solution

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• Cell Culture ContaminationCell culture contamination falls into six categories: bacteria, yeast, filamentous fungi and moulds, mycoplasma, viruses, and cross-contamination by another cell line. Bacteria, yeast and fungi announce themselves within one to five days through turbidity, a pH shift or visible particles under the microscope, and are handled by discarding the culture and finding the technique failure that let them in. Mycoplasma, viral contamination and cell line misidentification produce no visible change at all and are found only by testing — PCR or DNA stain for mycoplasma, STR profiling for identity. The correct response to any confirmed biological contamination is to discard the affected culture, decontaminate the incubator, and restart from a clean frozen stock.
• Puromycin for Mammalian Cell SelectionPuromycin is an aminonucleoside antibiotic from Streptomyces alboniger that mimics the aminoacyl end of tRNA, enters the ribosomal A site and causes premature release of a truncated peptide chain, killing cells that cannot inactivate it. In mammalian cell culture it is used as a selection agent at 0.5-10 ug/mL, most commonly 1-2 ug/mL, and it kills non-resistant cells faster than any other common selection antibiotic -- typically within 2-5 days. Resistance is conferred by the pac gene encoding puromycin N-acetyltransferase, which acetylates the drug and renders it inactive. Because the effective concentration depends strongly on cell line, cell density and serum content, a kill curve must be run for each new cell line and each new lot of puromycin.
• Penicillin-Streptomycin and Antibiotic-Antimycotic in Cell CulturePenicillin-streptomycin, universally shortened to pen-strep, is supplied as a 100X sterile solution containing 10,000 units/mL penicillin G and 10,000 ug/mL streptomycin, diluted 1:100 into medium to give a working concentration of 100 U/mL penicillin and 100 ug/mL streptomycin. Penicillin blocks bacterial cell wall synthesis and covers mainly Gram-positive organisms; streptomycin binds the bacterial 30S ribosomal subunit and covers mainly Gram-negatives, so the pair gives broad antibacterial coverage. Antibiotic-antimycotic (anti-anti) is the same combination plus 25 ug/mL amphotericin B at 100X, giving 0.25 ug/mL in use for antifungal coverage. Neither controls mycoplasma, and major cell banks including ATCC recommend against routine antibiotic use because it masks low-level contamination rather than preventing it.
• Mycoplasma Testing in Cell CultureMycoplasma testing detects Mollicutes species that contaminate laboratory cell cultures without producing turbidity, pH change or any other visible sign, which is why a culture can carry 10⁷–10⁸ organisms per mL and still look healthy. The practical methods are PCR or qPCR (roughly 1–10 CFU/mL, results the same day), enzymatic luminescence assays (about 20 minutes, moderate sensitivity), fluorescent DNA staining read against an indicator cell line (about 10²–10⁶ CFU/mL depending on whether an enrichment step is used), and direct broth-and-agar culture, which remains the reference method but takes up to 28 days. Test cultures on arrival, after quarantine and at least monthly thereafter; a confirmed positive is normally discarded and replaced from a clean frozen stock rather than treated.
• Selection Antibiotics for Mammalian Cell LinesSelection antibiotics are cytotoxic agents used to isolate mammalian cells that carry a co-transfected resistance gene, by killing every cell that does not. The five standard agents are G418/geneticin (100–2000 µg/mL, resistance gene neo), hygromycin B (50–1000 µg/mL, hph), blasticidin S (1–20 µg/mL, bsd or bsr), zeocin (50–1000 µg/mL, Sh ble) and puromycin (0.5–10 µg/mL, pac). The correct concentration is not the one on the datasheet but the one determined empirically for your cell line by a kill curve — the lowest concentration that kills 100% of untransfected cells within the agent's normal selection window, which is 2–4 days for puromycin and up to 14 days for G418.
• Antibiotics in Cell Culture: Working Concentrations and When Not to Use ThemAntibiotics in cell culture are antibacterial and antifungal agents added to growth medium to suppress microbial contamination; the standard combinations are penicillin–streptomycin at 100 U/mL and 100 µg/mL, gentamicin at 5–50 µg/mL, amphotericin B as an antimycotic at 0.25–2.5 µg/mL, and antibiotic–antimycotic 100X, which supplies all three at 100 U/mL, 100 µg/mL and 0.25 µg/mL when diluted. The professional consensus is that these should not be used routinely or prophylactically: they mask low-level contamination, select for resistant organisms, do nothing at all against mycoplasma, and can alter cell physiology and experimental response. Their defensible uses are primary tissue isolation, work with material from non-sterile sites, and short-term rescue of an irreplaceable culture — not the standard maintenance of established cell lines.