Antibiotic-Antimycotic Triple Solution 100X

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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.
• 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.
• 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.