Media supplement to prevent bacterial and fungal growth during routine cell culture.

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Bac-Off® Tonic for antibiotic treatment of media. This product was developed to address the problem of bacteria in cell culture. Cells have, as a natural and normal part of their constitutive programming, the ability to enclose bacteria adherent to their luminal surface in membrane, and sink them into the cytoplasm. Cells containing bac-vacuoles at high multiplicities are adversely affected. The eventual release of even a single viable bacterium from sequestration has predictable and dismal consequences. Bac-Off® is formulated at MIC (minimum inhibitory concentration) to inhibit the growth of most bacterial agents and control contamination in cell culture, without harming the cells. This quinolone antibiotic interferes with the chromosomal breakage-reunion reaction of bacterial gyrase and human topoisomerase II. Bacterial replication is coupled with and dependent on chromosomal replication, with as many as several waves of chromosomal replication present in log cultures. In the presence of Bac-Off® at MIC dosage, bugs are unable to relax replication supercoiling and are kicked into a prolonged G2 shunt. As a practical matter, bacteria released into the medium will be washed away during routine feeding operations.
Dosage of Bac-Off® was established by identifying the MIC for bacteria and the toxicity dosage for human microvascular cells. Nearly twenty-five individual isolates of ACBRI Primary Human Microvascular cells have been probed to date. Results indicate that the effective MIC is 2 µg/ml and that visible toxicity is not encountered until levels exceed ~10 µg/ml.

Store Bac-Off® at −20°C until ready to use. Add 1 ml of Bac-Off® to each 500 ml of medium. If an entire 500 ml unit of Cell Systems medium will not be used within 30 days, activate the medium with growth supplement and Bac-Off®, then aliquot and freeze in smaller units which will be used within 30 days (store at 2 to 8 °C).
Additional Information: Bac-Off® is delivered as a 500X concentrate which, at working strength (1:500 v/v dilution in complete medium) exhibits Minimum Inhibitory Concentration (MIC) according to standard protocols against the listed species of microorganism. Bac-Off® may also be used at double working strength (1:250 v/v) at which concentration it exhibits Minimum Bactericidal Concentration (MBC), which generally does not exceed the MIC by more than a factor of 2. Bac-Off® may also be used at ten times working strength (1:50 v/v) at which concentration it exhibits mycoplasmacidal properties. This dosage is well tolerated by most mammalian cells in vitro for short periods of time. Bac-Off® contains the synthetic fluoroquinone Ciprofloxacin, with a wide range of in vitro activity against a wide range of Gram-negative and Gram-positive microorganisms.

Active against the following microorganisms: Bacillus fragilis; Clostridium difficile; Staphylococcus spp.; Staphylococcus haemolyticus; Staphylococcus epidermidis; Staphylococcus saprophyticus; Staphylococcus hominis; Streptococcus pneumoniae; Streptococcus pyogenes; Pseudomonas aeruginosa; Haemophilus influenzae; Haemophilus parainfluenzae; Enterococcus faecalis; Escherichia coli; Enterobacter spp.; Enterobacter cloacae; Enterobacter aerogenes; Citrobacter spp.; Citrobacter diversus; Citrobacter freundii; Klebsiella spp.; Klebsiella pneumoniae; Klebsiella oxytoca; Moraxella catarrhalis; Morganella morganii; Proteus spp.; Proteus mirabilis; Proteus vulgaris; Providencia Spp.; Providencia stuartii; Providencia rettgeri; Serratia marcescens; Acinetobacter Lwoffii; Aeromonas hydrophilia; Campyhlobacter jejuni; Edwardsiella tarda; Legionella pneumophilia; Neisseria gonorrhoeae; Salmonella spp.; Salmonella enteritidis; Salmonella typhii; Shigella spp.; Shigella dysenteriae; Shigella boydii; Shigella flexneri; Shigella sonnei; Vibrio spp.; Vibrio Cholerae; Vibrio vulnificus; Yersinia spp.; Yersinia enterocolitica; Yersinia pestis; Bacillus anthracis.

The mechanism of action of Bac-Off® is distinct from that of penicillins, cephalosporins, aminoglycosides, macrolides, and tetracyclines. There are no reported instances of cross-resistance between Bac-Off® and other classes of antimicrobials. The inoculum size has little effect when tested in vitro.

The bacteriostastic ('working strength' 1:500) and bacteriocidal ('double strength' 1:250) activity of Bac-Off® results from inhibition of the bacterial enzymes topoisomerase II (DNA gyrase) and topoisomerase IV. These enzymes are necessary for bacterial replication, transcription, and repair. Mammalian topoisomerases are not affected by Bac-Off® at less than 7 to 10-fold working strength. This is a much wider gap between the dosage active against bacterial growth and the dosage toxic to mammalian cells than all other commonly used antibiotic classes. Bac-Off® is stable for at least six months at refrigerator temperatures, and may be frozen (<−20 °C) one time without loss of activity. No special precautions are necessary for disposal of Bac-Off® solutions at working or double strength.