Antibiotics

·         Store stocks long term at -20°C or short term (for actively-used aliquot) at 4°C. Concentrations given are 1000X for E. coli/Salmonella in rich media, unless indicated otherwise.

Ampicillin

·         Binds to and inhibits enzymes in the bacterial membrane that are involved in the synthesis of the cell wall (therefore only works on actively dividing cells, but is bacteriocidal to them).

·         bla provides resistance: its product, b-lactamase, breaks down ampicillin and other penicillin derivatives (b-lactams) in the periplasm. b-lactamase also breaks down ampicillin in the surrounding media. This may allow non-bla-containing cells to grow, resulting in the formation of “satellite colonies.”

·         Dissolve ampicillin (4°C) in water at a concentration of 50-150 mg ml^-1 and filter sterilize.

Carbenicillin

·         Carbenicillin is recommended for use instead of ampicillin in liquid cultures when it is important to maintain a plasmid in as many cells as possible (e.g. overproduction of a protein encoded on a plasmid) because ampicillin selection tends to be lost in cultures as the drug is degraded. Carbenicillin is less sensitive than ampicillin to low pH and to the activity of β-lactamase and so using carbenicillin reduces the loss of drug resistance. For the same reason, the use of carbenicillin instead of ampicillin in solid media helps prevent the growth of satellite colonies.

·         Dissolve carbenicillin (4°C) in water at a concentration of 50-150 mg ml^-1 and filter sterilize.

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Chloramphenicol

·         Interacts with 50S subunit of ribosome. It inhibits protein synthesis by blocking the peptidyltransferase step and is bacteriostatic.

·         cat provides resistance: its product, chloramphenicol acetyltransferase, alters chloramphenicol in the cytoplasm so that it is no longer active. Its expression is catabolite sensitive: increased cAMP levels lead to increased chloramphenicol acetyltransferase synthesis.

·         Dissolve chloramphenicol (room temperature) in 100% ethanol at a concentration of 10-30 mg ml^-1 (use lower levels for single- or low-copy resistance genes and higher level for multi-copy plasmids) and filter sterilize.

Erythromycin

·         Inhibits protein synthesis by binding to the 23S rRNA molecule of the 50S subunit, blocking exit of the peptide chain. Bacteriostatic. Gram positives accumulate ~100x more erythromycin than Gram negatives and so erythromycin is not very effective for Gram negatives.

·         Dissolve erythromycin (room temperature) in 100% ethanol at 10 mg ml^-1 (500X) and filter sterilize.

Kanamycin and Neomycin

·         Kanamycin and neomycin are structural analogs that inhibit protein synthesis at the step of translocation by binding the 30S subunit of ribosome, eliciting miscoding; they are bacteriocidal against aerobes and facultative anaerobes.

·         Resistance is provided by aminoglycoside phosphotransferases (phosphorylation of kanamycin and neomycin prevents their entry into cells), aminoglycoside acetyltransferases, or aminoglycoside nucleotidyltransferases.

·         Dissolve kanamycin and neomycin (room temperature) in water at a concentration of 50 mg ml^-1 and filter sterilize.

Gentamicin

·         Binds 30S ribosomal subunit; blocks initiation of translation and causes misreading of mRNA; bactericidal against aerobes and facultative anaerobes

·         Resistance is provided by an enzyme that inactivates aminoglycosides (like gentamicin) by covalent modification.

·         Dissolve gentamicin (4°C) in water at a concentration of 30 mg ml^-1 and filter sterilize.

Nalidixic Acid

·         Inhibits DNA gyrase; bacteriostatic.

·         Mutations in gyrase confer resistance

·         Dissolve nalidixic acid (-20°C) in water at a concentration of 20 mg ml^-1, adjust the pH to 11 with NaOH, and filter sterilize.

Rifampicin

·         Inhibits b-subunit of RNA polymerase. It is bacteriostatic.

·         Rifampicin sensitivity is dominant when it is conferred by mutations in the b-subunit of RNA polymerase. Resistance genes (dominant) are available.

·         Dissolve rifampicin (-20°C) in methanol or dimethylformamide at a concentration of 50 mg ml^-1 (500x) and filter sterilize (ensure filter is compatible with solvent).

Spectinomycin

·         Inhibits tRNA translocation from A to P sites in 30S ribosomal subunit. It is bacteriostatic

·         Mutations in rpsE confer resistance, as does the gene aad (aminoglycoside adenyltransferase—see also streptomycin).

·         Dissolve spectinomycin (4°C) in water at a concentration of 50-100 mg ml^-1 and filter sterilize.

Streptomycin

·         Inhibits protein synthesis by binding to S12 protein; bacteriocidal.

·         Streptomycin resistance is conferred by mutations in rpsL or by aminoglycoside adenyltransferase (aad—see also spectinomycin).

·         Dissolve streptomycin sulfate (4°C) in water at a concentration of 25-100 mg ml^-1 and filter sterilize.

Tetracycline

·         Inhibits protein synthesis by the binding to the ribosomal 30S subunit’s acceptor site and preventing the binding of aminoacyl-tRNA. It is bacteriostatic.

·         Tetracycline resistance is conferred by the antiporter TetA (TetR is a repressor of tetA transcription; when bound to tetracycline, this repression is relieved) or decreased permeability of the cell to the drug. Tetracycline resistance is inhibited my Mg²⁺. For the tetRA system, multiple copies of the genes provide lower levels of resistance than when only one copy is present.

·         Dissolve tetracycline (-20°C) in 50% ethanol at a concentration of 15 mg ml^-1 (it does not precipitate as quickly at lower concentrations) and filter sterilize.