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.
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chloramphenicol in the cytoplasm so that it is no longer active.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.
Post Comment
No comments