Media amendments

Antibiotic concentrations and stock solutions

This table lists standard concentrations of antibiotics commonly used in microbiology laboratories. These concentrations apply to E. coli. Antibiotic selection in other organisms may use different concentrations, so check primary literature for more info.
Reagent Abb Stock Conc. Working Conc. Dilution Solvent MSDS Notes
Ampicillin Amp 100 mg/mL 100 g/mL 1,000 ddH2O link Ampicillin degrades quickly in both plates and stock solutions. Culture plates with Amp can be stored at 4C for about 2 weeks. Stock solutions can be stored at 4C for 2 weeks but can last as long as 4-6 months when stored at -20C. In general, use carbenicillin for ampicillin selection, because it is more chemically stable.
Carbenicillin Crb 100 mg/mL 100 g/mL 1,000 50% EtOH link Carbenicillin can be used in place of Ampicillin at the same working concentration. Carbenicillin is more stable than Ampicillin, and is generally preferable.
Chloramphenicol Cam 20 mg/mL 20 g/mL 1,000 EtOH link  
Cycloheximide Chx 100 mg/mL 100 g/mL 1,000 100% DMSO link Anti-fungal, toxic to humans, use PPE!
D-Cycloserine Dcs 4 mg/mL 4 g/mL 1,000 ddH2O link Degrades noticeably over the course of 1 week at 4C. D-cycloserine is best used within two days of pouring. A single small-scale experiment found 6-10x as many E. coli colonies on 8-day old MG-Dcs plates that had spent 3 of those days at -20C and 5 at 4C, compared to what was found on 1 day old MG-Dcs.
Erythromycin Ery 250 mg/mL 250 g/mL 1,000x EtOH link May crystallize when added to liquid culture. Still works. Selection strength may vary in non-model bacteria
Gentamicin Gen 20 mg/mL 20 g/mL 1,000 ddH2O link For non-canonical experiments:
Use 20 g/mL in culture from 20mg/mL 1000 stock
Kanamycin Kan 50 mg/mL 50 g/mL 1,000 ddH2O link Plates start to lose sensitivity after 2 weeks; recommended Kanamycin plates only last a few weeks at 4C; best to make fresh Kan plates every 2 - 4 weeks.
Nalidixic Acid Nal 30 mg/mL 30 g/mL 1,000 1M NaOH link  
Novobiocin Nov 200 mg/mL 200 g/mL 1,000 ddH2O link  
Rifampicin Rif 50 mg/mL 100 g/mL 500 MeOH + drops of 10M NaOH link Toxic to humans, use PPE! Rifampicin is TOXIC to humans, light-sensitive, and degrades in solution rather quickly. It needs to be stored wrapped in foil and replaced every 2 months.
Spectinomycin Spec 60 mg/mL 60 g/mL 1,000 ddH2O link Common to see background growth of cells on selective plates. Can use at 2x concentration or higher.
Streptomycin Str 100 mg/mL 100 g/mL 1,000 ddH2O link  
Tetracycline Tet 10 mg/mL 10 g/mL 1,000 70% EtOH link Light sensitive, stability < 2 weeks
Trimethoprim TMP 10 mg/mL variable? variable DMSO link Both 10 and 20 g/ml listed for use. Use as counter selectable marker for mutations in thyA requires presence of thymine/thymidine in media
Zeocin Zeo 25 mg/mL 25 g/mL 1,000 ddH2O link Some labs report using a higher concentration for selection.
10 g/mL is specifically used for growth of amberless strain A37

Other common media amendments

Reagent Abb Stock Conc. Working Conc. Dilution Solvent MSDS Notes
5-fluorouracil 5FU 13 mg/mL 13 g/mL 1,000 DMSO link 100mM stock concentration 100M working concentration
Anhydrotetracycline ATC 0.1 mg/mL 100 ng/mL 1,000 DMSO link Can also make a 1mg/mL stock and dilute 1:10 for a final concentration of 0.1mg/mL
3′-Azido-2′,3′-dideoxythymidine AZT 10 mg/mL 200 g/mL 50 ddH2O link Can substitute pharma grade Zidovudine. Soluble up to 20 mg/mL in ddH2O. Very unstable in solution. Store at 20C and activity will still decrease over weeks of storage.
Bromo-chloro-indolyl-galactopyranoside X-gal 40 mg/mL 80 g/mL 500 Dimethylformamide (DMF) link X-gal is a histochemical substrate for β-galactosidase, which cleaves X-Gal to yield an insoluble blue precipitate.
Diaminopimelic acid DAP 60 mM 0.3 mM 200 ddH2O link Use 5 l stock per 1 ml of media. Dissolve ~30min shaking at 37C
Isopropyl β-D-1-thiogalactopyranoside IPTG 0.1 M 0.1 mM 1,000 ddH2O link Light sensitive, MW: 238.31 g/mol
Triphenyl Tetrazolium Chloride TTC 5% (50 mg/mL) 0.05% (50 g/mL) 1,000 ddH2O link Used in growth/sugar-fermentation indicator agars. Filter sterilize. Light sensitive. Store stock solution at 4C wrapped in foil. Use at ten-fold higher concentration in motility agar (0.5%).

Working concentrations are for liquid or solid media.

Preparation

* Generally, prepare 30-50 mL of solution in a 50 mL conical tube. Then, filter sterilize solutions by pushing them through a 50 mL syringe fitted with a 0.22 m filter made of a material appropriate for the solvent. Compounds dissolved in Ethanol or DMSO do not require filter sterilization. Aliquot into 1.5 mL portions in 1.7 mL tubes using the repeat pipettor. Label with a permanent marker (one that won't wash off with ethanol). Store at -20C in the dark.

Non-canonical amino acid stock solutions

Amino Acid
(Abbreviation)
CAS Number Solvent MW
(g/mol)
[Stock] [Stock]
(mg/mL)
[Culture] Notes
Y 60-18-4 Water 181.19 10mM 1.812 250M Requires heating to 70C to dissolve
Stock precipitates out of solution at or below room temperature
IodoY 70-78-0 Water 307.09 10mM 3.071 250M Dissolves at room temperature with ~1 hour of shaking
AminoY (FAA) 300-34-5 196.203
AminoY (HS) 23279-22-3 Water 287.14 10mM 250M Dissolves readily without shaking or heat
BromoY 38739-13-8 Water 260.0846 10mM 250M
ChloroY 7423-93-0 Water 215.63 10mM 250M
NitroY 621-44-3 Water 226.2 10mM 2.262 250M Will dissolve with heating to 50C, no shaking
ONBY (FAA) 184591-51-3 316.31 Light sensitive
ONBY (S) 207727-86-4 50% DMSO
50% Water
(exothermic)
352.77 50 mM 250M Light sensitive
Requires adding OH- to dissolve
AzF 33173-53-4 10% DMSO
90% Water
206.204 10mM 2.062 250M Light sensitive
Requires heating and shaking to dissolve
CNF 167479-78-9 Water 190.2 10mM 1.902 250M Dissolves at room temperature with ~15 minutes of shaking
L-DOPA 59-92-7 Water 197.1879 10mM 1.972 250M Oxidizes quickly and turns black in solution
5HTP 4350-09-8 Water 220.2246 10mM 2.202 250M

FAA: free amino acid
S: salt
HS: hydrated salt
Topic attachments
I Attachment Action Size Date Who Comment
elsedocx Making_stock_antibiotics_and_other_reagents.docx manage 89.7 K 11 Jan 2019 - 21:56 Main.KateElston Easy spreadsheet that tells you exactly how to make the most common stock chemicals

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Contributors to this topic edittopic JeffreyBarrick, CraigBarnhart, AurkoDasgupta, SeanLeonard, GabrielSuarez, JordanMonk, DanielDeatherage, AlvaroRodriguez, DaciaLeon, SimonDAlton, ElizabethRobinson, DennisMishler, KateElston, BrianRenda
Topic revision: r62 - 08 Jul 2020 - 18:23:58 - Main.JeffreyBarrick
 
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