Antibiotic Stock Solution Preparation Protocol
Antibiotic Stock Solution Preparation Protocol
1. Purpose:
This protocol outlines the steps to prepare antibiotic stock solutions used for bacterial culture experiments. These solutions can be added to culture media to select for bacteria or plants expressing resistance to specific antibiotics.
2. Materials:
* Chemicals:
- Antibiotics:
- Ampicillin (50 mg/ml)
- Basta/Glufosinate ammonium (40 mg/ml)
- Carbenicillin (100 mg/ml)
- Chloramphenicol (35 mg/ml)
- D-cycloserine (10 mg/ml)
- Erythromycin (20 mg/ml)
- Gentamycin (25 mg/ml)
- Hygromycin B (50 mg/ml)
- Kanamycin (50 mg/ml)
- Nalidixic acid (30 mg/ml)
- Neomycin (10 mg/ml)
- Rifampicin (10 mg/ml)
- Spectinomycin (50 mg/ml)
- Streptomycin (100 mg/ml)
- Tetracycline (10 mg/ml)
- Triclosan (10 mg/ml)
- Trimethoprim (10 mg/ml)
- Solvents:
- Distilled Water (H₂O)
- Ethanol (EtOH), Methanol (MetOH)
- DMSO (Dimethyl Sulfoxide)
- NaOH (for adjusting pH)
* Equipment:
- Pipettes (adjustable for µl & ml)
- Sterile falcon tubes (15 ml and 50 ml)
- Analytical balance
- Magnetic stirrer with stirring bar
- pH meter with calibration solutions
- Autoclave
- Aluminum foil (for light-sensitive antibiotics)
* Personal Protective Equipment (PPE):
- Lab coat
- Gloves
- Safety glasses
- Face mask (optional)
3. Procedure:
1. Preparation:
Ensure all glassware, pipettes, and tubes are clean and decontaminated. Set up the workspace in a laminar flow cabinet or a clean, sterile surface.
2. Antibiotic Dissolution:
For each antibiotic, prepare the stock solution using the following procedure:
Select the appropriate antibiotic and refer to the working concentration from the table below:
Antibiotic Stock Solution (mg/ml) Solvent Working Concentration (µg/ml) Ampicillin 50 Distilled Water (H₂O) 50 Carbenicillin 100 H₂O or 50% EtOH 100 Chloramphenicol 35 EtOH or MetOH 35 D-cycloserine 10 0.1M NaPO₄ pH 8.0 10 Erythromycin 20 EtOH 20 Gentamycin 25 H₂O 25 Hygromycin B** 50 H₂O 35 Kanamycin 50 H₂O 50 Nalidixic acid 30 H₂O (pH 11 w/ NaOH) 30 Neomycin 10 H₂O 800 (80 µl/ml) Rifampicin** 10 Methanol (MetOH) 10 Streptomycin 100 H₂O 100 Tetracycline*** 10 70% Ethanol (EtOH) 10 Triclosan 10 Ethanol (EtOH) 10 Trimethoprim 10 DMSO 10 Using the analytical balance, accurately weigh the amount of antibiotic powder in grams, according to the required stock concentration from the table.
Dissolve in the appropriate solvent (as indicated in the table). For light-sensitive antibiotics (marked with ** or ***), cover the tubes in aluminum foil once dissolved.
3. Sterilization and Filtering:
Most antibiotic stock solutions should be filter-sterilized:
Prepare a sterile syringe filter attached to a 0.22 µm filter membrane (preferably nylon if using DMSO; avoid cellulose acetate membranes with DMSO).
Filter the antibiotic solution into a sterile 50ml falcon tube. Use a pipette to transfer the solution for filtering if volume is small.
4. Aliquoting, Storage, and Labeling:
Once filtered, aliquot the solution into sterile tubes (1 ml per tube is common for ease of use).
- Label each tube with:
- Antibiotic name
- Date of preparation
- Concentration
- Storage conditions (light sensitive, -20°C or +4°C as indicated in the table).
- Store the antibiotic stock solution:
- Most solutions are stored at -20°C in aliquots.
- Working stocks for antibiotics like Kanamycin, D-cycloserine may be stored at +4°C.
5. Use in Media Preparation:
When preparing media with antibiotics:
- Thaw the aliquot and vortex if needed.
- Add 1 ml of the antibiotic stock to 1 liter of media (for a typical 1000X dilution).
- Ensure media has cooled to ~55-60°C post-autoclaving before adding the antibiotic to avoid inactivation.
4. Light-Sensitive Antibiotics:
Wrap the following antibiotics in aluminum foil after dissolving, as they are light-sensitive:
- Hygromycin B
- Rifampicin
- Tetracycline
5. Storage & Notes:
- Solutions with solvents like DMSO require specific storage temperatures and filters.
- Always prepare unstable antibiotic solutions fresh to avoid degradation over time.