Preparing Antibiotics Stock Solution and Ampicillin Agar Plates

AMPICILLIN 

Beta-lactam-antibiotics are not very stable when dissolved. Slow but steady degradation happens even when frozen to -20°C. Therefore commercial beta-lactam-antibiotics are sold as dried powder and reconstituted just before use! If possible adjust stock solution pH to stability optimum and choose suitable storage temperature! Due to enhanced solubility Ampicillin-Na is used for cell culture instead of Ampicillin. Optimal efficacy and stability for Ampicillin is at pH=4-5! If you try to adjust Na-Ampicillin solution to pH=5 you will unfortunately and probably get precipitation. Literature recommends pH=6.8 for enhanced stability of Na-Ampicillin solutions! Especially if you use demineralizised water from a ion exchanger, which sometimes has pH=9-10 adjusting the pH can be important! Preferred storage temperature is -70°C!

See also here: Effects of Storage Temperature and pH on the Stability of Eleven P-Lactam Antibiotics in MIC Trays. JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1985, p. 366-370.

And even more:  Interestingly also undissolved crystal Na-Ampicillin seems to degradate at 4°C under dark storage conditions!! In our lab old Na-Ampicillin from 4°C was dissolved in pH=9 water and for horrible onr month cloning, transfection and ligations failed completely. We bought new and problems where gone. Please save yourself from this fate!!

KANAMYCINE

I also observed impaired activity of Kanamycin after several months at -20°C! 

50 mg/ml is suitable concentration for stocks. Add 200 µl to 100 ml LB-Agar/LB-Medium (100 µg/ml). 200 µg/ml for Ampicillin works also fine.

Preparation of Agar Plates

1.  Agar Recipes:

   10 g of NaCl
   10 g of Tryptone
   5 g of Yeast Extract
   20 g of Agar
   Adjust pH to 7,0 (with 5 N NaOH if necessary)
   Add deionized Water to a final Volume of 1 Liter.

2. Autoclave (121°C program for solutions, put autoclave-temperature-sensor into water!)
3. Let it cool down in a water bath (adjusted to 55°C)!
4. Add Ampicillin-Na or Kanamycin stock solution to an end concentration of 100 µg/ml
5. Stir or shake accurately to get a homogenous antibiotics distribution!
6. Pour about 25-30 ml in a 10 cm dish
7. Let agar solidify at room temperature, then put to 4°C

Stability of Agar plates: Of course stability problem is the same for medium and plates! Always add antibiotics to LB-Medium freshly and short before use! It is often said, that Ampicillin plates are stable for 4 weeks at 4°C. In fact already after 1 week you can observe reduced selectivity! Therefore I always plate out 25 µl of antibiotic-stock together with bacteria! Picking colonies without wanted content is very annoying.