Preparation of Antibiotic Stock Solutions and Agar Plates

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Antibiotic Stock Solutions

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!

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.

Gentamycin

Gentamycin 10 (C/P) Add dry after autoclaving. Antibiotics should be added to lower than 60 C broth, or filter sterilized. "C" refers to chromosomal resistance; "P" plasmid based resistance.

Gentamycin（庆大霉素、正泰霉素、艮他霉素）对多种革兰阴性菌及阳性菌均有抑菌或杀菌作用，对金葡菌、表皮葡萄球菌、大肠埃希菌、肺炎克雷伯菌、变形杆菌、沙门菌属、志贺菌属、肠杆菌属及绿脓假单胞菌均有良好的抗菌活性，对布鲁菌属、鼠疫杆菌也有抗菌作用。庆大霉素配制成溶解可在冰箱中长期保存，不必像其他抗生素一样必须新鲜配制。

Preparation of Agar Plates

1. Agar Recipes: 10 g of NaCl, 10 g of Tryptone, 5 g of Yeast Extract, 20 g of Agar

2. Adjust pH to 7,0 (with 5 N NaOH if necessary)

3. Add deionized Water to a final Volume of 1 Liter.

4. Autoclave (121°C program for solutions, put autoclave-temperature-sensor into water!)

5. Let it cool down in a water bath (adjusted to 55°C)!

6. Add Ampicillin-Na or Kanamycin stock solution to an end concentration of 100 µg/ml

7. Stir or shake accurately to get a homogenous antibiotics distribution!

8. Pour about 25-30 ml in a 10 cm dish

9. 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.