Mycoplasma, Cell Culture, Antibiotics, Fluoroquinolone, Cipro, Ciprofloxacin, dissolving Cipro

"Cipro" is a trademark of the Bayer corporation

I created this page because I do a lot of cell culture (primary and immortal cell lines...mouse and human) and we have a terrible time with mycoplasma infections (detected via the Gibco MycoTect kit). We have decided to use Ciprofloxacin (Cipro) to remedy the situation. We purchased Ciprofloxacin powder (not, I repeat NOT Ciprofloxacin:HCl) from Sigma (their subsidiary, Fluka). Everyone and all of the literature on this explains that Cipro must be dissolved in deionized, distilled, sterile water. But my experience, and later my reading from the MERCK index showed that Cipro was absolutely insoluble in water and ethanol. So I decided to dissolve it using an acid solution and it worked, and it has worked, and the cells are doing wonderfully. Below is the protocol:

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Actual working condition Cipro (1000X)...

1) Measure out 0.05 grams of Cipro and put into a 15 ml Falcon tube

2) Measure out exactly 5.0 ml of Deionized, Distilled, sterile water and put into the same tube as Cipro.

3) Close Cap and vortex for 15-30 seconds (not to dissolve but to mix the powder into solution).

4) Pipette 30 MicroLiters (0.03 mL) of 6.25 Normal (6.25 Molar) hydrochloric acid (HCl) into the water/cipro solution.

5) Vortex on high for about 2 minutes. The solution should start to go clear and the Cipro powder should start to dissolve. A tough pellet might form at the bottom of the cone of the tube. If this happens, the pellet will not go into solution until you forcefully invert the tube seveeral times to make the pellet come off of the bottom of the tube, and immediately vortex to break it apart (it will then easily go into solution). If there is still powder that will not go into solution, add 1.0 microL more HCl to the solution and vortex again. Repeat as necessary (giving minutes between Acid additions) until all powder goes into solution.

6) Pipette out ~10 microL of the dissolved Cipro solution and add to a pH paper to check the acidity of the solution. It should not be below pH 5.0. If it is, add a little dilute base (NaOH or NaHCO3) to buffer it, or make a new batch of Cipro.

7) This solution is 1000X (10,000 microGrams per milliLiter) Cipro and can be used directly for cell culture (use 1.0 microLiter per milliLiter of your cell culture media). You should add the Cipro to a tube of media, vortex it, then use that to feed the cells. Never put Cipro directly into the flask or onto cells. It must be diluted out well into buffer before being added onto cells. The working dilution is 1X (10 microGrams per MilliLiter).

8) This solution can undergo freeze-thaw without worry of degradation of the working product. Stored at room temperature, it will last for about three weeks. Frozen at -20 degrees C to -80 degrees C, it will (and has for me) lasted for 6 months (+).