Urgent amp agar plate question! - Help with stupid question (Jun/20/2007 )

Just curious. If you keep for months, wouldnt amp degrade? So it will give chance for lotsa (i mean lotsa) satellite colonies to grow on it. That's for my case after using those old stock of amp plate. I heard carbenicillin would be a better choice. More stable and longer half life for degradation.

---

The same happened to me, when I use old amp plates, I have satellite colonies very soon. That’s way I have added some extra amp sometimes.

I never head about carbenicillin. Is it commonly used instead of amp??

I search about it and sounds better than amp, but I don’t know anybody who used it. Why?

The antibiotic carbenicillin, an ampicillin analog, is a commonly used selection agent that binds and inhibits enzymes involved in the synthesis of the bacterial cell wall. It is active against most isolates of Pseudomonas aerogenosa and certain indole-positive Proteus strains that are resistant to ampicillin. The gene conferring resistance to ampicillin and its analogs, ampr, codes for the enzyme β-lactamase. Carbenicillin is less sensitive to β-lactamase than ampicillin. In addition it has a superior stability at low pH. Experiments have shown that the use of carbenicillin in place of ampicillin helps prevent overgrowth of satellite colonies. Effective concentration: 50 to 100 μg/ml.

---

I think I know why:

Here:
Sigma Ampicillin: 5 g 59 USD
Sigma Carbenicillin: 5 g 660 ISD

---

I stored them at 4c face down.

---

What is a satellite colony and how do they look like?
Thanks.

---

Small colonies that appear around a large colony. They are late colonies, appearing several hours after the first wave of colonies have grown. They are always found around a large colony and never on their own.

---

Hmm.. now we know the price difference. But I think carbenicillin is way better in term of storage life.

---

if you have plates w/o any amp, you can better define the end conc instead of there are are non-hydrolyzed rests of amp; determine precisely the volume of agar; spread out amp diluted in LB (200-500 µl)

---

Okay, what happens is that Ampicillin is actively degraded by bacteria with an Amp resistance gene. So where these bacteria grow, you will form little pockets of agar containing no amp at all. The colonies who lack the resistance gene can then grow there, surrounding the original colony. These are called satellite colonies.
Now, to make things worse is that when Amp is depleted you no longer have what is called "antibiotic pressure", meaning that the colonies that have the plasmid might actually loose the plasmid since it's no longer necessary. Selective pressure will favor those without the plasmid (energy conservation).
This is why you should always make sure you have enough Ampicillin in all your bacterial media, at all times.

However, I have used months old amp plates without issues. There seems to be a lot of disagreement with this.

---

I believe it is all down to the concentration of ampicilin in use. In my lab, we use ampicilin at a concentration of 25ug/ml. However I have seen people and protocols use concentrations as high as 100ug/ml. The bla resistence gene is the same under both conditions.

Thus a 100ug/ml ampicilin plate will tolerate very significant ampicilin degredation before any noticible effect is seen. Losing 75% of the ampicilin will have no effect as far as selection goes.

On the otherhand, the 25ug/ml plate my lab uses will probably not tolerate the same lost; quantity or percentage wise.

---