University of Agriculture Faisalabad
Question
Asked 11 April 2015
What are satellite colonies? Why do they grow on LB amp plates?
Most often I observe sattelite colonies (small E. coli type colonies) after 8-10 hr of transformation. What are these why they don't grow every time.
Most recent answer
Air bubbles can be minimized by avoiding over drying of the media
Popular answers (1)
University of Delhi
They typically appear because beta-lactamase secreted by the transformed vector cells degrade the antibiotics around the transformed cells. Thus leading to growth of non-transformed colonies. Satellite colonies also appear when pH of medium is below 7 as beta-lactamase is inactive below pH7 and other thing is bacterial growth makes media pH acidic a bit. We usually keep pH of ampicillin media 7.4 and avoid overgrowth of cells.
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All Answers (16)
University of Delhi
They typically appear because beta-lactamase secreted by the transformed vector cells degrade the antibiotics around the transformed cells. Thus leading to growth of non-transformed colonies. Satellite colonies also appear when pH of medium is below 7 as beta-lactamase is inactive below pH7 and other thing is bacterial growth makes media pH acidic a bit. We usually keep pH of ampicillin media 7.4 and avoid overgrowth of cells.
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Centro de Investigaciones Biológicas del Noroeste S.C.
Satellite colonies appear since beta-lactamase is secreted by the transformed bacteria with the vector harbouring this gene selection marker and the non-transformed cells take advantage of this fact to grow up in the selection media.
Sometimes increasing ampicillin concentration (100 to 120 ug/mL) is more than enough to decrease the appearance of satellite colonies. And also, is recomendable not to leave more than overnight or 16 hours your plates with this antibiotic in the growth chamber, since you leave more than 36-48 hours your plates with this antibiotic the risk to find satellite colonies increases a lot.
If your cloning a gene but this is not your final vector to express your gene of interest, I also suggest to change for another vector with kanamycin as gene selection marker since this is not secreted by the transformed cells.
Regards,
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Hi Rakesh,
We did not see a picture of your plate. The definition of satellite colonies is: "Satellite colonies are very small colonies that have not taken up the plasmid and that form around a large colony that has taken up plasmid." (from the link provided by Michael). Please see the attached picture. Did your colonies look like that?
"With 8-10 hours after transformation" as you described in your question and you observed some tiny 'satellite colonies'. 8-10 hours is considered a normal overnight growing period time. I barely see satellite colonies forming on an ampicillin plate with overnight growing, unless your plates were different. However, sometimes we did see some tiny colonies scattered on plates after overnight incubation (don't know what caused that). So, you need to know what situation was yours. A prolong incubation can cause satellite colonies (as mentioned in Aaron's answer).
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Amity University
dear all
i am continuously getting these kinds of colonies(picture attached) while doing transformation. i think these are some kind of microaerophilic colonies. if anyone can help me out, i would be very thankful.
Anubhav Mittal
Serum Institute of India Limited
Dear Mittal,
I think it is air bubbles in your plate not bacterial colonies. I am also getting these types of air bubbles in plates even in control while using disposable petri-plates. Try using glass petri-plates. Hope this will help you.
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Institute for Medical Research, Malaysia
Hi Mittal,
Those are obviously air bubbles trap in the media.
I always get those 'colonies' as well.
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Arrakis Therapeutics
I am here because i got similar colonies "air bubbles". Now I know I have no actual colonies
Hi Mittal,
Except the air bubbles trap in the medium, I do see some colonies in the plate (if you look carefully). Next time when you take pictures of such plate, you can use a black paper or black cloth as background.
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The Francis Crick Institute
Use Carbenicillin instead. It is a lot more stable and cleaner than Ampicillin
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Pasteur Institute of Iran
Dear Kumar
I know it is somehow late to answer this question ;) however, I reply this to those who have the same problem. Actually, In addition to the acidic pH and expired LB-amp agar, most of the time when you prepare fresh Lb-amp a little moisture remain on LB-amp (most of the time it is not visible to the eyes ). The secreted beta-lactamase could spread easily in adjacent colonies (un-transformed bacteria) and they could survive thanks to the beta-lactamase and creat microsatellite colonies. The solution is to spread the transformed bacteria for a longer time until the spreader rod move a little harder on the plate (air dry the moisture). also, you can put the newly-made plates on the incubator for 5 hours then use.
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