When I pick a single from a freshly streaked selective plate and incubate a starter culture of 5mL LB medium containing the appropriate selective antibiotic for 8H at 37 ℃ with vigorous shaking(300 rpm), the tube is still clear which explain that my bacteria is not grown.
Sometimes you get small "satellite"-colonies right next to "real"-colonies that are growing on agar-plates containing antibiotics yet do not possess the resistance genes by themselves. Make sure your selective plates are not too old, the agar-plates were not incubated too long at 37°C and select a truely single colony that is not growing next to other colonies! Maybe resistant colonies degrade the antibiotic in their immediate surrounding and that leads to the growth of these "satellite"-colonies? Maybe increase the antibiotics concentration on your selective plates a bit?
Best regards,
Christian
which bacteria used for testing? wich selective agar used? pls control selective antibiotic and if bacteria stay in 4 c you can use brain heart broth / blood agar for enrichment
Sometimes you get small "satellite"-colonies right next to "real"-colonies that are growing on agar-plates containing antibiotics yet do not possess the resistance genes by themselves. Make sure your selective plates are not too old, the agar-plates were not incubated too long at 37°C and select a truely single colony that is not growing next to other colonies! Maybe resistant colonies degrade the antibiotic in their immediate surrounding and that leads to the growth of these "satellite"-colonies? Maybe increase the antibiotics concentration on your selective plates a bit?
Best regards,
Christian
I agree with Christian: depending on what antibiotic you use, satellite colonies become more of a possibility as the antibiotic is depleted by bacterial growth. Selecting an isolated colony is your best bet. As for why the culture in medium doesn't show growth after 8 hours, it could be impacted by what exactly you're growing. Certain genes may slow down cell growth, or even kill the cell if the product is toxic in high enough quantities. Back when I used to work in a lab specializing in vaccine R&D, certain genes from various viruses caused the bacteria to grow slowly, taking up to 2 or 3 days to grow to an appreciable amount, and at that point you risk the same problem as you do from the agar plate, where the excess time lets the antibiotic be broken down by the present bacteria and then those without the gene of interest./AB resistance can take over. It can get pretty complicated!
You could always insert another gene for antibiotic resistance if you want to be very selective - gentamicin, ampicillin, and a few others are common ones that come to mind. Hope this helps!
Why do you use such a high speed? We usually use from 180 to 220 rpm. Check if that is the problem. I would repeat the O/N cultures but more colonies in case it was just that one, set up at least three more cultures. Check that your antibiotic is also not contaminated, so culture bacteria in absence of antibiotic too.
1. If you do the regular way: freshly streaked on selective plate for overnight growing and see the colonies, and then placed in 4C for 2 days, most bacteria should not die in 2 days in 4C, and it is rarely to see 'Satellite colonies' form in such a short period of time (on Ampicillin plates, for example). Just in case you are new to the satellite colonies, I attach a picture for you (see attachment).
2. Other causes can be:
(1) For some reasons, the bacterial colony did not get into the LB medium during inoculation.
(2) If the incubator is shared, the temperature can be changed by other people and forget to change it back after they done (have seen this in my career). So, check temperature setting when you on to it. Also, put a thermometer in the incubator to see the real temp in case the heater was broken.
(3) Wrong antibiotic was added and killed the bacteria.
(4) Right antibiotic was added, but the concentration is too high. In this case, the culture in the tube may still be 'clear' (no sign of growing) in 8 hours, but they may show 'turbid' in the culture much later.
Repeat the experiment Weng..You will surely get a turbid broth..As the bacteria may not die when kept in 4oC.I usually keep my bacteria in 4 for one month.There may not be any probs.But it case of some Vibrios.It start dieing after one and a half month.Most bacteria was stable upto many months.This problem occurs to me too.I think your loop may be hot while picking the colonies.That Killed the bacteria.
Regards
Noble
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