Sudden growth on streak plates - (Dec/04/2018 )

Hi,

I'm currently trying to isolate bacteria in pure culture from sponge samples. I started by plating a small amount of homogenised sponge sample on an agar plate and incubating it at room temperature as I was instructed, no problems. Then I picked individual colonies from these plates and streaked each one onto a fresh agar plate using the 13 streak method, again, no problems. I will have normal growth (text-book streak plate colonies) on this first streak plate, and I would then transfer a colony to a second streak plate. I then incubate all plates overnight and when I check them in the morning, the first streak plate seems to have sudden, and very random, new colonies all over the plate (see yellow colonies in picture) .

The new colonies are always the same morphology as at least one of the colonies that was previously on the plate, so I am not convinced it is contamination, however I am concerned with the rate of growth. As I am culturing marine bacteria, the initial plates take over a week to show any growth and even then it is usually only a small amount. Then once they have been opened they suddenly are covered in randomly placed colonies. 

I have been very careful to work aseptically (this is not my first time in a microbiology lab and I am familiar with bacterial cultivation), and have carried out all of the above under a clean bench. I have also left open agar plates under the clean bench to try and see if it is an issue with the bench itself, but this doesn't seem to be the case. I am not sure why I am getting these strange growth patterns. I am a little concerned as I have never seen this before and it keeps happening on multiple plates, and while it is happening I cannot be sure that I have isolated anything in pure culture. 

Can anyone shed a little light on what the issue may be? If there even is an issue at all...

Many thanks,
Laura

What medium are you using?

Hi Laura,

An interesting problem. I don’t have a simple answer but maybe some additional control tests you can try. I think you are saying that the late-appearing, random colonies are possibly growing in places where your streaking loop hasn’t touched, is that correct?  Try streaking only on half a plate; do two identical plates each streaked (with the same inoculum) only on one side. Incubate, and after you see growth, open ONE of them and pick a colony for restreaking, then close it again.  Do not open the other of the pair of plates, and continue to incubate it side by side with the one you opened and closed.  Is there a difference in how they look after the additional incubation?  Does the one you never opened get new growth on it like the one that was opened?  Does any of the new growth on either plate occur on the half where you never touched the loop?  You have already left a plate open in the clean bench (hood?), but you might also add a plate with “mock streaking,” where you streak with a sterile loop and incubate that just to make sure nothing is coming in by the loop handle or something.

I don’t know much about marine bacteria but there are mycobacteria that can go airborne; I wonder if you have something like that.  Or if the late-appearing, random colonies stay in the area where you streaked, maybe they are just something with an extra-long incubation period. Or they could be “microcolonies” of one of the original organisms.  There are many possibilities.  Try to have fun figuring it out! And I’d open it only under the hood if I were you, too.

To add to the questions - can you mark the colonies you on the plate so that you know where the colonies were before you opened the plate?

Are these anaerobes by any chance?