5 replies to this topic

[czernobill]

Hi,

I isolated some corynebacteria from bird. I streaked them on LB+Tween agar several times and there was good growth after 72h of incubation. Then, I sealed the plates with parafilm and stored them at 4°C for 3 months.
Now, I have serious problems in  cultivating them. They do not grow on LB+Tween plates. Even in LB+Tween broth there is no obserable growth.

Is there a general method for re-activating these bacteria.
Next time, I will try to grow the bacteri in liquid medium, add 50% glycerol and store them at -80°C. Maybe this helps.

Another question is how long I can store sheep blood at 4°C before adding them to liquid agar?


Thanks a lot!

Edited by czernobill, 08 November 2012 - 07:21 AM.

[El Crazy Xabi]

I don't know about the blood sheep but not all bacteria are keen to be stored.

For how long did you incubate this time? They may take quite longer than in active culture transfers

It happened something similar to me with fungal isolate I stored for >10 months but after several attempts finally, and I don't know why, it grew.

I'm not completely sure, but trying to incubate the original plate for a 24 h or so, so the cells may reactivate and then try to replate them to a fresh plate.

[Phil Geis]

Why Tween 80 - as an additional source of fatty acids (as used for culture of cutaneous islates) or as a neutralizing agent?  Tween 80 is also a surfactant and may have affected membrane integrity.  Also contiued esterase-driven buildup of fatty acids might also have affected viability.

Scrape off the entire culture remnant and culture in broth  - with and without Tween 80 - incubate with agitation.  You're not going to "reactivate" cells, you're looking for any suvivor.

[El Crazy Xabi]

I also thought that Tween 80 could be a source of stress, but it seems it enhances the growth of Corynebacterium species

[czernobill]

Hi,
thanks for your suggestions!
Before storing the bacteria on plate I tested LB with vs without Tween. On plates lacking the substance there was no growth. There is is quite a large number of publications where they used Tween as additive for culturing Corynebacteria.

Quote

Scrape off the entire culture remnant and culture in broth  - with and without Tween 80 - incubate with agitation.  You're not going to "reactivate" cells, you're looking for any suvivor.]

I am a little worried about taking the entire remnant from the plate. If this will not succeed, I would have nothing left. Until now, I have been trying to take about 10 colonies and inoculate a plate or liquid medium. This has not been working.

I followed Xabis advice and put the original plates to 37°C for three days and again inoculated a plate. Now, I am waiting for any colonies.
I emoved the original plates from the incubator and instead of putting it to 4°C, I put it into a jar inside my drawer.

Edited by czernobill, 13 November 2012 - 09:26 AM.

[Phil Geis]

I am familiar with Tween 80 facilitating growth - also found with species of human cutaneous oirgin.  The point was that the same material might be responsible for loss of viability over time.
You need to do enrchment. You're unlikely to raise cells from the dead - rather you want to find the potentially few viable cells in the mass.