It is going to kill me

[Nmr]

Hi friends...
It is going to kill me....my bacteria not grown yet.. with my minimal media that i prepared using MCA as a carbon source..

any suggestions from you guys that may help me to find out the problem..

thanks

[JackBean]

if you don't tell us, what bacteria it is, we probably won't be able to give you any advice

[Nmr]

actually... i dont know the strain of the bacteria. I'm going to identify it.

I have a sample from soil and this soil supposed to have bacteria that able to degrade the MCA which is only the carbon source.

[JackBean]

then I would suggest to measure pH and ionic strength of the soil as well as composition of micronutrients, so that you can have appropriate media

[magicsiew]

Can try with PYG media + FBS, or just PYG alone...

[Nmr]

Hello guys again...

the problem that i have to isolate bacteria from the soil that i believed it is contaminated with Chloroaliphatic compounds such as MCA, DCA, TCA.
so i have been preparing the media since 3 weeks ago and i got nothing. until now i prepared 3 different forms of minimal media 2 broth and one agar which are containing the above chloroaliphatic compounds as carbon source and energy. then i put the sample in 2 different ways first i put 5 gr of the soil directly to the broth medium in flask then incubated for one week after that i strike on the agar plate that contain the same ingredients of the broth medium i got very very light growth, but i feel not happy with it.
the other way i spread the suspended soil with D.W on the agar plate directly. then incubated in 32 C as the outdoor temperature.

Note: the PH of my meduim is 6.5 the temperature in 32 as outdoor temperature the the shake is 122 rpm.
So,guys any suggestions for my case...and to make easy for help me...do you think that ...1- the problem with the sample.
2- with my medium that i prepared .
3-with the conditions ( ph, temp, air..etc).
4- others.
Guys time running out of me...so any suggestions and solutions of my case

thank you..

[canalon]

1- Your sample seem to contain something since you have growth in your first technique. Evn though for some reason your are not happy with it. In fact the 2 steps technique appear cleaner to me as you are diluting the soil more, hence removing other trace carbon contamination. And I would not resuspend the soil in distilled water, rather in your broth (with or without carbon source), that would probably be less stressful than pure water.

2- Might be. Degrading bacteria are not necessarily able to grow on minimum media. They might need other things present in soil on top of your carbon source to grow (iron, maybe as heme; Nitrogen source; whatever else)

3- You are working with soil bacteria, aeration is not continuous and even in surface soil there are plenty of nearly anaerobic pockets in the soil. I would also try anaerobic and microanaerobic conditions to see if there is something. The rest seems OK but you might want to test diverse conditions

So in short you are looking for an elusive bacteria that you have no clue about. You have to cater to many possible lifestyles in hope of finding the right one. you cannot content yourself of one condition.

Otherwise, try SIP: enrich the soil in 13C MCA/DCA/TCA, let grow for a correct time (might be variable, too long and the degrader will have strating sharing the goodies around, too short and there won't be enough DNA to separate). Extract DNA and separate 13C labelled DNA from regular 12C and do 16S identification of the 13C labelled bacteria. Use that to tailor your growing conditions to the species/genus that you now suspect of using your nCA.

[Nmr]

Thank you my friend for your tips and clues...and i will keep trying hopefully to get something .