Hi,

If you will use fluorescent protein for colocalisation which is same in fluorescent nature then you can not distinguish between same and different bacteria.

You should use different Fluorescent protein ( GFP, YFP) for the study of colacalisation of different protein.

Now the question is which gene you will use for fusion with GFP or YFP. The gene should be consecutively express and should be on cell surface like cell membrane protein.

Bye..

Hi Salim!

Thanks for your response.

Biswadeep--by co-localization I assume you mean evaluate whether you have multiple bacterial endophytes. I would start by first examine bacteria on plants. Do water washes (with a little tween detergent; 2 drops in 100 mls H2O) from the surface and plate this onto TSA or other bacterial media. This will tell you what is on the surface. Then surface disinfect the plant tissues to remove surface bacteria. If these are leaves or herbaceous plant parts you can surface disinfect with 1% sodium hypochlorite for 5 minutes with agitation. If the tissues are seeds or woody stems more rigorous surface disinfection is needed--perhaps 3% sodium hypochlorite for 20 or more minutes--with constant agitation. After rinsing off the sodium hypochlorite--you can triturate or grind the tissues to small pieces and suspend in sterile water. The suspension should contain the plant tissues and bacteria in small enough pieces that you can plate onto TSA or other media. If you diluted the suspension of ground tissue enough you should have bacteria separated out enough to obtain pure cultures. Then id your bacteria using 16S sequences. Using variations of this technique--you could even estimate the number of bacterial cells per unit of mass of plant tissues. It's a really simple method but if done carefully--you could infer some important information. Good luck with it. Jim White, Rutgers University

James, Thank you for your response and idea. Actually I have strains of bacteria and want to see how they localise themselves within the xylem. Help me out how to tag my strains with gfp to study them?

Biswadeep I assume you want two localise two/more bacteria not different strains of same bacterium. I would suggest co-localise two or more bacteria through IHC (with fluorophore tagged secondary antibody) using multiple antigen labelling method. Use commercially available primary antibody against the surface protein of the bacteria of your interest than use flurophore tagged secondary antibody say for example FITC. Observe the sample in fluorescent microscope. You can also try Grams staining.

Good luck.