luminescence measurement of Mtb or BCG - (Dec/06/2007 )
Hi, everybody,
How was you day? not bad, fine, good or very good?
Any one here use luciferase gene engineered Mtb or BCG for measurement its viability? I just have done some experiment to study the role of some cytokines in the innate immunity system in response to Mycobacterium infection. I use luciferase tagged Mycobacterium bovis BCG to infect macrophage, then measure the luminescence to quantify the bacteria. However, I found that this method is not so accurate. the results have big difference with the results of plating. I am wondering what's the problem? since there so many papers reported this method. I think it should be reliable.
Any advices will be appreciated.
many thanks.
first of all.... we have the same name! 
second... luciferase always needs to have a control. typical controls are beta gal, or to co-transfect a cat plamid. ther is also the renilla v firefly method. have a look at the other papers and see what they used as a control.
V
Hi Yvette
I also work with TB and BCG for macrophage infection. I also use the luciferase activity to see the proliferation. I use the plasmid pSTM1 for luciferase activity and it work well and consistent with plating.
If I can help you, you can ask me precise question.
biofred
second... luciferase always needs to have a control. typical controls are beta gal, or to co-transfect a cat plamid. ther is also the renilla v firefly method. have a look at the other papers and see what they used as a control.
V
Thank you vetticus3. maybe you borrowed part of my username. No problem, you can use it freely, there are no copyright conflict.
I don't understand what you said about the control of luciferase very much. aren't there any control in papers measurement Mtb with luminescence.
I also work with TB and BCG for macrophage infection. I also use the luciferase activity to see the proliferation. I use the plasmid pSTM1 for luciferase activity and it work well and consistent with plating.
If I can help you, you can ask me precise question.
biofred
It is very funny that there was a message said that "Flood control is enabled on this board, please wait 20 seconds before replying or posting a new topic" when I reply the second times. I suppose that the system thought that I am supplying water.
Hi, biofred, thank you very much for your kind reply. I use the plasmid pSMT3LxEGFP which have been engineered with double mark------luciferese and EGFP. my problem is that my luciferase measurement result was not consistent with plating. I used 0.01% decanal as the substrate and measured luminescence with Berthold Luminometer Sirius . I found that the error of different measurement were so big. I measured the same sample for three time, however, there were big difference among the three results. As I know that the Mtb or BCG is apt to get together and form clamps, so it is not easy to make the bacteria diffuse equably. I sonicated the sample and the same problem still exist. I am not sure if there are any problem with the luminometer. I heard that the Berthold tube luminometer is not so precising.
so, biofred, could you pls give me your protocol. what kind of luminometer do you use. what and how much substrate do you use.
Thank you very much.
I also work with TB and BCG for macrophage infection. I also use the luciferase activity to see the proliferation. I use the plasmid pSTM1 for luciferase activity and it work well and consistent with plating.
If I can help you, you can ask me precise question.
biofred
It is very funny that there was a message said that "Flood control is enabled on this board, please wait 20 seconds before replying or posting a new topic" when I reply the second times. I suppose that the system thought that I am supplying water.
Hi, biofred, thank you very much for your kind reply. I use the plasmid pSMT3LxEGFP which have been engineered with double mark------luciferese and EGFP. my problem is that my luciferase measurement result was not consistent with plating. I used 0.01% decanal as the substrate and measured luminescence with Berthold Luminometer Sirius . I found that the error of different measurement were so big. I measured the same sample for three time, however, there were big difference among the three results. As I know that the Mtb or BCG is apt to get together and form clamps, so it is not easy to make the bacteria diffuse equably. I sonicated the sample and the same problem still exist. I am not sure if there are any problem with the luminometer. I heard that the Berthold tube luminometer is not so precising.
so, biofred, could you pls give me your protocol. what kind of luminometer do you use. what and how much substrate do you use.
Thank you very much.
Hi Yvette, first at all I wish you an Happy new year
For luciferase experiment I use some strains transfomed with the pSTM-1 plasmid (Paper in attach and just in case: Assessment of Immunity to Mycobacterial Infection
with Luciferase Reporter Constructs, Snewin 1999). When I compared my result of plating and luciferase activity, I can see the same effect. Of course for luciferase activity you need to have a homogenous culture but it's the same for plating. For that we always add tween 80 (200µl/500ml) in our liquid medium. When I compared my triplicate, It's almost the same except some error of manipulation of course.
Concerning the substrate I use decanal 1% in ethanol. To measure, I put 100µl of culture in 2ml tube, add 900µl of PBS and in the end 100µl of substrate just before the measure. Our luminometer is a turner designs TD 20/20.
I hope it's can help you, I need more just ask!
Bye have a nice day
biofred
Hi, Biofred, I really appreciate your kind reply.
There are still some questions:
1. I use the stock of bacteria to infect macrophage. my protocol for infection is as below:
- defreeze bacteria
- spin 10 min 10.000 x g 4 °C
- decant supernatant and resolve in PBS, vortex
- sonicate for 20 sec in sonicator bath
- ready for injection
The bacteria suspension was incubated with macrophage in 24-well plate for 2 hours to infect macrophage. After incubating for 2 hours, I wash the cell with PBS for two times and I found that some bacteria clumps which were snatched by the macrophage can’t be washed. These extracellular bacteria can still propagate in the culture medium. After sonicating the bacteria, I have dipped a drop of bacteria suspension on a slide and watch with microscope, I found that bacteria were still clumps. That was to say that some macrophage can’t contact with bacteria and can’t be infected, however, some macrophage have been infected with a clump of bacteria. I suppose this is the reason why I can’t get consistent results for each experiment.
I think the ideal situation is to make the bacteria form a homogeneous suspension. So every macrophage have the same opportunity to be infected. The extracellular bacteria should be washed out after infection, then what we count is the intracellular bacteria (I have tried to use Gentamycin to kill the extracellular bacteria after infection, however, Gentamycin can affect the growth of bacteria).
How do you deal with the bacteria before infection. Do you have any idea of how to get the homogeneous suspension of bacteria for infection? Could you give me your protocol of infecting macrophage with Mtb or BCG?
2. For the measurement of CFU and RLU after infection, I always use 0.1% Triton to lyse the infected macrophage. I do not really understand what you said. Do you mean that add tween 80 (200µl/500ml) into the cell lysate then plating or measuring the RLU?
Thank you again and have a glorious new year!
There are still some questions:
1. I use the stock of bacteria to infect macrophage. my protocol for infection is as below:
- defreeze bacteria
- spin 10 min 10.000 x g 4 °C
- decant supernatant and resolve in PBS, vortex
- sonicate for 20 sec in sonicator bath
- ready for injection
The bacteria suspension was incubated with macrophage in 24-well plate for 2 hours to infect macrophage. After incubating for 2 hours, I wash the cell with PBS for two times and I found that some bacteria clumps which were snatched by the macrophage can’t be washed. These extracellular bacteria can still propagate in the culture medium. After sonicating the bacteria, I have dipped a drop of bacteria suspension on a slide and watch with microscope, I found that bacteria were still clumps. That was to say that some macrophage can’t contact with bacteria and can’t be infected, however, some macrophage have been infected with a clump of bacteria. I suppose this is the reason why I can’t get consistent results for each experiment.
I think the ideal situation is to make the bacteria form a homogeneous suspension. So every macrophage have the same opportunity to be infected. The extracellular bacteria should be washed out after infection, then what we count is the intracellular bacteria (I have tried to use Gentamycin to kill the extracellular bacteria after infection, however, Gentamycin can affect the growth of bacteria).
How do you deal with the bacteria before infection. Do you have any idea of how to get the homogeneous suspension of bacteria for infection? Could you give me your protocol of infecting macrophage with Mtb or BCG?
2. For the measurement of CFU and RLU after infection, I always use 0.1% Triton to lyse the infected macrophage. I do not really understand what you said. Do you mean that add tween 80 (200µl/500ml) into the cell lysate then plating or measuring the RLU?
Thank you again and have a glorious new year!
Hi Yvette,
To infect my macrophage I use an homogeneous (always add Tween 80 in you medium (200µl for 500ml of 7H9+OADC)) cultur of mycobacteria with an exponential growth like 0,6. I use different MOI and for that I can translate my OD to CFU. After preparation bacterial suspension I can infect my macrophage. To see if you bacterial suspension is homogeneous you can test the luciferase activity of triplicate and normally the result will be the same for all. I am doing 3h of incubation because I test different time and this one is the best. After that I remove the medium with the extracellular mycobacteria and wash 2 times like you. Normally like this you remove almost all mycobacteria. If you want you can also treat you macrophage with amykacin (who kill extracellular mycobacteria) but it's not really necessary.
To count my intracellular mycobacteria, I remove the medium and add 200µl of SDS 1% (for 24 well) and leave for 5'. You can see under light microscope your cell lysate. So you're ready to test luciferase activity. For the plating it's the same, just realise sequential dilution of your cell lysate, plate and wait.
Just a question why do you use this kind of protocol because for infection I always saw infection with liquid culture. Your mycobacteria need to recuperate before use. If you bacteria are not homogeneous all of your test will be disappointed.
Hope to help
biofred