What type of agar is best to grow my bacteria? - Sceince fair project on leaf bacteria, can someone help (Dec/01/2005 )
HI,
I am doing a science fair project on a topic that i found while researching. It concerns an idea that plant leaves have protective bacteria that not only prevent the passage of harmful environmental bacteria into the leaf but also kills these bacteria when the two come into contact. I'm not sure if this will make since to anyone but what i plan on doing is getting the thickest leaf I can find and then after sterilizing the exterior of the leaf bending it flat to allow for the exposing of the inside of the leaf. After doing this I will take a sample of the bacterial growth on the inside of the leaf and swab it onto a petri dish containg some type of agar. now this is where i run into trouble because i do not know what type of agar i should have in my dish. Seeing as how i do not know what types of bacteria there are inside the plant i was thinking about an agar that would give similar conditions to that of the plant so that the bacteria that i obtain will grow so that i can then find out what type of bacteria they are and transfer each seperate individual colony to a streak plate. Any way at the moment i am trying to figure out what type of agar is best to use for my project. Could someone please help me. I would really appreciate any type of feedback information. Thanks a lot for any help that i get.
Loulou
Why are yo not looking forbacteria on the surface of the leaf? I think that its more likely that the plant would have formed relationships with certain bactera that promote their adherence to its surface. These bacteria would then form a biofilm and protect the plant from other harmful bacteria. This is often seen on marine plants such as tunicates. Its worth a try but I suspect that you will not get much growth (if any) if you plate samples from the interior of the plant. I would look at using agar mediums that promote the growth of Psudomonas type organisms. Do a google search and you should get the composition. You could also try a rich medium like LB as a basal medium.
oh well what you are saying makes sense but I was thinking that looking at the interior would cover the types of bacteria that try to diffuse through the layers of the leaf exterior. It might make more sence to do both of these. I see your point but would this make my project less significant do you think. oh and thanks for the suggestions on agar; i tried to find some info on this topic but i must have been looking under the wrong places. Thanks again. Please feel free to make more comments.
Loulou
If you don’t know what you are looking for I would suggest a general medium such as TSA, if you can be more specific as to what you expect to find I may be able to suggest a more specific medium but TSA is a very general agar and should do fine.
Also I would agree with ML1975 you should think of looking at the surface of the leaf rather than the interior.
Thanks redwaler for your help. However, since i now have some ideas on agar if i were to look at the outside of the leaf then this would complicate my project a bit more. First of all how would i destinguish the bacteria that is harmful to the leaf to that which is helping protect it. I can't sterilize the outside of the leaf like i had origiinally planned because doing this would get rid of all the bacteria on the exterior of the leaf. I'd need a way of obtaining just the bacteria helping the plant keep other environmental bacteria out from entering into the leaf. Do you have any suggestions as to what I should do. I have a procedure written up on the way I had planned on doing my project but I am open to any ideas and this sounds plausible I just hope that it is possible to do. If you would like I can post a generalized procedure so that you can look at.
I would appreciate any type of input from anyone. Any type of ideas might be of help. Thanks again!
Loulou
do leaf cells contain internal bacteria that are beneficial? I am not an expert, but I would bet that your beneficial bacteria will be on the outside
bacteria inside leaf cells would likely not be beneficial, because they would be feeding on the leaf itself (the host) in order to survive. bacteria inside the cells would likely be pathogens and not commensals
good luck, your idea is great but I think execution will be tricky
I don’t think you would get any bacteria inside the leaf but im not certain, but as you said in your first post you want to assess the beneficial bacteria that are on the leaf surface that prevent the harmful bacteria from entering the cell in the first place surely if you look inside the cell you are not assessing what you set out to do. If you can post a procedure or send me a private message I will do my best to help you out.
Thanks everyone for the input. I am going to look into these ideas further. These ideas are all helpful, thanks again. Below is a general procedure that i have written up if anyone would like to look at it.
1. obtain the thickest leaf possible(aloe is one)
2. Now you will need to sterilize the outside of the leaf by dipping it into a solution of approximately a 3% bleach solution for a about a minute and then rinse this in some cool sterilized water.
3. bend the leaf in half so that is cracks exposing the insidelayers.
4. Using a sterile swab, rub the swab onto the exposed layers of leaf and then swab it onto plates. use at least two so that you have a backup in case one dish does not produce any colonies. The plates should have agar on them( i have not quite yet decided exactly what type of agar i plan to use)
5. because the leaf is currently at room temperature the plates should be incubated at room temperature also.
6. After 24 hours, look at plates and see which colonies grew the largest and also look at some of the smaller colonies too because i don't know exactly what bacteria it is that i am looking for so analyzing as many possibilities as possible would be best.
7. using some of these colonies make "streak plates" for individual colonies growing on the main dish.
8. after allowing these streak plates to grow, you can now produce "stock plates" so that you can use these plates to make further examination of the cells.
9. I would like to then make microscopic examinations of the stock plates with each type of bacterium but I'm not sure how.
10. Also i would need to try and identify some of these bacteria. Someone I know suggested trying to grow them on certain types of agar in order to help identify the type of bacteria( however in my opinion this is just guessing and checking which might take longer than i have)
11. once i get these bacteria identified which i am hoping will be fairly simple, i will test them with other types of bacteria and germs found in the environment that may be pathogenically spread and see if any of the bacteria that i cultured prevented the growth of these bacteria or if they possibly kill them off also. I have not yet got into the depth of this step just yet because i first need to get a better hold of what i am going to do in the first couple of steps.
What will probably be my biggest problems are: 1st how will i seperate the good cells from bad cells if i am to do what everyone is suggesting, 2nd how will i be able to identify the types of bacteria that i have. the agar type is only a small problem compared to the previous two. If you know anything about this i would greatly appreciate any help. Thanks
Loulou
I’ve send you a private message so I wont repeat what I put there but I’m not sure if I pointed this part out, you should look at all the growth and not just the large colonies as some will grow faster than others and you should also incubate the plates for longer at that temperature.
I don't know a lot about plant commensalism (my data once forced me to chase down the role Rhizobium plays in fixing nitrogen for legumes, and I once figured out an enzymatic pathway in Arabidopsis) but, as others have pointed out, you are making some pretty sweeping assumptions in your experimental design, such as:
- Bacteria on the outside surface of the leaf must be bad.
- Bacteria inside the leaf must be good.
- There are going to be bacteria found inside the leaf.
- These bacteria will be culturable on routine agar at room temperature.
- Any bacteria found inside the leaf must be there to protect the leaf from invading pathogens.
Let's formulate a good hypothesis first, then we'll all come up with an experimental design that'll help you test that hypothesis.