Problem with biofilm assay - plzzzzz i have problem (Aug/08/2010 )

HI every one
Iam so happy to be here
I have problem and i need u to helpe me plz i have done my experiments on the effect of antibiotic on bacterial cells within the biofilm i used two strains E.coli and sulphate reducing bacteria (SRB) i have the result that when they grow as pure culture the antibiotic can kill almost of them but when they grow as mix culture they survive
BUT when i did counting for both strains i have the planktonic cells more than biofilm cells (i added the antibiotic in the same day of inoculation)which against the theary
is my result wrong because iread that biofilm cells usually more resistance than planktonic cells who can explaine for me why planktonic more than biofilm cells in my case plz (i did this many time) and i had the same result

sorry about my English


i am waiting for ur replay as soon as possible

Thanks

sara1 on Sun Aug 8 14:56:10 2010 said:

How do you do counting for both planktonic cells and biofilm cells? using streak plate? We have to know how you do it before we can answer you.
p/s: where are you from?

counting
E.coli serial dilutions in MacConkey agar
SRB under the light microscope using chamber

And what are your planktonic cells ? and wich ones are the biofilm ones?


I suppose the SRB ones are the biofilm ones? and the ecoli the planktonic ones? or how do you test wich is wich?
How did you make the biofilm? how do you work??

How do you take samples?

because its important to know how to takes samples.

+ you need to give the bacteria time to make a biofilm...


Too many questions...

Describe the experiment more.

sara1 on Sun Aug 8 14:56:10 2010 said:

sara1 on Sun Aug 8 20:32:21 2010 said:

Do you re-test the survivial colonies as there are not contamination of other bacteria species which may be possible survived and degraded the antibiotics?

Lets say if you are not expect all your cells to die in both planktonic or biofilm condition after the addition of antibiotics, (And antibiotics seems to just "slow down" their growth for this special case), under planktonic condition all the growth and divided cells will still remain in the aqueous phase, whereas in biofilm condition (no shaking, leave on benchtop etc...) all your cells will settled down in the bottom of your plate, attached and form biofilm. In biofilm condition, your cells will grow (even if they really grow) very very very slowly (this is the nature of biofilm forming bacteria). Thus relatively, cells in your planktonic condition will surely have more cells count than your biofilm.

If you are doing efficiency of antibiotics, I would suggest you do a comparison between antibiotic treated/non-treated in planktonic , and treated/non-treated in biofilm, rather than "treated planktonic" vs "treated biofilm".

Hope this helps.

Adrian

Thanks alot Adrian for your explanation
it is realy helpful
and thanks for every one who replay
just one question
why antibiotic did not kill planktonic cells in mixed culture

sara1 on Wed Aug 11 23:05:24 2010 said:

In order to answer that you have to know the mechanism of your antibiotic in killing your cells.
Is it bactericidal (kills bacteria) or bacteriostatic (inhibit growth)?

It could be possibility that your bacteria's gene expression during planktonic condition had produced "something" that destroy the antibiotics... Could be due to bacteria quorum sensing as well... but I really not sure. Maybe there's anyone could enlighten us in this issue?

OR your results could be a very new and interesting findings...

There is nothing special about the fact that 1 type of bacteria will be destroyed when using antibiotics and that they survive when its a mixed culture.
This has indeed to do with for example quorum sensing.

Another remark is: how do you test this?

Because you tell nothing about the experiment itself.

You say that they die when using a non mixed culture, but how did you test this? And how did you test the mixed culture? Did you do both on plates? Or?

And the antibiotic did kill both types of bacteria? (because I have spoken to people using a type of antibiotic killing type A, but not type B and then stating that when using a mixed culture both types survived while it was only type B that survived and they forgot to test if type B was resistant to it..)

BTW: how do you test planktonic cells? Because for all you know you are taking samples that arent really representative for planktonic cells.