Biofilm in potable water - (Mar/16/2010 )

Hello!
I was hoping someone had experience with biofilms!
My thesis has changed a bit along the way and now I will be culturing the bacteria from the biofilm(from potable water), I have the plan ready for the agarplates but I have no idea how to treat the biofilm when it`s ready, I scrape it off and then..? How to break the EPS? What do I use to dilute it in without killing the cells?

Sorry for my bad english...

When I worked on biofilms, we scraped the biofilm from the plastic samples with a spatula moved the spatula a bit in some clean, sterile water and plated them out in non diluted or diluted form. ( you can simply dilute the water in wich you moved the spatula around to get the bacteria of the spatula)

In short:

take plastic rings from the water ( or whatever you use to get the biofilm growing)

use a spatula to get some of the bateria of the plastic rings

Put the spatula in some water, turn it around a bit

and dilute it or plate it out

I do need to say that we only let the biofilm grow for 1 max 3 days and we used plastic rings we placed in the water.

How are you getting (collecting) the biofilm?

Hi Pito and thanks for you help!

I use flowcells, if you have heard of them? http://cu.imt.net/~mitbst/Flow_Cells.html (in case you haven`t) so it should be easy to scrape it off, its just glassplates.
Water is running through the cells with about 10ml/min, for about 5 weeks(3 left) from the looks of it.

So it`s enough to just rinse it off with sterile water? No vortexing required? And I also dilute it in the sterile water?

yeah, just scrape it off and rince your spatula in some water (0.9%)

and then plate it out.

However: whats the object of your thesis?

Vortexing might be required, just try some with vortexing and some without and see if there is a difference.

( we sometimes placed the spatula in some water and then vortexed the little beaker with the spatula in it in stead of just moving the spatula around)

Ok, I will try that, thanks for your help!

I`m writing my thesis for a water treatment plant, they have this pilotplant running parallel to the main waterplant and there is a "little" problem with the biofilm. I collect the biofilm after each treatment the water goes through and compare the results (i take pictures of it every week and count the cells). It started with something else and now it`s mainly about the "life" inside biofilm. I will be plating the biofilm to find out how much coliforms, mold, Pseudomonas aeruginosa and Legionella there is. The 2 last ones are most likely not there because of the temperature of the water. But this is what they want me to investigate..

ah I see, anyway then you can use the method I described. The important thing is that you simply plate them out and identify them by plating out and "watching"what it is or are you going to sequence them?

I will have to count them, so i have to use serial dilution method. The agarplates i use will be specific, so if theres growth it will be only that one bacteria growing-or not.

Now i seem to have another pickle with the serial dilution, my supervisor said i should go from 1000cfu/g biofilm, is it usual to estimate it in grams?

You could indeed do that, however, how are you going to see how much grams you have? If you are going to take samples each day, it will be hard to get an amount in grams since the biofilm will be small and very thin.

I do not know the exact method you use.
Others use coverage (in combination with thickness).

It all depends on what you are cabable of measuring.
I suppose your supervisor knows what he/she is doing.

I use flowcells, the potable water is running through the cells and will be running for another 2-3 weeks, in the end when we think its enough, i open the cell, scrape off the biofilm, dilute it and plate it. Thats the plan at least, Im just all bewildered about how to do the first dilution..oh well, I`m sure my supervisor knows..i hope!

Thanks for you help!

You could put some cover slips in the flow cell that had been weighed before hand. Then you could pull them out, dry them, and weigh a second time to determine a bacterial load.