Protocol Online logo
Top : New Forum Archives (2009-): : Microbiology

How to remove attached bacteria (or biofilm) from metallic samples - (Apr/24/2013 )

Hi everyone,

I am doing E.coli attachment test on steel samples. I immersed my steel samples in solution containing E.coli for different time intervals from min to hours. Then i washed my samples with sterile milliQ water and dried them in air. Then I did microscopic analysis of the attached bacterial cells. After that I tried to ultrasonically clean my samples by immersing them in DI water for 5-10 min but I am not getting proper cleaning of my samples and bacteria are still there on my sample.

Does anyone have experienced of cleaning the bacteria from metal samples. Any help will be highly appreciated. Thanks

-Fiaq Khan-


We vortex our stainless steel discs with approximately 10 acid washed beads in a volume of PBS for 2 mins at 1800 rpm. It's a very effective method for removal of biofilm-encased cells.


Hi Rahaf,

Thanks for your reply. Please explain me a bit more that what is the size of your stainless steel discs and please also explain the term "10 acid washed beads". Thanks

-Fiaq Khan-


The discs we use are approximately 1.5 - 2 cm in diameter.

To acid wash the glass beads, we immerse about 50g of glass beads in 80 ml of 2M HCL for 1 hour, then wash with distilled water (about 10 times - 100ml each time) until the pH is neutral. This just ensures the removal of any debris from the beads. Then autoclave the beads before use. You can also purchase pre-acid-washed glass beads from sigma, so that can save you a bit of time.

Also, may I ask what growth conditions do you use for cultivating your biofilms (ie. media concentration, addition of supplemental nutrients)?



What type of sonication instrument? Probe or bath? Ultrasound baths need quite a long time 30 min minimum and MQ water is not the best actually. Proceed as if you wanted to clean the metal, use a pyrophosphate solution, or a surfactant.

You can also try enzymatic methods, probably proteinase K plus something else, better to know what's in your biofilm to better choose of enzymes.

Anyway, why do you want to remove the biomass? Depending what you wanna you should choose one or other method.

-El Crazy Xabi-

I am using Ultrasonic bath. I have used steel samples and performed attachment studies with E.coli bacteria. After 4 hours of attachment, I dried my samples and did microscopic analysis. Now I want to remove the attached bacteria from my steel surface without effecting the surface of steel.

-Fiaq Khan-

If you wanna keep the surface of steel intact, I wouldn't recommend to use the beads procedure or acid, they will alter it.

-El Crazy Xabi-

Can you describe the "steel"? In manufacturing applications, folks remove biofilm with caustic (dilute KOH or NaOH) with a very low concentration of EDTA. This would be only appropriate for stainless steel - esp. 316.

-Phil Geis-