This week’s article is an overview about a subject that is near and dear to our hearts here at MO BIO. That subject is BIOFILM!

After spending many months working with all types of biofilms and biomat samples from around San Diego and speaking with researchers all over the world, we understand the difficulty in determining the microbial diversity in these sample types.

In many ways, biofilms are similar to soils in that they are mixed microbial communities, with varying degrees of cell densities, moisture content, chemical composition and inhibitors.  Much like soils they can contain humic substances, metals, and salts, not to mention the polysaccharides, all of which can impact isolation and purification of nucleic acids.  However, the basic structural components of soils and biofilms are vastly different and require different approaches for optimized recovery of DNA and RNA.    

Here we describe some of the things to consider when working with biofilms.

Collection:

Collecting biofilms can be as easy as scraping the side of a very slimy rock or cutting a sample from a microbial mat or it can require the taking of both the biofilm and substrate it is attached too and separating the two by extensive mixing, homogenization, sonication, or chemical/enzymatic treatment.  Basically, the bigger and thicker the biofilm the easier it is to take a sample.  When biofilms grow thinly on substrates such as rocks, showers, or teeth, sample collection becomes more difficult.  The most commonly employed methods for separating the microbial community from the substrate in samples such as these are sonication, homogenization, and scraping.  Chemical and enzymatic treatments can be more specific and so may not work effectively on different types of extracellular compounds that may be present.

EPS: 

The extrapolymeric substances that bacteria secrete are important not only structurally but it can protect the bacteria from environmental stressors including application of antibiotics and cell lysis buffers.  The older and more developed the biofilm the more EPS and rigidity there is. Thicker biofilms may also have more sediment, salt and mineral deposits.  For the best lysis conditions, using less sample is key.  It is also worthwhile to take samples from more than one location due to potential differences in microenvironment that would influence the community composition.  For thinner biofilms, using more sample may be better as there may be fewer organisms within the community.  Additionally, if the substrate is small enough it may be possible to lyse the microbes directly from it by adding both the substrate and associated biofilm directly to the bead beating tube.

Lysis: 

Ensuring complete lysis is probably one of the most difficult things when it comes to biofilms.  A high powered bead beater can be used successfully, especially with microbial mats, but the more EPS that is present in the sample the more viscous the lysate will become if the bead beating time is too long.  As a result, as time increases the amount of lysate that can be removed and processed declines.  This will ultimately reduce overall nucleic acid yields.  Excessive homogenization is not a problem when using the vortex for lysis.  When combined with the PowerBiofilm™ lysis buffers which contain an EPS treatment and lysis enhancer, ten minutes of vortex lysing is sufficient and will result in yields similar to what can be obtained with high powered bead beating. 

Inhibitors:

Even with sufficient lysis, carryover of the degraded polysaccharide, humic substances, and other organic/inorganic compounds can occur.  One method for removing the degraded polysaccharide after the lysis step is Inhibitor Removal Technology (IRT).  Depending on the color and viscosity of the lysate (an indicator of the level of inhibitors) the IRT steps can be modified.  For relatively clear samples or samples that are known to have less EPS (and therefore, less viscosity), less inhibitor removal can be used.  For viscous samples or samples that have a lot of humic related brown color to them, more inhibitor removal can be used.  Two different amounts are suggested because in the absence of inhibitors some nucleic acid removal may occur.  Therefore, using the right amount of inhibitor removal will help to optimize overall yields.

Summary:

For the best inhibitor free nucleic acid yields, it is important to balance EPS content with cell densities.  This is done by selecting the right amount of sample to process and not bead beating for too long.  Selecting the right amount of inhibitor removal can also help to optimize yields.  MO BIO has many customers working with biofilms so it is a very exciting topic to us and one that we will be discussing more of in the future.    

For more information of biofilms and microbial mats visit these sites:

http://www.biofilmsonline.com/cgi-bin/biofilmsonline/index.html

www.biofilmbook.com

What is your experience with biofilms?  Do you have a great method to detach biofilms from their substrates or have an interesting sample that requires special handling?  Let us know!

-Heather