We speak with many scientists each week who are working with filtered water for the isolation of microbial DNA and RNA. Water samples are difficult because of their typically low biomass (depending on the water source) and because these samples are precious.

Why is molecular research on microbes in water difficult?

For some people, getting back to the original source of water may not be possible for months or even years. For example, we talk to scientists collecting samples at hydrothermal vents in the middle of the ocean, in the Antarctic, and in the Baltic Sea.  For some researchers, water samples may have been collected after a certain event, such as a flood or heavy rain and so the conditions of the water will not be the same in a week or even after a day. They need to get answers from every sample collected and they need it to be accurately reflect the current microbial content.

Choosing a Filter:

People who want to determine the microbial communities of collected water will filter them onto filter membranes. The typical size is a 47 mm membrane. This is large enough to have a good flow but small enough to work for DNA or RNA extraction. If the membrane is too small (25 mm), it may clog if the water contains higher levels of debris and if it is too big (142 mm), it will need to be sliced up in order to fit in standard 5 ml and 15 ml tubes.  Ideally, the less handling and manipulations going on with the water filter, the more microbial DNA and RNA can be recovered.

To help make sure that the 47 mm filter membranes are extracted the most efficiently without needing to be sliced into small pieces, MO BIO Labs uses a 5 ml screw cap tube (see picture right). This tube allows for full access of the microbial side of the filter to be homogenized with the garnet grinding resin. We have found after thorough testing that this tube allows for maximal recovery of DNA from all types of filter membranes.

Another question we hear from customers is how to choose a type of membrane. There are many choices from polyethersulfone (PES) to mixed cellulose esther, MCE (cellulose acetate and cellulose nitrate) to polycarbonate to aluminum oxide. Each of these membrane types handle a bit differently and will give slightly different results after extraction.  It is important to remember that the different characteristics of a membrane also reflect its use for other applications such as direct culturing (PES, MCE) or light and electron microscopy (polycarbonate, aluminum oxide).  Overall selection of a membrane for DNA and RNA isolation is more dependent on pore size, sample volume, and retention of inhibitors such as pesticides.  In other words, more than one membrane type may work for your application.

In our experience here is what we found:

Polyethersulfone: Are one of the toughest membranes and can be handled more than the others. They dry quickly under vacuum making them easy to fold without tearing.   Both 0.45 and 0.22 micron pore sizes can be used but a 0.22 micron pore size is best when you want to filter large volumes of water with low microbial biomass because they can handle the longer harder pressure of the vacuum. For nucleic acid extraction, we can get yields equivalent to the mixed cellulose esther with the PowerWater® DNA and RNA Isolation Kits.

Mixed cellulose esther (cellulose acetate and cellulose nitrate): Are best for when a 0.45 micron pore size is needed.  We recommend the use 0.45 micron pore size if your water has a lot of debris and tends to clog or filter very slowly with 0.22 micron pore sized membrane. Cellulose membranes tend to retain water making them a little more difficult to handle.  The video below will demonstrate how we handle them in our lab.

There are several published studies demonstrating that pesticides and herbicides can bind to cellulose acetate and cellulose nitrate so if you are using water that may contain pesticides and herbicides, avoid using cellulose membranes.

Polycarbonate: This type of filter can be more difficult to work with due to its thinness and the ease at which it can wrinkle.  A 0.45 micron pore size is commonly used to prevent clogging.  Unlike the PES and MCE membranes, microbes in your water sample will sit on top of the membrane rather then inside.  This leads to clogging faster but also retention of smaller particles that would have been able to pass through.  We have found that for isolating DNA, less extreme bead beating will give you higher molecular weight DNA. If your sample is used for PCR only, then the stronger bead methods should be fine although expect a lot of shearing.

Aluminum Oxide: This type of filter is also known as an Anodisc™ filter membrane (Whatman).   It handles like a thin sheet of glass and will break up easily in any bead tube. Most labs are not using these due to the difficulty in transferring them to storage tubes. These are used with samples containing very low biomass such as ocean water.  They come in both 0.45 and 0.22 micron sizes.  Similar to the polycarbonate, microbes are retained more on top rather than within the filter, leading to easy extraction of DNA and RNA but also increased shearing with bead beating.

How to Handle a Filter Membrane:

Many of you out there probably already have a good technique for folding your filter membranes and placing them into a tube. For those of you who are new, or experiencing problems with this, here is a video we made in our lab to demonstrate the technique. This is a 0.45 micron mixed cellulose esther membrane.  Demonstrating is Heather Callahan, Ph.D, the scientist who created the PowerWater^® kit series and speaking is our Technical Support Manager, Nik von Atzigen:

Summary:

We know how hard it is to work with different environmental water samples and how important it is to get every last drop of DNA or RNA. Our goal is to make sure you are successful at every step. When we developed the PowerWater^®and RapidWater^®products, we kept that in mind as we optimized each step; from the tube needed to grind in, to the matrix used for grinding, to the solutions used for removing inhibitors, to the binding chemistry, to the washing chemistry, and the final elution. If you have any questions on how to maximize your water filter DNA or RNA extraction, please send them to us and we’ll get back to you.  I can assure you, if you have a problem, we have the answer.

Ask the Water DNA and RNA Isolation Experts!

For more information on Water DNA and RNA Isolation products, go to:

PowerWater^® DNA Isolation Kits and RapidWater^® DNA Isolation Kits

PowerWater® RNA Isolation Kits

~Suzanne (follow me on twitter: http://twitter.com/SuzyScientist)

This entry was posted on Sunday, October 11th, 2009 at 12:10 pm and is filed under Tech Tips. You can follow any responses to this entry through the RSS 2.0 feed. You can leave a response, or trackback from your own site.