So as you’ve surmised from the last posts, I’m doing a water study in Kampala…but what do I do with the water? Why do I do it? How do I do it?
Step 1 (a): Sterilization
First things first, I prepare a 10L bucket of 10% bleach solution. You can do this using household bleach or about 7g of Calcium Hypochlorite dissolved in water. We use bleach because it degrades RNA and DNA, the genomic material of all living organisms. This is important because our microbial analysis methods detect the genome sequence of diarrhea-causing pathogens, like Salmonella Typhi, which causes Typhoid fever, or Vibrio Cholerae, which causes Cholera. I put the flasks and the filtration units in the bleach and wait for 30 minutes so the chlorine has a enough time to actively degrade all the nucleic acids.
After 30 minutes, I shake off the filtration units and dump the bleach out of the flasks, and then dechlorinate! I dechlorinate – or neutralize the bleach – using sodium thiosulfate. This step makes sure that there is no residual chlorine left on the equipment. If there’s residual chlorine, I’ll accidentally disinfect the water sample,degrading the microbes we’re trying to find! So after I take the equipment out of the bleach, I rinse it down with sodium thiosulfate dissolved in deionized water, i.e. purified water.
Step 1 (b): Coagulation
Many of the collected water samples are very turbid – meaning they’re very cloudy or appear dirty. To aid in virus and bacteria capturing, we add Magnesium Chloride to the sample at a 25mM concentration. Magnesium Chloride is a salt solution that helps make particles in the water clump together. If the pathogens clump together, and clump to other soil particles, they’ll get trapped in the filter more easily and we can detect them! To coagulate (clump together) we add the Magnesium Chloride and mix vigorously for 30 seconds followed by 30 min of rest with 15 min interval gentle mixes.
Step 2: Water Filtration/Concentration
Now we’re ready to vacuum filter the water sample! The filtration unit is fit snuggly into a polypropylene vacuum flask that is attached to a Sparkfun 12V vacuum pump. The pump is wired to a car battery adapter that plugs into a 12V port on a GoalZero Sherpa100 Power Pack (i.e. a battery). Click on those link to get a better close up of the products used! This whole set up requires no power, as the power pack can be recharged using a set of solar panels (not shown here). Major thanks to Jake Metz for being the brains behind the vacuum pump/power supply design! 🙂 The best part – I can use the power pack to charge my computer, light a lamp, or charge my iPhone!
The vacuum pump has two small ports – one ‘inlet’ and one ‘outlet’. The inlet is the suction, and is connected to the clear tube in the above photo. The outlet releases the suctioned air from the inlet. The other end of the tube is connected to the flask, so the vacuum pump pulls all the air out of the flask creating a vacuum and forcing the water from the filtration unit through the filter membrane. I usually add a cup-full of sample water to each filtration unit before adding a membrane and beginning filtration. I do this to allow the filtration unit and flask to become “wetted” with the sample water and remove any left over sodium thiosulfate or bleach. After this step, I discard the filtered water and add a membrane to the filtration unit! Sometimes it’s hard to visualize where the membrane goes, so here’s a photo of a recently filtered membrane.
To filter the water, I simply pour the water into the filtration cup, and as the vacuum pulls the water down, I add more. I do this until I’ve filtered ~2L or until the filter clogs and will not filter anymore water. Clogging generally happens with nondrinking water samples because they are so turbid.
You’ll notice in the above photo that I have 2 flasks. Why would I need two if I just use one filtration unit per flask? Well, the filtration is sequential. The water that is filtered through the first membrane is filtered through a second membrane, and then a third! Sound confusing? No worries – check out this nifty diagram.
Hopefully this makes more sense now. 🙂 The first membrane is used to trap particulates like dirt and large objects. The second membrane is used to trap bacteria. And the last membrane is used to trap viruses. They each vary in size and material based on their capturing capacities.
Step 3: Stabilization & Storage
Once all the water has been passed through, a genome stabilizer is added to each membrane. This ensures that whatever genomic material (DNA, RNA) on the filter, stays there and stays stable until we’re ready to analyze it. RNAlater is a high salt solution that can be used to stabilize RNA in cells, tissues and on filter membranes for a long period of time. Stored at -20C, the nucleic acids can last a very long time – which is important for me since we’ll be doing the analysis at the University of Illinois in ~8 months.
After the RNAlater is added, the filter is placed in sterile 200ml Whirlpak bag, sealed with tape and stored at -20C. Later, at the University of Illinois, we will use a genome extraction kit to remove the nucleic acids from the membrane and analyze for sequences matching to diarrhea causing pathogens like rotavirus, cholera, adenovirus, salmonella, and norovirus.
That is the water sampling process in a nutshell – hope you enjoyed it! 🙂 Please feel free to ask questions or comment on any portion of the protocol. Happy to explain further, and promote discussion!