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Mud, mud, glorious mud!

May 14, 2009

I’ve been looking forward to being able to write a “sediment blog” – because it means things are progressing with the sediment experiments!

On Monday, the divers collected 10 large sediment cores for the ocean acidification experiments. These cores have come from the sediment around the pier, which is right in front of the marine lab, and we now have 20 cores which are spread over the 5 pH treatments being used for the other studies. Fred and Pieter will be performing incubations throughout the experiment, to look at the oxygen consumption and nutrient fluxes. This gives us information about the biological processes that are going on within the sediment (such as respiration and growth). Measurements of temperature, total alkalinity, pH and dissolved inorganic carbon will also be taken from the overlying water to monitor the environmental conditions in the cores.

The divers returning from a sampling trip. Photo: Bonnie Laverock

The divers returning from a sampling trip. Photo: Bonnie Laverock

At the end of the experiment, samples will be taken for analysis of nitrification and denitrification. These processes are carried out by bacteria, and are important processes in marine sediment because they allow nutrients to be cycled back into the water column in a form that other organisms can feed on. We’re interested in how ocean acidification might affect this nitrogen cycle in the sediment. Therefore, Karen and I will also be looking at the bacteria that are present in the sediment. We’ll be looking at how the overall diversity of bacteria is affected by ocean acidification, and also whether the genes responsible for nitrogen cycling are affected.

Pieter and I have already taken some sediment samples so that we can have a look at diversity in normal sediment – this will give us a good baseline so that we can tell whether the communities have changed at the end of the experiment. We’ve got two lots of sediment so far: some really smelly, muddy sediment from the pier in front of the marine lab, and some sandy sediment from a site further downstream, called Brandal. As you can see, they are quite different, so it will be interesting to compare the bacteria in there!

Muddy sediment from the pier (left) and sandy sediment from Brandal (right). The interface between the oxic layer (light-coloured) and the anoxic layer (dark-coloured) is an important site for nitrification-denitrification reactions.

Muddy sediment from the pier (left) and sandy sediment from Brandal (right). The interface between the oxic layer (light-coloured) and the anoxic layer (dark-coloured) is an important site for nitrification-denitrification reactions. Photos: Bonnie Laverock

So how do we look at the bacteria in the sediment??

Most bacteria cannot be grown in the laboratory, so we have to use molecular methods to look at their internal molecules, such as proteins, DNA, or RNA. To look at diversity (what organisms are there and how many), we look at the differences between the DNA, just as we might look at a human’s DNA to investigate that person’s ancestry. To look at genes which control a specific function, such as nitrogen cycling, we can look at RNA – this is similar to DNA, but it tells us which genes are being made into proteins – so we’re looking at the genes that are actually being used in the environment. We can use “fingerprinting” techniques to look at the structure of a bacterial community, and how it changes.

Extracting DNA – a classroom experiment

You can extract DNA in your classroom from a piece of fruit or vegetable – peas, onions and bananas usually work pretty well. Follow the instructions below … the way in which scientists extract bacterial DNA is slightly different, but it uses the same principles (cell wall disruption, salty environment, protein breakdown and alcohol precipitation) …

Banana DNA (left) and bacterial DNA from a sediment sample (right). Both have been beaten to release DNA from cells, prepared in a salty environment, had their proteins removed, and have been precipitated in alcohol. Photos: Bonnie Laverock.

Banana DNA (left) and bacterial DNA from a sediment sample (right). Both have been beaten to release DNA from cells, prepared in a salty environment, had their proteins removed, and have been precipitated in alcohol. Photos: Bonnie Laverock.

Step 1: Dissolve 3 grams of salt in about 100 millilitres of warm water (distilled water is best if you have access to a lab).
Why? The salt provides a “good environment” for the DNA so that it is more likely that the DNA will come out of the cells and into solution (DNA is negatively charged, and salt is positively charged)

Step 2: Add a mashed up banana to a food blender, pour the salty water over the top, and blend for 5 to 10 seconds. Pour through a sieve into a glass.
Why? The blender helps to break open the cells which contain the DNA.
 
Step 3: Add 2 to 3 teaspoons of normal washing-up liquid and stir gently (we don’t want bubbles!).
Why? Washing-up liquid works by breaking up the fat and grease stuck to our dishes. We have fatty molecules in our body – they are called lipids, and they hold cells together. The washing-up liquid helps to break open cells by attacking these lipids – this releases the DNA from the cells.
Optional extra step: If you want, you can also add pineapple juice to your mixture!
Why? The pineapple juice contains a compound called bromelain, which is an enzyme that breaks down proteins. Adding this to the mix prevents your DNA from being contaminated by cell proteins.
 
Step 4: Use “rubbing alcohol” (or ethanol if you’re in a lab) and pour it very slowly down the side of the glass so that you can see it floating on top of the soapy mixture. Let it sit for a few minutes and watch what happens. The stringy whitish stuff you can see rising upwards is the banana’s DNA!
Why? DNA dissolves in water, so you cannot see it when it is in the soapy mixture. When you add the alcohol, the DNA rises upwards because it is less dense than water, soap, and the other cell goo. The reason you can see it now is that DNA does not dissolve in alcohol, so it becomes a gloopy solid as it leaves the watery layer.

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