It is time again for a Winogradsky column update! It has been three weeks since we last shared with you our Winograsky column progress. If in that time you’ve forgotten what a Winogradsky column is and our goals for them I urge you to visit: Adventures with Winogradskies and Further Adventures with Winogradskies to refresh your memory.

After 7 weeks:

We are finally starting to see some pretty pink layers! Many of the darker colored bacteria from week 4 are gone from these columns… could it be that our columns are undergoing microbial succession?? Could Winograsky columns be a good model system for studying microbial succession?

Strangely (or not so strangely?), it is our large containers that show the most variation between replicates from the same sample location. We have everything from green-as-grass and pretty-in-pink to black-as-coal microbes. The container above on the far right is the one that we’ve been simulating soil conditions in by covering the bottom half of the container – could the lack of pink and purple microbes be due to this?

Above you can see the variation in vials taken from different sampling locations. The microbes in these vials seem to be growing at a slower pace than those in the large container – we also see this with our small micro-centrifuge tubes (not pictured) which haven’t changed significantly form week 4.

Experimental Tubes ( (where we added either Potassium Nitrate or Ammonium Acetate) – After 6 weeks:

On the left are the tubes we added Potassium Nitrate too and on the right are the tubes we added Ammonium Acetate too. The Potassium Nitrate tubes have green microbes growing in firework like patterns up the sides of the tubes! The Ammonium Acetate tubes have pinkish-brown microbes clouding them up! It is amazing how different the Potassium Nitrate and Ammonium Acetate tubes are given that they came from the same original sample!

The two above tubes were kept in the dark via aluminum foil. The left tube has had Potassium Nitrate added to it and the right tube has had Ammonium Acetate added. It is hard to see but the Potassium Nitrate tube has a lot of gas bubbles in the diatomaceous earth in the bottom of the column.

Left to Right: Ammonium Acetate Added (Kept in Dark), Ammonium Acetate Added, Nothing Added – Potassium Nitrate Added (Kept in Dark), Potassium Nitrate Added, Nothing Added

The differences between the different treatments (light with chemical vs. dark with chemical vs. no chemical ) is most significant when you observe the tubes right next to each other. It will be interesting to investigate the community composition differences between all these tubes! I wonder what types of communities we’d see with different pH’s, salt concentrations or other added chemicals… Until next time, stay tuned!

Our Winograsky columns have come a long way from our previous post two weeks ago. To review what a Winogradsky column is and our goals for them please see: Adventures with Winogradskies.

After 4 weeks:

Green layers have started to develop on the top of some of the tubes and to a less visible extent on some of the larger containers.

In some tubes we are starting to see defined layers. Layers seem to vary between tubes containing seawater from different sample locations as well as between tubes with water from the same location.

Unfortunately, our tiny microcentrifuge tubes haven’t changed much in the past two weeks.

4 Weeks vs. 1.5 Weeks:

Definitive layers are less visible in our large containers (perhaps due to their non-cylindrical shape?), but there is significant visible growth compared to the containers at 1.5 weeks. Additionally, upon closer inspection both red and green splotches can be found in the container.

Simulating Soil Conditions – 4 weeks:

At first glance, the container that we covered to simulate soil conditions looks similar to the uncovered columns. However, there is a small white strip at the top of sediment that we don’t see in the uncovered columns. There also appears to be a biofilm growing along the sides of the container.

As well as the beginning of a layer floating on the top of the seawater in the container.

Experimental tubes at 3 weeks (where we added either Potassium Nitrate or Ammonium Acetate):

From left to right: Ammonium Acetate Added, Potassium Nitrate Added, Nothing Added

We decided to keep one vial where Ammonium Acetate was added and one vial where Potassium Nitrate was added in complete darkness.

From left to right: Dark Potassium Nitrate Added, Light Potassium Nitrate Added, Light Nothing Added

Notice the difference in the amount of bubbles between the vials under different light and chemical conditions. All three of these vials have seawater from the same location.

Often when microbiologists begin to explore an unknown (or relatively unknown) environment, they begin by using classical microbiological techniques to try to characterize the communities of microbes living in that environment. These classical techniques are often referred to as “culture-based” because they are oriented towards the goal of trying to grow (or culture) microbes in the lab. Although culture-based techniques can be limiting (it’s nearly impossible to culture every single organism in any given environment), they are very useful for laying the foundation for the non-culture-based techniques the Seagrass Microbiome project will be using.

As a result, we in the Eisen lab have been playing around with some classical microbiology techniques alongside our non-culture-based explorations. One of the coolest techniques we’re using is Winogradsky columns. Winogradsky columns are essentially microbial terrariums. The basic recipe is as follows: take a clean tube, add a few essential chemicals, spike in some wild microbes, close the lid and let natural nutrient cycles take over. If you’ve done everything right, in a couple of weeks, you should start to see layers of microbes each living in a different mini niche within your mini ecosystem.

Two weeks ago we did just that. We’ve been following the progress of the tubes on the side as we pursue other non-culture-based projects. We’ve also been exploring different ways of making the columns to figure out the best way to get clean layers while still preserving the integrity of the column. Below are some pictures of our progress to date:

Two Days in:

One week in:

1.5 weeks in:

9 Days in:

2 weeks into the experiment: 

_________________