Category Archives: Teaching and Research

Environmental virology, Microbial Ecology, Teaching and Research, Winogradsky

Check out our newest paper in PLoS ONE!

Temporal and Spatial Distribution of the Microbial Community of Winogradsky Columns

Winogradsky columns are model microbial ecosystems prepared by adding pond sediment to a clear cylinder with additional supplements and incubated with light. Environmental gradients develop within the column creating diverse niches that allow enrichment of specific bacteria. The enrichment culture can be used to study soil and sediment microbial community structure and function. In this study we used a 16S rRNA gene survey to characterize the microbial community dynamics during Winogradsky column development to determine the rate and extent of change from the source sediment community. Over a period of 60 days, the microbial community changed from the founding pond sediment population: Cyanobacteria, Chloroflexi, Nitrospirae, and Planctomycetes increased in relative abundance over time, while most Proteobacteria decreased in relative abundance. A unique, light-dependent surface biofilm community formed by 60 days that was less diverse and dominated by a few highly abundant bacteria. 67–72% of the surface community was comprised of highly enriched taxa that were rare in the source pond sediment, including the Cyanobacteria Anabaena, a member of the Gemmatimonadetes phylum, and a member of the Chloroflexi class Anaerolinea. This indicates that rare taxa can become abundant under appropriate environmental conditions and supports the hypothesis that rare taxa serve as a microbial seed bank. We also present preliminary findings that suggest that bacteriophages may be active in the Winogradsky community. The dynamics of certain taxa, most notably the Cyanobacteria, showed a bloom-and-decline pattern, consistent with bacteriophage predation as predicted in the kill-the-winner hypothesis. Time-lapse photography also supported the possibility of bacteriophage activity, revealing a pattern of colony clearance similar to formation of viral plaques. The Winogradsky column, a technique developed early in the history of microbial ecology to enrich soil microbes, may therefore be a useful model system to investigate both microbial and viral ecology.

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Microbial Ecology, Teaching and Research, Winogradsky

Published! Paper on the microbial community in Winogradsky columns

Check out the paper in PLoS ONE! 

Ethan Rundell ’13 worked in my lab for two years, his work culminating in first authorship on this paper.

Abstract

A Winogradsky column is a clear glass or plastic column filled with enriched sediment. Over time, microbial communities in the sediment grow in a stratified ecosystem with an oxic top IMG_4059layer and anoxic sub-surface layers. Winogradsky columns have been used extensively to demonstrate microbial nutrient cycling and metabolic diversity in undergraduate microbiology labs. In this study, we used high-throughput 16s rRNA gene sequencing to investigate the microbial diversity of Winogradsky columns. Specifically, we tested the impact of sediment source, supplemental cellulose source, and depth within the column, on microbial community structure. We found that the Winogradsky columns were highly diverse communities but are dominated by three phyla: Proteobacteria, Bacteroidetes, and Firmicutes. The community is structured by a founding population dependent on the source of sediment used to prepare the columns and is differentiated by depth within the column. Numerous biomarkers were identified distinguishing sample depth, including Cyanobacteria, Alphaproteobacteria, and Betaproteobacteria as biomarkers of the soil-water interface, and Clostridia as a biomarker of the deepest depth. Supplemental cellulose source impacted community structure but less strongly than depth and sediment source. In columns dominated by Firmicutes, the family Peptococcaceae was the most abundant sulfate reducer, while in columns abundant in Proteobacteria, several Deltaproteobacteria families, including Desulfobacteraceae, were found, showing that different taxonomic groups carry out sulfur cycling in different columns. This study brings this historical method for enrichment culture of chemolithotrophs and other soil bacteria into the modern era of microbiology and demonstrates the potential of the Winogradsky column as a model system for investigating the effect of environmental variables on soil microbial communities.

Rundell EA, Banta LM, Ward DV, Watts CD, Birren B, Esteban, DJ. (2014) 16S rRNA Gene Survey of Microbial Communities in Winogradsky Columns. PLoS ONE 9(8): e104134. doi:10.1371/journal.pone.0104134

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Microbial Ecology, Teaching and Research, Winogradsky

Watching Bacteria Grow: Winogradsky Panel Day 49

Various green bacteria are growing now all over the panel.  These are likely to be cyanobacteria and some green sulfur bacteria.

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You may also notice the addition of clamps along the bottom.  On Day 33, I arrived to find a puddle below the panel and all of the water drained out.  Luckily, the crack was too small to let any soil out.  The clamps are holding well so no more leaked out after I refilled it.  Perhaps I can claim that this is an experiment in shocking the ecosystem with a drying and aeration event?  I didn’t notice any significant changes in the following days and weeks, except the continued growth of green and yellow bacteria, so I suspect the shock wasn’t too detrimental.

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In the last several days, a filamentous green colony has developed.  Note the large gas bubbles around it.  This is likely a cyanobacteria like Oscillatoria, so the gas would be oxygen generated through its plant-like photosynthesis.  IMG_6120IMG_6141The two images above show the top right corner of the panel at day 35 and then day 49.  Notice how the black mud is slowly being covered by the growth of a very diverse mixture of microbes.

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Microbial Ecology, Teaching and Research, Winogradsky

Watching Bacteria Grow: Winogradsky Panel Day 30

The pace of the changes in the Winogradsky panel has slowed, but now some green microbes are really noticeable.  There are shades of bright blue-green, yellow-greens and yellow.IMG_6117IMG_6118IMG_6120IMG_6121

The range of greens is due to different combinations of pigments in photosynthetic bacteria.  I had a recent conversation with someone who didn’t realize that organisms other than plants can photosynthesize.  Many bacteria can, and like plants, they use a variety of different pigments to capture energy from light and use it to drive metabolic reactions in the cell.  The green is from chlorophylls, the same as plants use.  These are found in the cyanobacteria, which have the deeper blue-green color.  But there are many others too:  the “purple bacteria” have bacteriocholorophills and carotinoids, giving them a purple, yellow or brown color, and the bacteriochlorophylls of the “green sulfur bacteria” produce green or brown colors.  The role of these pigments is to absorb different wavelengths of light to harvest the energy.

Phototrophy in cyanobacteria is most similar to that of plants.  In fact, the chloroplast, the organelle found in plant cells that carries out photosynthesis, is very similar to cyanobacteria.  Most likely, early in the evolution of life, a larger cell engulfed an ancestral cyanobacterium and a symbiotic relationship evolved, eventually becoming plants.  Like plants, cyanobacteria use water in the process of capturing light energy, producing oxygen.  Some of the gas pockets seen in the Winogradsky might be oxygen.  Other bacteria use a slightly different process, and use hydrogen sulfide instead of water, and produce sulfur.  The sulfur forms globules, which are stored inside or outside the cell.

Not all color is from pigmentation of cells however.  The red-oranges in the Winogradsky panel are likely due to a combination of pigmented cells but also due to the oxidation of iron.  Some bacteria, like Chromatium, are able to use iron in phototrophy, instead of hydrogen sulfide or water.  They oxidize the iron in the soil, and oxidized iron has a red-orange color (the rust on a car is oxidized iron).

 

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Microbial Ecology, Teaching and Research, Winogradsky

Watching Bacteria Grow: Winogradsky Panel Day 14

There are many interesting changes over the last few days. The black areas in the sulfur enriched mud are coalescing, and there are some light pinkish-red colonies developing at the surface. The olive-green band next to the orange is either disappearing or getting covered up as the orange stripe expands.

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The unenriched mud is developing more visible colonies too, as various browns and some very pale greens start to appear.  A few emerald greens are now visible, most of them very faint but there is one larger bright one with a big gas bubble.IMG_6014
It turns out that the bloodworms (midge larvae) are not dead.  In the past, when I have prepared columns (instead of a panel), they all die within the first few days.  Here, they seem to be doing quite well.  Notice the worm tracks in the top left (photos taken Day 11).  It will be interesting to see how the bacterial colonies develop in this area in the presence of this disturbance.

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Teaching and Research, Uncategorized

It’s a slime mold, not a cupcake

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Slime molds look kind of like vomit but they are pretty awesome.  In fact, one of them is affectionately nicknamed “dog vomit slime mold.”  While vacationing in British Columbia, my 2 year old son, exploring in the trees and shrubs, exclaimed “cupcake!” Knowing it was highly unlikely that there would be a cupcake there, and not wanting him to eat whatever he found, I rushed over to see a pine cone covered in a thick layer of whitish yellowish stuff.  It did, in fact look a little like a piece of rather unappealing cake with thinly smeared white frosting and  sprinkles of pine needles and soil from the forest floor.

Looking around, I found several other similar things growing, and some that looked quite different.  One looked like coral, another had bubbles of clear, sticky liquid.  As  I observed them over several days, they changed, all eventually looking alike, and after more days, dried up completely to a pile of dusty powder that easily fell apart when disturbed.  These were all the same slime mold, at differnt stages of its lifecycle. I’ve sent these photos to various slime mold experts, and it has been tentatively identified as Brefeldia maxima, the “tapioca slime mold.”

Slime molds are indeed slimy, in the picture at the left you can see the slime trail it leaves behind as it moves, but they are not mold.  They are actually amoeba.  These amoeba can exist in the single celled form but under the right conditions, the single cells will amass together. In some slime mold species, called plasmodial slime molds, the cells will actually fuse together to form a single, giant multinucleated cell. Others, called cellular slime molds, form a multicellular mass. Eventually, the slime mold will differntiate and form fruiting bodies, complex strructures in which spores will develop. Spores can then be released to disseminate the slime mold to new locations. What amazes me is the complexity of this process and the blurring of the lines between unicellular and multicellular organisms. You have a single celled organism, in which many individuals can come together into a multicellular (or single giant cell) form, now behaving as one organism that can differntiate to form complex structures like fruiting bodies and spores. Clearly, we can’t be thinking of microorganisms like these as “simple.”

Some recent studies further demonstrate the complexity of slime molds. The slime mold Physarum was used to map an optimal network between points, in this case the Tokyo metropolitan area. Food sources were laid out corresponding to the communities surrounding Tokyo, and Physarum was placed at Tokyo. The slime mold extended plasmodia, or branches, out to the food sources. At first the branches fan out randomly, making many connections, but eventually most plasmodia disappear, leaving only the most optimal connections between the food sources. The final map of the plasmodia turns out to be quite similar to the Tokyo area metro system. Physarum can also do this with the US interstate highway system, and the Canadian highway network (um, isnt there just one highway in Canada?) So an amoeba can simulate a networks developed by human engineers!

In my lab, a student is exploring using Physarum for a similar purpose. Vassar is looking ahead 50 years or so to plan development of the campus. Can we use the slime mold to provide suggestions to the landscape architects on where to place the pathways between buildings? Can the slime mold help me find the best way across campus? We will keep you posted on what we find!

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Teaching and Research

How do you read popular science articles critically?

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It is increasingly important for us to become critical readers. It is easy to find information on any scientific topic, but it is often easier to find poor quality information than high quality, accurate information. Is it really a promising cure for cancer? Is the vaccine actually going to work or even come to market? Is this really the next outbreak that threatens us all? How do we make sense of this information? It is not always easy to distinguish the good articles from the bad.

Last semester in my Microbial Wars class, students selected news articles and presented the article along with a discussion of its merits and problems. Over the course of the semester, the students identified several features that helped in critically evaluating the articles. Below are some of the major points they came up with.  You can test your critical reading skills on this article on the HPV vaccine or this one on a transmissible H5N1 influenza virus.

1. Source: does the publication tend to publish reliable and accurate articles?

2. Author: does the author of the article have a science background? Have they written other science articles? Since a journalist has to interpret and explain the research, their ability to understand the research and its context are important, otherwise they may simply be repeating back information from press releases or other sources without critical evaluation of the information.

3. Is the original source easy to find? Are there links, or sufficient details to know who did the original research so that if you want to find the original source, you could do it fairly easily.  Do the sources or studies that the article cites or links to actually support their statements, and are they reliable?

4. Is the research being described published in a peer-reviewed scholarly source? If its not peer reviewed, beware! Is it based on conference proceedings? Conference presentations and abstracts are very minimally reviewed, and should not be considered equivalent to a published study. Often the data from a conference proceeding hasn’t been seen by most scientists or the journalist, so use great caution when reading about this kind of unpublished information.

5. We talk a lot about articles being sensationalized, but what does that mean? It means presenting information in a way that provokes interest or excitement, at the expense of accuracy. Watch out: this happens a lot, even in the most reputable news sources and with top writers. Is the article making unsubstantiated claims? Consider carefully what the research has shown or what specifically was tested. Are the statements made in the news article accurately reflecting what the research shows, or is it taking it several steps beyond the results of the research? Remember that scientific research tends to move in slow, baby steps, not leaps and bounds.

6. Balance: What makes a balanced article? Should different perspectives be included? Should contrary opinions be presented? If so, how much weight should be given to “dissenting” views? A common problem is presenting two sides of a debate as if both sides are equally supported. This can lead to a false sense of how much debate there really is among those in the field.

7. Motivations: what are the motivations of the people quoted or referenced in the article? Are those individuals likely to have an unbiased perspective on the research? You will sometimes see comments or quotes from researchers not involved in the particular study being discussed; these can often provide a good perspective.

8. Headline: headlines are written to draw readers in, and so are likely to be sensationalized or inaccurate. (They are also often written by somebody other than the author of the article). Does the headline accurately portray what is in the article? If you only remember one key message from the article, it probably shouldn’t be the headline.

Considering these points when reading an article may help in reading it critically. No single point will make or break an article, but this is at least a good place to start.

Thanks to the students of Bio/STS 172 for interesting discussions and developing this list!

Any other suggestions?

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Epidemics, Teaching and Research, Uncategorized

I’ll take my milk pasteurized, thanks

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A recent article in Vassar’s newspaper, The Miscellany News, discussed the Vassar Raw Milk Co-op, which brings unpasteurized milk to Vassar College (Co-op offers raw milk delivery service, Nov 10,2011). The article raises several important questions about raw milk, pasteurization, and sustainable agriculture, but some of the information presented is incorrect. Importantly, Vassar’s Raw Milk Co-op website, to which readers are directed, has a great deal of misleading, incorrect or unsubstantiated information.

In the production and packaging of milk, it can become contaminated at virtually any stage of the process. That contamination, when it is by organisms like E. coli or Listeria, is what causes milkborne illness. Pasteurization, a process of heating milk to reduce the levels of microorganisms present, will kill those contaminating bacteria if they are present. The risk in drinking raw milk is due to the fact that if it does become contaminated, you will be consuming those pathogens. In a farm environment, it is safe to assume that contamination will, at some point, happen. Pasteurization is one check that we have to protect us from that.

The majority of cases of milkborne illness result in diarrhea and/or vomiting. Occasionally the symptoms can be more severe, such as in last week’s outbreak in California, in which five children have become sick. Three have been sent to the hospital with hemolytic uremic syndrome, which can lead to kidney failure. These cases have led to the recall of the organic raw milk, contaminated with E. coil 0157:H7, which has been linked to the outbreak. There are outbreaks associated with pasteurized milk as well, usually due to post-pasteurization contamination, such as at the packaging stage. However, it is estimated that only 1% of people consume raw milk, but from 2000-2007, 75% of outbreaks were associated with contaminated raw milk.

In NY consumers can choose to buy raw milk from a farm if they determine that they are comfortable with the level of risk. But it is also important that they know the facts behind the reported benefits to balance their decision. Many proponents of raw milk claim that industrially raised, antibiotic laden cattle given GMO corn feed produce milk that needs to be pasteurized because it is inherently of poor quality and unsafe, but that organically raised pasture fed cows produce milk that is safe. That is simply untrue. Pasteurization was developed in the mid 1800s to eliminate pathogenic and spoilage microorganisms, not to fix the problems of industrialized agriculture. There is no credible scientific evidence to support the suggestion that organic pasture-fed cows generate safer milk than cows from industrial farms. Contamination by pathogenic organisms comes from fecal matter, the environment, the handlers, packaging, storage, and undetected infections in the animals, and is unrelated to diet and housing conditions.

Additional claims such as that pasteurized milk causes allergies, asthma or other conditions are not supported by the scientific literature. The claim that raw milk is better for individuals who are lactose intolerant is not supported by scientific data either, and represents a clear misinterpretation or misunderstanding of available information. The level of nutrients in raw compared to pasteurized milk is not significantly different, invalidating yet another central claim in support of raw milk.

There is an abundance of misinformation on the topic of raw milk. Unfortunately, groups like Vassar’s Raw Milk Co-op perpetuate this misinformation. At Vassar, we often say “go to the source.” This is an opportunity to practice that principle. Rather than seeking confirmation of one’s beliefs in the websites of others, we must check the original research. What is actually supported by credible scientific investigations? Is the information you are reading being correctly interpreted? Are you getting the whole story or just fragments?

There are many good things about the locavore and Slow Food movements. Supporting small local farms and sustainable agriculture, humane treatment of animals and having the sense of community achieved from getting to know the farmer who raises your food, are important to me and many people. But you don’t need to consume raw milk to do that. These issues are distinct from the question of pasteurization. If you want, you can even get the raw milk and pasteurize it yourself. Just heat your milk on the stove to 63C for 30min before drinking it.

Thanks to the students in STS/Biol 172 for discussion and research on this topic

A few links:
FDA Milk Safety

CDC Milk Safety

Review on Milk Safety and Pasteurization

A recent review of the scientific literature. Note that they included many studies of poor design, which should have been ignored.

Slightly updated and edited on December 1, 2011

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Teaching and Research

Microbiology can be dirty

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Students are starting to repopulate the campus and the relaxed pace of the summer is being quickly replaced by frenzied preparation for the start of the semester. This semester, I will be teaching the intro to microbiology course that I have taught every year. In the lab, we do some bioinformatics analysis of metagenomic data from a Winogradsky column. A Winogradsky column is a clear cylinder full of pond mud that is used as an enrichment culture to grow bacteria that cant be grown under normal laboratory conditions. To make a Winogradsky column, you collect mud from a pond or riverbank. (For those of us that are used to the cleanliness of working in a sterile hood, that means you have to take off your lab coat, get down on your hands and knees, and scoop up handfuls of goopy stinky slimy stuff from the the edge of a pond. I wear latex gloves.) You then add it to a plexiglass cylinder along with a source of cellulose (I use leaf litter) and additional sulfate to promote enrichment for microorganisms involved in the sulfur cycle. Over a period of months, layers of microorganisms requiring a range of environmental conditions develop in distinct niches with distinct populations participating in diverse metabolic activities. As various metabolites in the column are used, byproducts are produced, and the environment in the column changes. As a result of changing concentrations of oxygen, hydrogen sulfide, and variations in metabolites, different microbes will thrive and create their own niche.

Although you can see some changes occurring in the first few days, it takes several weeks or months for it to develop so I always set it up before the semester starts. This year, I had the help of my daughter (age 5) who was eager to get her hands into the gooey muck. We took the mud from the edge of Vassar Lake, a pond on campus. In the pictures you can see the changes that take place over time. The column starts out as grey silt, while the column on the left is 1 year old. The patches of colours are the different communities of bacteria.

(OK, so there is no virology in this post but it sure would be interesting to analyze the viral population of the column in addition to the bacterial population. What is the role of bacteriophages in the community dynamics and nutrient cycling in the Winogradsky column?)

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Evolution, Teaching and Research

Variola virus evolution

Why do some people get severely ill with an infection while others catch the same virus but don’t get sick? There are many factors that can influence the progression and outcome of disease, but they can be lumped into four basic categories: host, agent, transmission and environment. For example, infection with Variola virus results in smallpox, but case fatality rates in different outbreaks range from very low to as high as 30%. Its likely that many factors contribute to this variability, but it’s likely that differences in viral strains is one of them.

Summers are quiet here at Vassar. There are no summer classes but we do have a program to support undergraduate research (“URSI”) so that students can gain some research experience and professors can get cheap labor. This summer I had a student working with me who is a Biochemistry major and Computer Science minor (called a “correlate” here). She can write code and I cant, so I had her working on a bioinformatics project. We were interested in investigating the difference between poxviruses that cause high mortality rates in humans (like some strains of Monkeypox virus and most strains of Variola virus) and those that dont. I recently published a paper along with another undergraduate student showing that certain genes in poxviruses are under Darwinian selective pressure. We wanted to test the hypothesis that the selective pressure differs between virulent and avirulent strains. She used several approaches involving analysis of synonymous and non-synonymous mutation rates to see if amino acid altering mutations were fixed at different rates in virulent and avirulent viruses.

As she crunched away at code writing and data analysis and discovered one of the joys(?) of science: failing to support your hypothesis. We could not find evidence that selective pressure differed between virulent and avirulent strains. Although no Vassar student wants to fail, failing to support a hypothesis is not actually failure. Rather, its an integral part of the scientific process, something that comes from the successful execution of an experiment that tests your hypothesis. The scientific method is actually quite humbling: you set up experiments that will tell you if you are wrong, and as a scientist, you have to get used to proving yourself wrong.

So now we must ask a new question: since some poxvirus genes show evidence of selective pressure, and that selection is not related to virulence, what is the cause of that pressure?

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