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Microbes
For most of Earth’s history, life consisted solely of microscopic organisms and in many respects these microbes still dominate the Earth today 1 . Microbes include bacteria, archaea, fungi, protists, green algae, and plankton. Viruses are also sometimes considered to be microbes. The ~5 X 10 30 microorganisms that inhabit our planet play an important role in the cycling of carbon, oxygen, nitrogen, phosphorus, sulfur and other elements which are essential to life 2 . Two important parts of the cycling process are called assimilation and decomposition . During assimilation, microbes convert inorganic elements into forms that are useable by other microbes, plants, and animals. During decomposition, microbes break down organic matter and recycle it so it can once again be made available to other organisms. Photosynthetic microorganisms were responsible for initially pumping oxygen into the atmosphere and today these microbes carry out almost 50% of all the photosynthesis on our planet 3 . Microbes can also change the weather and affect climate by sequestering CO
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and releasing small particles that aid cloud formation.
Microbes are easily the most abundant and diverse life form on the planet 4 . Although microbes cannot be seen with the unaided eye, there are so many of them that their combined weight is estimated to be more than all other organisms combined. Microbes can be found almost everywhere on the planet including soil, air, and water. They can be found in the upper reaches of our atmosphere and 6 km below the Earth’s surface. They live everywhere macroscopic organisms are present and they can survive extreme temperatures, pressures, and pH levels that other organisms cannot tolerate. They can be found growing in extremely cold places such as Antarctica and extremely hot places such as the volcanic pools at
Yellowstone National Park and deep sea thermal vents where temperatures can reach 350˚C. Microbes are also found on and within larger organisms including humans. The number of microbes that live on and inside of the human body (~10 14 ) exceeds the total number of human cells (~10 13 ) by a factor of ten 5,6 .
Microbes that live on or in humans can aid digestion, make vitamin K, help develop the immune system, fight off disease-causing microbes and detoxify harmful chemicals 2 . Studying microbes in the human gut will likely lead to new ways to diagnose, treat, and prevent disease 7 .
Human industries including agriculture, medicine, biotechnology and the food industry have greatly benefited from the use of microbes. In agriculture, certain crops are seeded with specific strains of nitrogen-fixing bacteria to increase yields. Additionally, a microbe that causes plant disease,
Agrobacterium tumefaciens, has been used to create genetically modified crops that are more drought and pest resistant and have higher yields. In medicine, hundreds of drugs that are available today were originally derived from microbial sources and many of these drugs (especially antibiotics) are mass-
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produced for human use by microbes. Medical microbes are used to produce insulin, interferon, human growth hormone, vaccines and many other useful compounds. Biotechnology has used microbes to generate important chemicals, enzymes, and alternative fuels such as hydrogen, methane, and butanol 4 .
Microbes are also used in bioremediation projects designed to clean up oil spills, gasoline leaks, sewage, and industrial discharges 4 . In the food industry, microbes are instrumental in making bread, cheese, yogurt, wine, and beer. Without microbes you could not have something as simple as a roast beef sandwich. This is because microbes produce the cheese, make the vinegar for the pickles, and help the bread to rise. In addition, microbes contribute to the beef by enabling cows to digest grass and other plant matter they depend on to survive.
Not all microbes are beneficial however. A minority of microbes are pathogens that cause a wide range of ailments, which effect humans, animals, and crops. This has led some to incorrectly fear all microbes.
Such fears have led to the development and use of antimicrobial soaps and cleaners and the excessive use of antibiotics. Both of these practices select for resistant microbes and facilitate the evolution and spread of resistance genes. Excessive use of antimicrobials and antibiotics may counter-intuitively speed up the development of resistant pathogens and lead to the development of ‘superbugs’ for which there is no treatment. Many ‘superbugs’ already exist including the SMS-3-5 strain of E. coli that is tolerant of or resistant to high concentrations of 32 different antibiotics 8 !
For more information on microbes please see:
Intimate Strangers: Unseen Life on Earth Video http://www.microbeworld.org/index.php?Itemid=194&id=259&option=com_content&view=article
Understanding Microbial Life http://www.lifeworksfoundation.com/news/microbe-ecology-film.php
Soil Microbes www.agron.iastate.edu/~loynachan/mov/
Size and abundance of microorganisms www.pmbio.icbm.de/mikrobiologischer-garten/eng/index.php3
Microbes in the News www.microbeworld.org
MANGA/ANIME Microbes
Search microbe theater on microbeworld.org or www.youtube.com/show/moyashimon?s=1 http://en.wikipedia.org/wiki/Moyashimon
Online Textbook of Bacteriology http://textbookofbacteriology.net/index.html
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Metagenomics
The majority of life’s diversity lies in the microbial world. Historically, scientists have studied microbes by collecting samples from the environment and growing them in nutrient rich media. They would then examine cultures of specific organisms in isolation. Unfortunately, <1% of all microorganisms on Earth can be cultivated in this manner 4,9 . Only about 5,000 species of bacteria have been classified but scientists think there are probably several million species out there. Many of these unculturable microbes depend on each other to survive and thus cannot live in isolation. For these reasons, existing genomics data on microorganisms has historically been highly biased to organisms that we can easily culture. While much has been learned using this paradigm for studying the microbial world, it has left us with an incredibly incomplete understanding of microbial species and the communities in which they live.
Fast, cheap sequencing technologies and the ability to obtain DNA samples from a variety of microbial habitats has given rise to the new science of metagenomics . Metagenomics is the study of all the DNA in a given environmental sample (also known as the metagenome ). Metagenomics has given rise to metatranscriptomics and metaproteomics that examine all the RNA transcripts and proteins present, respectively, in an environmental sample. Metagenomics can indicate what an organism is capable of doing (i.e. what genes it has) while metatranscriptomics and metaproteomics will tell us what the organism is actually doing at any given time (i.e. what RNAs and proteins are being expressed).
Importantly, these techniques do not require microbes to be cultured, so scientists can get a better picture of the microbial diversity of an area while simultaneously studying thousands or millions of species in their natural habitat. The ultimate goal is to understand how different members of the microbial community can interact, change, and perform complex functions 4 . Metagenomics has been used to study the microbes present in the ocean, soils, sewage, coral reefs, whale carcasses, thermal vents, hot springs, and microbial communities associated with humans, termites, aphids, and worms.
The National Research Council has recently concluded that the emerging field of metagenomics promises to revolutionize research in microbiology and will likely contribute to research in nearly all biological fields 4,10 . Metagenomics will likely yield many practical applications in life sciences, earth sciences, medicine, alternative energy, environmental remediation, biotechnology, agriculture, biodefense, and microbial forensics 11 .
“We are in the midst of the fastest growing revolution in molecular biology, perhaps in all of life science, and it only seems to be accelerating”
-JC Wooley 1 .
Minnesota Mississippi Metagenomics Project
“The Mississippi is well worth reading about. It is not a commonplace river, but on the contrary is in all ways remarkable.” –from Life on the Mississippi by Mark Twain
Out of all of the rivers on Earth, the Mississippi is one of the largest and most important. It serves as a transportation system and provides drinking water to >50 cities (~18 million people). It also is an important habitat for fish and wildlife, a source of recreation for millions of people, and an important source of nutrients for the Gulf of Mexico. Despite the importance of the river, we know little about its most common inhabitants: the Mississippi microbes.
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The College of Biological Sciences was recently awarded a grant to study the metagenome of the
Mississippi river in Minnesota, putting the University of Minnesota on the forefront of metagenomics research. The goal of the Minnesota Mississippi Metagenome Project (M3P) is to understand the function and diversity of microbial life in the Mississippi River and how humans impact it. The overarching hypothesis is that humans do impact the structure and function of the microbial community and that this impact is magnified downstream as the Mississippi accumulates water and pollutants from its tributaries and confluences. The M3P project seeks to understand how the microbial community changes with time
(e.g. over days, years and seasons), space (e.g. different locations and depths), and environmental conditions such as water pH and temperature. It also seeks to determine how the input of chemical pollutants, pesticides, pharmaceuticals, and nutrients from run-off or sewage affects microbial diversity and function. Furthermore, it seeks to understand the levels and the source of pathogens and fecal bacteria in the river so that sources of these organisms can be identified and corrected. In addition to understanding what microbes live in the river and how they are affected by human activity, another goal of this project is to screen this resource for interesting biological activities such as cellulose-degrading enzymes important for biofuel production or proteins involved in antibiotic resistance.
Currently 40 L (~10 gallons) water samples are collected from 10 or more sites once per year so that the resulting metagenomes can be compared. The water samples are filtered to obtain organisms 0.45-5 µm in size. Metadata (“data about the data”) including location, time, season, current speed, temperature, pH, and levels of pollutants is also collected. The metagenomics data obtained will be used to educate the public, and help guide regulations and policies to protect this important resource.
The M3P project has produced and will continue to produce a huge abundance of data that will take decades to fully analyze. Because of this, there are opportunities for students to get involved and learn about cutting-edge research techniques being used to explore the microbial world. You could potentially be the first person ever to discover an important fact about the microbes in the Mississippi. For those interested in continuing exciting hands-on research in the new field of metagenomics, please consider taking one of the two metagenomics courses available: Biol 4950 Exploring Mississippi Metagenomics and Biol 4850 Introduction to Metagenomics. Ask your professor about who you can contact to contribute to this project while getting real lab experience.
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Figure 1. M3P Project sampling sites . Pristine water samples from Lake Itasca will be compared to downstream water samples that are potentially impacted by human activities. Samples are also taken before and after confluences with major tributaries to understand their impact on the microbial population of the Mississippi river.
For more information please see: http://www.cbs.umn.edu/main/news/inthefield/m3p.shtml
Where do river microbes come from?
Asking how microbes get into the Mississippi river is a bit like asking how rain-water gets there.
Microbes are pretty much everywhere so they enter the river from a variety of sources. Many microbes enter the Mississippi at its headwaters near Lake Itasca. Other microbes flow in from the tributaries such as the Minnesota River, the Zumbro River, and the St. Croix. During rainstorms, microbes wash into the river from the surrounding landscapes along the length of the Mississippi. Humans also contribute to the microbes which flow into the Mississippi; run-off from cattle and swine ranches and (treated) nutrient rich sewage from sewage treatment plants enter the Mississippi river and alter the microbial landscape.
Microbial populations in aquatic environments are known to fluctuate temporally and spatially due to varied input sources 12 . Because the river is constantly flowing, free-swimming bacteria are inevitably swept along with the flow of the river towards the Gulf of Mexico. Some bacteria are more stationary by avoiding regions with strong currents or by anchoring themselves to rocks, fallen logs, or the river bottom. No one really knows how much the microbial populations change over time in the Mississippi.
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Metagenomics can help answer questions such as: Does the number and abundance of species remain fairly constant or do they change with the season? How do current, depth, temperature, light, and chemicals such as pollutants affect microbial ecosystems? How does human activity alter microbial populations? These are important questions as changes in the microbial populations may affect larger organisms such as clams, crayfish, insects, fish, birds, mammals, and people.
Metagenomics also has an important role to play in medicine and public health. Scientists are particularly interested in elucidating the sources and abundance of potentially harmful organisms such as E. coli and
Salmonella that can sometimes cause human infections. Antibiotics are the prime line of defense against bacterial infections, but unfortunately more and more microbes are evolving resistance to these drugs largely due to the widespread use of antibiotics in medicine (for humans and pets) and in animal feed for cows, pigs, and poultry. Remember that all of these organisms have populations of microbes living in their guts. When these microbe populations are repeatedly exposed to antibiotics, those in the population that already are resistant will be favored (natural selection) and the population’s genetic makeup will change with time (evolution). Many resistant microbes are not harmful to their host, but it becomes a problem when they transfer their resistance genes to organisms that cause disease. Because the feces of these organisms contain intestinal microbes, and because animal waste frequently runs-off into rivers, there is an increasing concern that resistant microbes are being released into waterways humans depend on for drinking water, transportation, and recreation. Metagenomics and microbial source-tracking may be able to identify the source of some river microbes so that problems could be corrected before it is too late. This type of analysis can provide information about where antibiotic resistant bacteria may have come from. For example, you may find that organisms located at a specific sample site came from cattle, so you could compare samples from nearby cattle ranches with your sample to identify the source of contamination.
You may think that by now, most of the species on Earth have been discovered and described.
Surprisingly, recent estimates suggest that it would take an army of 303,000 taxonomists 1,200 years and
$364 billion dollars to characterize Earth’s remaining undiscovered species, and that doesn’t even include microbes 13 ! To date there are about 1.5 million organisms have been named and described and scientists speculate that millions more remain to be discovered and classified.
The science of finding, describing, and naming organisms is called taxonomy . The process of sorting organisms into different groups called taxa (singular, taxon ) is called classification . Historically organisms were classified based on their appearance but now most scientists usually classify organisms by comparing their DNA with that of other organisms. Organisms which have similar DNA are more closely related to each other than to organisms which have very different genomes.
Modern classification schemes create hierarchical trees to show the evolutionary relationships
( phylogeny ) between organisms. Phylogenetic trees are constructed using information from the fossil record, comparative anatomy, physiology, morphology, and molecular data including DNA sequences.
Phylogenetic trees are similar to family trees in that both allow you to trace a lineage; however, while family trees can trace the lineage of an individual, phylogenetic trees can provide information about the evolution of a species. A common approach to produce a phylogenetic tree is called cladistic analysis .
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Cladograms are phylogenetic trees made up of a series of two-way branching points. The terminal ends of the branches of a phylogenetic tree represent species that are alive today. Intersections in the branches represent points of divergence from a common ancestor. The lower you go in the tree, the more distant in the past the divergence occurred. Clades are groups delineated by the two branches and their common ancestor.
Figure 2. Hominoidea Cladogram.
In the Hominoidea cladogram shown above, you can see that chimpanzees and bonobos are part of the same clade. If we go further down, we can see chimpanzees, bonobos, and humans are all part of the same clade as well. What other clades can you find? All organisms within a clade contain a shared derived character . For example, all mammals possess hair . Shared primitive characters are like shared derived characters except they occurred much earlier in evolutionary time and are therefore present in a much larger taxon (e.g. vertebrae are present in all vertebrata). Using this type of classification allows us to assign organisms into distinct groups based on their physical or molecular characteristics. Carefully constructing and examining clades can sometimes reveal unexpected relationships between organisms.
Did you know that crocodiles are more related to birds than they are to lizards? There is now much evidence to support this counterintuitive idea, as well as the idea that birds evolved from dinosaurs.
Just like biology itself, the science of taxonomy is evolving over time. In the 1800s natural historians declared that all organisms fit into two kingdoms: plant and animal. This view was altered in 1969 when
Robert Whittaker proposed a five-kingdom classification system which included monera, protista, plantae, fungi, and animalia kingdoms. New evidence from analyzing DNA, RNA, and protein sequence data suggests that a three domain model is more appropriate. A domain is more inclusive than a kingdom and each domain can encompass several subkingdoms.
The domains of life are the first group in the hierarchical system in which organisms are classified. There are currently nine different taxonomic groups of classification called taxa : domain , supergroup , kingdom , phylum , class , order , family , genus , and species ; Organisms are arranged in these groups based on common ancestry and similar traits found in each group. For example, the class of mammalia
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contains many creatures from great whales to tiny shrews, which all possess hair and mammary glands to produce milk for their young. Domains contain many species while each sub-category contains fewer and fewer organisms until you get to the species level where you are left with just one.
Figure 3. Eukaryotic Kingdoms and Supergroups.
As new information is uncovered, the boundaries drawn between groups of organisms may change again.
Metagenomics will help to cluster and split new and existing groups of organisms in ways not predictable with today’s information. This should not be viewed as a source of confusion but instead is a shining example of how science works. Science is based on evidence and old ideas and biases may be changed or abandoned when new evidence comes to light.
All known organisms have a scientific name. Scientists use these names to provide every organism with a
(somewhat) descriptive name that is universally used in countries around the world. Using universal names makes it easier for scientists to communicate their findings with their peers around the globe. Did you know that the Chinese word for ‘cat’ is ‘mao’? Although the US and China use different colloquial names for ‘cat’, scientists in both countries can easily understand one another by using the scientific name: ‘ Felis catus’ . Note this name has two parts. The first part is called the genus (plural, genera ) name and this portion of the scientific name is capitalized. The second part is called the species name or the specific epithet . Together these can be used to create a unique name for each species. It is convention to italicize the entire scientific name. The scientific name is a combination of the last two rungs on the ladder of classification (domain, kingdom, phylum, class, order, family, genus, species). As another example, E. coli is a scientific name we are all familiar with. The ‘ E.
’ in
E. coli is short for Escherichia , the genus in which E. coli is classified. We will see this method of classification again later during a lab on bioinformatics.
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In addition to their scientific name, organisms are classified based on how they obtain carbon, energy, and hydrogen/electrons. Autotrophs obtain their carbon from CO
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while heterotrophs obtain preformed organic carbon-containing molecules from other organisms. Phototrophs obtain their energy from light while chemotrophs obtain their energy from the oxidation of organic and inorganic compounds. To obtain hydrogen and electrons, lithotrophs (“rock-eaters”) use reduced inorganic molecules while organotrophs use organic molecules. Some organisms which obtain energy from inorganic sources and carbon from organic sources are called mixotrophs because they combine autotrophic and heterotrophic metabolic processes. These categories can be combined to define the major nutritional types of microorganisms. For example, cyanobacteria are considered to undergo photolithotrophic autotrophy and are often called photoautotrophs. Fungi and protozoa undergo chemoorganotrophic heterotrophy and are often simply called chemoheterotrophs.
You may also come across the terms prototroph and auxotroph . A prototroph is a microorganism that requires the same nutrients as most naturally occurring members of its species. An auxotroph however has a mutation which prevents it from synthesizing one or more of its important nutrients. For example, an organism may be considered an auxotroph if it is unable to synthesize certain amino acids when the “wild type” organism of the same species can. Auxotrophs can only survive if they can obtain the nutrients that they cannot produce from their environment. Scientists often use auxotrophs in the lab to study microbial genetics.
Today you will be observing microbes in commercially prepared pondwater. While looking a examining your water sample, you are likely to find a wide variety of microbes. Because so many of Earth’s microbial species remain uncharacterized it is even possible that you will be the first to discover a new species! Some common species of microbes are listed and described below. Note that the names given here, such as ‘Amoeba’, are genus names which encompass many subspecies which often have unique characteristics.
Amoeba (Kingdom: Amoebozoa, Phylum: Tubulinea)Although A moeba have been historically classified as a protist, new evidence suggests they may have more in common with animals and fungi.
Amoeba are amorphous blobs that move using pseudopodia (false feet). The organism’s cytoskeleton produces these extensions for locomotion and for engulfing food. Amoebae are freshwater protozoans which feed on bacteria and smaller protozoans. They are surprisingly similar to a human white blood cell in terms of appearance, locomotion, and feeding habits. While they appear simple, amoeba possess highly structured and coordinated organelles including a nucleus, contractile vacuole, and a food vacuole.
Euglena (Kingdom: Euglenozoa, Phylum: Euglenida)
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Euglena are interesting eukaryotes which have some characteristics of both plants and animals. They have chloroplasts like plants and can produce energy from sunlight. They also are able to move and absorb nutrients from the environment, much like animals. They have a red eyespot which allows them to sense light to optimize its photosynthesis. They also have two flagella (one small and one large) to pull themselves along. These flagella are often difficult
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to see. Both the eyespot and flagella are at the anterior (front) end of the organism. Euglena are the contortionists of the microbe world and can bend their body to squeeze through small openings. The kingdom of Euglenozoa contains two groups which use flagella for locomotion: Euglenoids and
Kinetoplastids. The parasite Trypanosoma sp.
, which causes African sleeping sickness, is an example of a
Kinetoplastid.
Euplotes (Kingdom: Alveolata, Phylum: Ciliophora)A ciliate with an oval shaped transparent body surrounded by a rigid pellicle. It uses cilia to guide food particles into its oral groove. Its large macronucleus looks like a letter “C”. It can sometimes be observed walking along objects such as green algae.
Paramecium (Kingdom: Alveolata, Phylum: Ciliophora) - is a one celled organism which has almost twice as many genes as humans do, most likely due to a past genome duplication which was not followed by cell division. Paramecium are covered by cilia which allow it to swim ten times is body length in one second. These cilia are responsive to electric fields and, in 2010, a group at Stanford was able to steer a live paramecium to create a Pac-man like game they called PAC-mecium. Although paramecia typically reproduce asexually, they do periodically exchange small capsules of DNA with a partner cell.
Paramecium are related to Blepharisma (see below). Both are predators that feed on bacteria and small protists by using cilia to sweep prey into their oral groove.
Blepharisma (Kingdom: Alveolata, Phylum: Ciliophora)- Like Paramecium and Stentor , Blepharisma belong to the kingdom of Alveolata because these species have small membrane-bound cavities (alveoli) just under their cell surface. The function of these mysterious cavities is unknown . Blepharisma and
Paramecium are also related to apicomplexans, an immobile parasitic group of organisms that include
Plasmodium sp . which cause malaria and kill more than 2 million people each year. Blepharisma won’t hurt you, but it is a predator which feeds on small protists and bacteria. It also is cannibalistic and can grow to be very large, (sometimes up to 400-500 µm in length). You can easily recognize Blepharisma because it will often appear to be red or pink in color due to a pink pigment held in vesicles beneath their membranes.
Stentor (Kingdom: Alveolata, Phylum: Ciliophora)
filter feeding, heterotrophic ciliate protists. Their appearance is somewhat variable but most varieties are trumpet or bowling pin shaped. They are among the largest unicellular organisms. Like many freshwater organisms Stentor contains a contractile vacuole to combat osmosis. They anchor themselves to solid objects and feed on passing food particles. They can also contract their bodies when disturbed but stretch out while feeding. Stentor are often clear but they can exist in many colors including green, blue, and purple. The green color is the result of ingested green algae which have a symbiotic relationship with Stentor . Stentor are also studied for their amazing regenerative abilities. Even 1/100 th of a Stentor can regenerate an entire organism!
Chlamydomonas (Kingdom: Viridiplantae, Phylum: Chlorophyta)Unicellular flagellates, these green algae can move towards the light to find the best places for photosynthesis. They have an ovular shape with two flagella at their anterior end which they use to pull themselves through the water. Recently,
Craig Ventor, best known for helping to sequence the human genome, has teamed up with Exxon Mobil to convert green algae like Chlamydomonas into next-generation biofuels.
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Volvox (Kingdom: Viridiplantae, Phylum: Chlorophyta)Green algae which forms spherical colonies.
Each colony is composed of 2016 flagellate cells similar to Chlamydomonas . It normally reproduces asexually but can reproduce sexually in response to adverse environmental conditions (e.g., their pond is drying out). This ancient species appears to be an early attempt at multicellularity. Volvox belong to the phylum Chlorophyta. Other types of Chlorophyta are phytoplankton, photosynthetic organisms that float near the surface of the water in lakes, oceans, and ponds and form the foundation of most aquatic food chains.
Spirogyra (Kingdom Viridiplantae, Phylum: Streptophyta)A filamentous freshwater green alga which has its chloroplasts arranged in a spiral pattern. Green algae like spirogyra use chlorophyll a and b and cartotenoids to capture light for photosynthesis. Additionally, they have a cell wall composed mostly of cellulose just like plants. Recent DNA evidence suggests that green algae are more closely related to plants than to any other group and some taxonomists have placed them in a new kingdom called
Viridiplantae that includes all traditional plants and green algae.
Gloeocapsa (Kingdom: Cyanobacteria, Phylum: Chroococcales) - Bacteria commonly found in the soil and in the water. They are photoautotrophs which are able to produce organic compounds using the energy from sunlight and an electron donor such as water or hydrogen sulfide. Their name comes from the fact that they are able to use chlorophyll a and a blue pigment called cyanin to undergo photosynthesis. In the past, they were also classified as “blue-green algae” because of these two pigments.
Members of this group have been undergoing photosynthesis for 3.5 billion years and they are responsible for providing us with an atmosphere abundant in oxygen. Some cyanobacteria are also known to produce a toxin called BMAA, high levels of which appears to cause brain damage in mice, monkeys and humans.
There is also some evidence showing that BMAA toxin produced by these cyanobacteria may contribute to some cases of ALS, Alzheimer’s, and Parkinson’s disease 14 .
For more information see:
Pac-Mecium : http://www.switched.com/2011/01/13/biotic-video-games-pac-mecium-stanford/
Types of Microbes: http://archives.microbeworld.org/microbes/types.aspx
Find Full Scientific Classification: http://www.ncbi.nlm.nih.gov/taxonomy
Amoeba Video: http://www.youtube.com/watch?v=7pR7TNzJ_pA&NR=1&feature=fvwp
Euplotes Video: http://www.youtube.com/watch?v=MdJMJr_QFXM
Stentor Video: http://www.youtube.com/watch?v=pds8w7C9FEw&feature=related
Euglena Video: http://www.youtube.com/watch?v=ZHZZKwrYm4g&feature=related
Site Selection and Filtering of Metagenomic Samples
When designing a metagenomic study, you first must identify where you would like to sample. Are you interested in studying oceanic microbes, river microbes, or soil microbes from your backyard? Your choice of sample site will greatly affect what types of information you can gain. After you have selected your general area of study, you will then want to vary the conditions under which you collect your
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samples. For example, if you are taking your samples from the Mississippi river you might choose to sample at a number of different locations so your results can be compared. You also may choose to sample at different depths, or at different times of the year. Each choice will allow you to make a different kind of comparison.
Typically, microbial samples collected from seawater, soil, the human gut, or other locations are first filtered and then processed to extract the DNA. Analyzing every kind of microbe in your sample
(eukaryotes, bacteria, and archaea) would be technically challenging and time consuming. Filtering your sample will provide you with what you are interested in studying while removing most everything else.
The filtering scheme you select will directly affect the type of results you obtain. For example, many studies specifically look at bacteria because they are ubiquitous, relatively uncharacterized, and because their small genome size makes later sequencing and analysis easier. If you are focusing your studies on bacteria, you should select filters to remove the larger protists and the smaller viral particles. Choosing the correct filter size can be problematic as small protists and large bacteria overlap in size. In addition, small bacteria which form large filaments may also be filtered out inadvertently. For these reasons you should always be aware of the limitations your filtering method imposes upon your study.
1.
List and observe broad classes of microbes and their characteristics.
2.
Use a microscope to estimate microbe size.
3.
Explain how different microbes can be helpful or harmful to humans.
4.
Define metagenomics and explain the types of questions it can help address.
5.
Explain how filtering works and why it is important for metagenomics studies
6.
Describe how the surrounding landscape influences the microbial populations in the Mississippi river.
7.
Outline the importance of microbes to the river ecosystem.
8.
List three reasons why it is important to study river microbes using metagenomics.
Student collected microbes
Commercially prepared pond water
Compound microscopes (w/ 100X objective)
Microscope slides
Fixed bacteria Slides
Live bacteria cultures
-Rhodospirillum
-Micrococcus
Coverslips
Swinnex filter holder (25mm) (autoclaved)
50 ml syringes
Sterile tap water
Filter forceps
Methyl cellulose
1X PBS
Ethanol lamps
Immersion oil
Bench paper (water proof)
Methylene green stain
Methylene blue stain
Lens paper
Lens cleaning solvent
Mixed cellulose ester filters (0.45 & 5 µm)
Microcentrifuge tubes
Glass pipettes + bulbs
150 ml beakers (3-4 per group)
1.5 ml microcentrifuge tubes + tube racks
15 ml conical tubes
Goggles
1cc syringe
Glass cleaner
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STUDENTS SHOULD WORK IN GROUPS OF TWO
COLLECT YOUR PONDWATER SAMPLE BEFORE COMING TO LAB :
Collecting your own microbes can be a lot of fun because you may find some surprising things. At least one water sample is needed for each group of two students. If there is no ice on the water, you are encouraged, but not required, to obtain microbes from your favorite outdoor water source. You may find water in lakes, ponds, rivers, streams, or even mud puddles. When selecting a water source be adventurous but be safe.
Alternatively, you may obtain water at any time of the year from the small indoor ponds in the CBS greenhouse on the St. Paul campus. When taking water from the CBS Greenhouse, be careful not to disturb the aquatic plants, turtles, coy, or the guppies.
Before collecting your sample, rinse out a small container (such as a water bottle) several times. Use the squeegee/scraper (located along the wall as you enter the room or behind the door) to scrape along the pond walls to re-suspend the microbes. To collect a sample, simply fill your container with the water sample of your choice. If it is cold outside, shelter your bottle inside of your coat as the cold temperatures outdoors might kill your microbes. Store your sample at room temperature. Remove the cap to expose it to air and sunlight. Fresh samples work best but you may store samples for one day.
WARNING: The floors may be slippery so watch your step.
DAY OF THE LAB:
Today you’ll be working with three distinct samples: (1) The sample you collected, (2) a sample of commercially prepared pond water, and (3) slides of live and fixed bacteria. You will observe eukaryotic microbes in the commercially prepared pond water, observe bacteria on the stained slides, and filter/centrifuge bacteria from your collected water sample.
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Figure 4. Outline of Microbial World Lab.
This figure shows the samples you’ll be working with and what you’ll be doing with them.
Learning about sample collection and filtering is important because how you collect and filter your sample can greatly affect your results. For the M3P Project, ~40 gallons were filtered from each of the 10 sites using vacuum pumps and a variety of filter sizes. The goal of the M3P project is to examine diversity among bacterial species.
You will be filtering bacteria from the water sample you obtained (e.g. CBS Greenhouse pond-water).
Filtering will help to separate bacteria from viruses and eukaryotes present in your sample. Filtering also concentrates the microbes making them easier to study. The bacteria you filter will be stored and used in the DNA extraction lab that will be done later during the semester.
1. Prepare your syringe and filter holder by thoroughly rinsing with tap water.
2. Write “0.45 µmSCS ” and your initials on one 1.5 ml microcentrifuge and one 15 ml conical tube.
(SCS stands for Student Collected Sample). Label the microcentrifuge tube only on the top of its lid.
Label the conical tube on its side or the top of its lid.
3. You will be serially filtering your water sample, which means you will run your water through each filter in a specific order. You will use the 5.0 µm filter first, and 0.45 µm filter last so that you can filter out progressively smaller microbes. NOTE: WHITE FILTERS ARE SANDWHICHED BETWEEN
BLUE PAPER. DO NOT USE BLUE PAPER.
A.
Secure the 5.0 µm filter into the Swinnex filter holder.
B.
Remove the plunger from the 50ml syringe.
C.
Attach the Swinnex filter holder to the 50 ml syringe using the luer lock connection.
D.
Hold the filter/syringe over a 150 ml labeled beaker (e.g. labeled “Filtrate 1”). Invert your water sample a few times in case some microbes settled to the bottom. Pour ~50 ml of your sample into the 50 ml syringe and reinsert the plunger.
E.
Press the plunger down using consistent pressure but do not use excessive force.
F.
Remove the Swinnex filter holder from the 50 ml syringe (release the luer lock connection), and place it on a paper towel.
G.
Remove the plunger from the 50 ml syringe.
H.
Re-attach the Swinnex filter holder to the 50 ml syringe using the luer lock connection.
I.
Hold the filter/syringe over the 150 ml labeled beaker (“Filtrate 1”). Pour another ~50 ml of your sample into the 50 ml syringe and reinsert the plunger.
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J.
Press the plunger down using consistent pressure but do not use excessive force.
Most of the eukaryotes present in your sample are now trapped on the 5.0 µm filter. Prokaryotes are in your “Filtrate 1” beaker.
4. Remove the 5.0 µm filter from the Swinnex filter holder using forceps and discard it in a waste container. Before filtering the color of the filter was ____________ but after filtering it became
______________.
5. Next, rinse off the 50 ml syringe and Swinnex filter holder with tap water, and then secure a 0.45 µm filter into the Swinnex filter holder.
A.
Remove the plunger from the 50 ml syringe.
B.
Attach the Swinnex filter holder to the 50 ml syringe using the luer lock connection.
C.
Hold the filter/syringe over a new 150 ml beaker labeled “Filtrate 2”. Pour ~50 ml of your
“Filtrate 1” sample into the 50 ml syringe and reinsert the plunger.
D.
Press the plunger down using consistent pressure but do not use excessive force.
E.
Remove the Swinnex filter holder from the 50ml syringe (release the luer lock connection), and place it on a piece of paper toweling.
F.
Remove the plunger from the 50 ml syringe.
G.
Re-attach the Swinnex filter holder to the 50 ml syringe using the luer lock connection.
H.
Hold the filter/syringe over the beaker labeled “Filtrate 2”. Pour another ~50 ml of your ”Filtrate
1” sample into the 50 ml syringe and reinsert the plunger.
I.
Press the plunger down using consistent pressure but do not use excessive force.
6. This time, remove your filter, and place it directly into a 15 ml conical tube labeled “0.45 µm-SCS” and your initials.
A.
Add 2-3 ml 1X PBS
B.
Replace the cap and mix vigorously for 2 minutes to resuspend the microbes that were trapped on the filter back into solution.
C.
Pour 1-1.5ml of the liquid into a microcentrifuge tube labeled with “0.45 µm-SCS” and your initials.
7. Centrifuge and store the bacterial sample :
A.
Centrifuge the bacteria in microcentrifuge tube labeled with “0.45 µm-SCS” and your initials for
10 minutes at 5,000 X g (~7,500 rpm).
B.
After the cells are spun down you may see a tiny cell pellet in your tube, or it may be too small to see. In either case, decant your tube to drain the water.
C.
Give the pelleted sample to your TA (make sure your initials are on the tube).
D.
These bacterial samples will be frozen and stored , for DNA extraction in the “Extracting the
Microbial Metagenome” lab to be done later in the semester .
Questions
1.
What is the purpose of filtering?
2.
How does filter size affect your sample?
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The goal of the second part of this lab is to observe and characterize the microbes that are found in the commercially prepared pondwater and to examine both live and fixed bacteria.
A. Commercially Prepared Pondwater
1. Fill a beaker 2/3 of the way full of tap water and label it “Rinsing Water”. This water will be used to rinse out your dropper before each time you take a new sample. You may store your dropper in this beaker so that it does not become contaminated with bench-top microbes.
2. Use the dropper to take one drop of water from the commercially prepared pond water. Place a drop of this sample water onto a glass slide and return any excess to the container holding the commercially prepared pondwater. Rinse out your dropper and leave it in the rinsing beaker.
3. Place a coverslip on the top of the water drop as demonstrated by the TA. It is easiest to place one side of the coverslip on the slide near the drop, lean the coverslip towards the drop and then release it allowing the coverslip to gently fall over the water droplet. Prepare at least three separate slides.
4. You may now view this slide under the compound microscope.

Focus your microscope lens on the correct plane. One common mistake is to focus on top of the coverslip instead of underneath it. It can be helpful to initially focus while looking at the very edge of the coverslip so you can more easily see which layer you are looking at. The top of the cover slip usually has a number of clear, immobile pits or pieces of debris. These will often have jagged edges and will sometimes have a diamond shape. In contrast, if you are correctly focused underneath the slide, you should see moving bubbles and microbes, including some of which are pigmented. These microbes are usually round or smooth and not irregular and jagged like the ghostly entities seen on top of the coverslip.

Use the low power objective lens first (4X). The low power lens shows an area with a greater depth of field, and diameter of field; therefore objects are easier to locate.

It is helpful to start with a large immobile organism such as a filamentous green alga.

After locating your organism with the coarse adjustment knob: o Sharpen the image using the fine adjustment knob. o Make sure that the slide is positioned so that the specimen is in the center of your field of view. o Turn the higher power lenses (the 10X and then the 40X) into place. Use only the fine adjustment knob to sharpen the image when these lenses are in place.
o Faster organisms can be slowed down by adding a drop of methyl cellulose to your slide.
Adding a drop of methylene green or methylene blue will stain the DNA and make the nuclei easier to see. Unfortunately, this kills the organisms. o Prior to turning the 100X lens all the way into place, add a small drop of oil onto the coverslip above where your sample is located. Continue turning the 100X lens into place. o After viewing the sample at 100X, turn the lens and remove the slide. Be careful not to expose the other objective lenses to the oil.
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o Remove the oil from the slide with Kimwipes.
5. Repeat this process for each of your three sample slides. (You may look at more than three slides if desired.) Make sure everyone in your group gets to use the microscope and see the different types of organisms you find.
6. Use the table at the end of this lab to record your observations. For each microbe do the following:
A. Give the Domain, Kingdom and Genus names of these species.
B. Draw and label a picture of the organism and provide its estimated size.
C. Describe its form of motility (e.g. cilia, flagella, pseudopodia)
D. Describe any internal structures that are detectable (e.g. contractile vacuole, nucleus, chloroplasts, food vacuole).
E. Indicate the organism’s carbon and energy source.
Photos of common microorganisms can be found on a laminated handout. If you cannot identify the organism using the card, books of dichotomous keys are available in the front of the lab room.
After lab you are encouraged to go online to see pictures and videos of live microbes to compare with what you found during lab.
B. Bacteria Slides
1. Find live cultures of Rhodospirillum and Micrococcus . Add a drop of these cultures to a microscope slide and cover with a coverslip.
2. View the slide(s) under the compound microscope using the oil immersion 100X lens. Be careful not to get oil on the other lenses.
3. Draw and record characteristics of the bacteria you observe.
4. Remove the oil from the slide using Kimwipes.
4. Clean the slide with glass cleaner, dry with a paper towel and return it to its tray.
6. If viewing a second slide, repeat this process with that slide.
1. Depending on the type in your room, select fixed slide(s) of:
17

A.
Bacteria Types Gram Stained (slide contains both coccus(round) and bacillus(rod-like) shapes).
OR
B.
Mixed Bacillus Gram Stained and Mixed Coccus Gram Stained.
2. View the slide(s) under the compound microscope using the oil immersion 100X lens. Be careful not to get oil on the other lenses.
3. Draw and record characteristics of the bacteria you observe.
4. Remove the oil from the slide using Kimwipes.
4. Clean the slide with glass cleaner, dry with a paper towel and return it to its tray.
6. If viewing a second slide, repeat this process with that slide.
References
1. Wooley, J.C., Godzik, A. & Friedberg, I. A primer on metagenomics.
6 , e1000667 (2010).
2.
Stark, L.A. Beneficial Microorganisms : Countering Microbephobia.
9 , 387-
389 (2010).
3. Pedros-Alio, C. Genomics and marine microbial ecology.
9 , 191-7 (2006).
4. Jurkowski, A., Reid, A.H. & Labov, J.B. Metagenomics: a call for bringing a new science into the classroom (while it’s still new).
6 , 260-265 (2007).
5. Savage, D.C. Microbial ecology of the gastrointestinal tract.
31 , 107-
33 (1977).
6. Berg, R.D. The indigenous gastrointestinal microflora.
4 , 430-5 (1996).
7. Gill, S.R.
Metagenomic analysis of the human distal gut microbiome.
312 ,
1355-9 (2006).
8. Fricke, W.F.
Insights into the environmental resistance gene pool from the genome sequence of the multidrug-resistant environmental isolate Escherichia coli SMS-3-5.
190 , 6779-6794 (2008).
9. Chistoserdova, L. Recent progress and new challenges in metagenomics for biotechnology.
32 , 1351-9 (2010).
10. Sciences, N.A. of & NRC
.
171 (National Academies Press: Washington, DC, 2007).at
<http:/www.nap.edu/catalog.php?record_id=11902>
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11. Jurkowski, A., Reid, A.H. & Labov, J.B. Metagenomics: a call for bringing a new science into the classroom (while it’s still new).
6 , 260-265 (2007).
12. Wu, C.H.
Characterization of coastal urban watershed bacterial communities leads to alternative community-based indicators.
5 , e11285 (2010).
13. Mora, C., Tittensor, D.P., Adl, S., Simpson, A.G.B. & Worm, B. How Many Species Are
There on Earth and in the Ocean?
9 , e1001127 (2011).
14. Pablo, J.
Cyanobacterial neurotoxin BMAA in ALS and Alzheimer’s disease.
120 , 216-25 (2009).
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Eukaryotes:
Domain
Kingdom
Genus
Drawing Estimated size:
Length (
Width (
Motility Internal
Structures
Detectable
Carbon and
Energy source
20

Prokaryotes:
Domain Drawing
21
Estimated size:
Length (
Width (
Arrangement:
(singular, chains, clusters)
Gram stain:
[positive
(purple), or negative
(pink)]

NOTE TO LAB STAFF AND TA’s:
1. STUDENTS MUST OBTAIN SAMPLES BEFORE COMING TO LAB.
2. Note: There are plenty of microbes in the CBS greenhouse pond water but sometimes students have difficulty finding them. Make sure they filter their sample until the filter clogs to increase the microbial density enough so that microbes are easy to see.
4. Encourage students to look at their own samples after they’ve examined the commercially prepared pond water to make sure they’ve seen some stuff first.
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