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Tuesday, October 7, 2008

Using DNA Sequencing to Identify the Bacteria Living on my Hands

Update: A more thorough study done at the University of Colorado took samples from 51 college students and they identified 4742 different species of bacteria and found that women harbored a greater variety. I used to work at the Microscale Life Science Center at the University of Washington, where they build tools for biologists to study mammalian cells at the single cell level. Here, here, and here are some papers describing some of the biology and engineering. The MLSC is a multi-disciplinary team made up of electrical and mechanical engineers, biologists, chemists, and pathologists. The devices I helped build were really neat and allowed our biologists to answer questions about cells that we never could even ask before! Bacterial contamination is a very serious problem with our lab-on-a-chip devices. If a bacterial outbreak invades the mammalian cells we are experimenting with, the results will be compromised and a lot of valuable time will be wasted. As part the engineering team, I had to identify how much bacteria was around, where it was, and how to keep it out of our sterilized experiment. I learned that bacteria floats around in the air and congregates on dust particles, and that everything in the room has some bacteria on it unless it hasn't been sterilized a couple of minutes before. It turns out that our body (and everything we touch) is the main source of bacteria contamination. Even if you're wearing Latex gloves to handle an experimental device, if you touch your face or a tweezers that was previously touched by un-gloved hands, it will get contaminated.



Realizing this, I became very curious about which species of bacteria actually were using my body as a host. Using the equipment in our lab, I transferred some bacteria from my hands to Agar plates, isolated and grew individual bacteria colonies for each species, sequenced their DNA, and imaged them using a scanning electron microscope (SEM). From the DNA sequences, I was able to tell exactly what species they were by comparing the sequences to an online DNA database, and learn more about them. The following is a recount of what I did.

Step 1: The first step was to culture some bacteria on an Agar plate in order to isolate the different species. Touching the Agar jelly on a plate was enough to transfer the bacteria from my hand to the nutrient-rich Agar where colonies quickly grew from single bacterium cells. You can see eight different bacteria cultures from my hand. In actuality there are probably a lot more additional types, which would have grown on different Agar substrates. The bacteria varied in color and appearance, but they all had one thing in common – they stink! I guess that’s what showers are for – to wash away the stinky excrement from the bacteria living on us! By the way, I touched the Agar with my hands AFTER washing them with soap and water. These bacteria or similar species are on all of us and are just part of our natural healthy body and actually repel "bad" bacteria by taking up virgin real estate on our skin and digestive system.

Step 2: Most biologists who work with these types of bacteria all the time could probably identify them based on their appearance, but this was new to me so I needed a more reliable method - I sequenced a small part of their DNA! To do this I used a method called polymerase chain reaction (PCR) to exponentially multiply a section of the 16S ribosomal RNA gene commonly found in all bacteria. Slight variations in the DNA sequence act as a fingerprint and can be used to identify individual bacteria species. Out of five bacteria types I did PCR on, only two of them turned out for whatever reason.

Step 3: I then sent the PCR products from the two successful amplifications to the University of Washington DNA Sequencing Facility. Each one only costs $6 to be sequenced. When they received my samples, they add a bunch of chemicals including some fluorescent nucleotides. After that they run it through their Sanger sequencer, which reads the fluorescent signal from the DNA as it moves through a glass capillary. From the plot, the sequence of G, A, T, and Cs, which are the building blocks of DNA can be determined and exported as a text file. The Ns are unknowns:




GATGCGCGNCTACCATGCAGTCGAACGATGAAGCCCAGCTTGCTGGGTGG
ATTAGTGGCGAACGGGTGAGTAACACGTGAGTAACCTGCCCTTAACTCTGG
GATAAGCCTGGGAAACTGGGTCTAATACCGGATAGGAGCGTCCACCGCATG
GTGGGTGTTGGAAAGATTTATCGGTTTTGGATGGACTCGCGGCCTATCAGC
TTGTTGGTGAGGTAATGGCTCACCAAGGCGACGACGGGTAGCCGGCCTGA
GAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGG
AGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGAC
GCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGTAGGGAA
GAAGCGAAAGTGACGGTACCTGCAGAAGAAGCACCGGCTAACTACGTGCC
AGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTATCCGGAATTATTGGGC
GTAAAGAGCTCGTAGGCGGTTTGTCGCGTCTGTCGTGAAAGTCCGGGGCTT
AACCCCGGATCTGCGGTGGGGTACGGGCAGACTANAGTGCANTAGGGGAG
ACTGGAATTCCTGGTGTAGCGGTGGAATGCGCANATATCAGGAGNAACACC
GATGGCGAAGGCAGTCTCTGGGCTGTACTGACNCTNAGANCCNAAANCNT
GGGANCNAACAGAATNAAATACCCTGGTANTCCATGCCNTAACCNTTGGCA
CTAGTGTGGGANCCNTTCCCCGNTTCCNGCCNCACCTAAANCATNAATTGC
CCCCCNTGGGAATACGNCCCCAAGGCTAAAACNCAAAGAAATTGACGGGG
GGCCCCCCCAACCGGNNGNAACATTGCGGATTTNTTNCAATNNCACCCCNA
AAAAACNTTACNAAGGNNTNAACNTTNTTCCCAAACCCCCNNAAAAAAANCG
GTTTNCCCCTTTNGGGGCGGGTTTCNCNNGGGGGNNCATGGTTTNNCCCC
CCCCCNC


Just to put things in perspective, the human genome contains 20,000 to 25,000 genes and approximately 3 billion base pairs. So if each cell contains 3 billions base pairs and a human body has about 50 trillion cells, that’s a lot of base pairs we’re walking around with! A bacteria genome has a lot of base pairs as well. But this small sampling of sequence from this one gene can be enough to identify what type of cell it is.

Step 4: After you have the sequence of a section of the 16S ribosomal RNA gene from the bacteria of interest, you have to compare it to known DNA sequences. Luckily people have been sequencing all sorts of DNA for over ten years now, and there is database where all the sequences are stored. Searching this database to find out what bacteria I had was the easiest part of this experiment. All I had to do was copy and paste the sequence above into a page of the website and click a button. Eight seconds later it showed a list of all the genes in the database that matched my sequence with a score as to how well it matched. The website returned this match, a 799/859 (94%) base pair match, and tells me that my sequence is from a Micrococcus Luteus partial 16S rRNA gene!

Step 5: Further utilizing the power of the internet (www.wikipedia.org), I quickly looked up some information on the first bacterium. This is what it said:

"Micrococcus luteus is a Gram positive, spherical, saprotrophic bacterium that belongs to the family Micrococcaceae. An obligate aerobe, M. luteus is found in soil, dust, water and air, and as part of the normal flora of the mammalian skin. The bacterium also colonizes the human mouth, mucosae, oropharynx and upper respiratory tract. Although M. luteus is non-pathogenic and usually regarded as a contaminant, it should be considered as an emerging nosocomial pathogen in immunocompromised patients. M.luteus is resistant to reduced water potential and can tolerate drying and high salt concentrations. M. luteus is coagulase negative, bacitracin susceptible, and forms bright yellow colonies on nutrient agar. To confirm it is not Staphylococcus aureus, a bacitacin susceptibility test can be performed..."

The other one turned out to be Bacillus Subtilis:

"Bacillus subtilis is a Gram-positive, catalase-positive bacterium commonly found in soil. A member of the genus Bacillus, B. subtilis has the ability to form a tough, protective endospore, allowing the organism to tolerate extreme environmental conditions. Unlike several other wellknown species, B. subtilis has historically been classified as an obligate aerobe, though recent research has demonstrated that this is not strictly correct. B. subtilis is not considered a human pathogen; it may contaminate food but rarely causes food poisoning. B. subtilis produces the proteolytic enzyme subtilisin which has been shown to be a potential cure for certain types of cancer. B. subtilis spores can survive the extreme heating that is often used to cook food, and it is responsible for causing ropiness in spoiled bread..."

Seems like there’s a lot more known about the second bacterium that the first one. It amazes me there could be so much history and information for something living on my body that I never even realized was there.

Step 6: Even though I reached my objective of the experiment already, I couldn’t pass up the opportunity to use one of the scanning electron microscopes (SEM) on campus. I worked with a grad student who uses the SEM all the time and he helped me collect some images. First we had to sputter a 6 nanometer layer of gold onto the bacteria for image improvement. That’s a picture of the inside of the SEM as shown on a monitor. The inside of the SEM is first drawn to near-vacuum pressure. Then with a little work with the SEM software and a little searching around for some good specimens, the images emerge.








Here’s a picture of the Micrococcus Luteus. There are two clusters of 4 cells. I heard clumping is a typical behavior of these cells. Each cell is about 500 nanometers in diameter. The cell culture of these bacteria is the yellow-colored group at about 3 ‘o clock in the second figure of the blog.












Here’s a picture of Bacillus Subtilus. Unfortunately the images aren’t as clear as the previous one but you can still make out the rod-shaped bacterium in the center. The cell culture of these bacteria is the milky-colored group at about 1 ‘o clock in the second figure of the blog.







In conclusion, I’m happy to have been introduced to some of the creatures that call my body home. As long as they are there and not causing any harm, it’s fine with me. In fact, I bet they do a good job at keeping harmful bacteria away by crowding the surface of my body so much that the bad ones have too hard of a time elbowing their way in.

Special thanks to Elizabeth Skovran for showing me how to do all this, John Lund for helping with the SEM imaging, Dr. Babak Parviz and Dr. Mary Lidstrom for supplies and equipment use.

more SEM images

5 comments:

Sheryl Torr-Brown said...

Great post--thank you

Michael Kucher said...

With regard to slower boots after installing a cloned drive, I was able to fix that on my Unibody MacBook by flashing the PRAM Command+Option+P+R.

Tim Molter said...

Michael, Nice I'll give that a try next time!

aquira said...

thanks michael...
this post is really helpful. I have task from my professor to find out out how to analyze DNA sequences if we have the bacteria. and u have the steps...

thnk u ^_^

Tim Molter said...

aquira, awesome! glad you found this info useful. I knew someone would eventually. ;)