1. Library West
Where did we streak and how many
unknowns were collected and tested?
1--Books 1: 2 unknow ns
2--Books 2: 2 unknow ns
3--Chair Couch 1: 1 unknow n
4--Chair Couch 2: 2 unknow ns
5--Table 1: 2 unknow ns
6--Table 2: 2 unknow ns
7--Keyboard 1:2 unknow ns
8--Keyboard 2: 2 unknow ns
9--Starbucks 1: 1 unknow n
10--Starbucks 2: 2 unknow ns
11--Wooden Chair 1: 2 unknow ns
12--Wooden Chair 2: 1 unknow n
Total plates for isolation:37;16 to grow and 21 to
isolate
Observation/ Results: Not good isolationsstreaks the
1st timeso wedid another streak for isolation and used
aseptic techniqueas much as possible
Schedule of Procedures:
November 5th: Streak for isolation
November 12th: Re-streak for isolation
November 19th: PCR day. Only did PCR for significant
growth. Send off for results.
December 3rd: Obtain and review results.
So, what grows in our library?
1-1 Terribacillus aidingensis strainMP602
Terribacillus aidingensis MP602 was isolated from Cryptomeria fortunei in
Tianmu mountain in Zhejiang, China. This bacterium showed high
antagonistic activity against certain phytopathogenic fungi and has the
potential in application as a biopesticide.
1-2 Bacillus methylotrophicus strainJS25R
A methanol-utilizing, plant-growth promoting bacterium.
2-1 Curtobacterium
There is enormous heterogeneity of these bacteria, presently comprising
more than a dozen medically relevant genera with more than 100 species
in total. It is a genus of bacteria of the order Actinomycetales. They are
Gram-positive soil organisms found in seeds or wilting plants.
2-2, 10-2 Bacillus megaterium
Is a rod-like, Gram-positive, mainly aerobic spore forming bacterium found
in soil, seawater, sediments, dried food, honey and milk. Produces B12 and
penicillin amidase. It is used in AIDS diagnostics; rarely causes disease or
infection.
3, 4-2, 5-1, 8-1 Micrococcus luteus
Being Gram-positive, they appear blue-violet when stained using a Gram-
stain technique. An aerobe and is normally found living in the human
mouth, mucosal linings of the upper pharynx, and respiratory tract. Found
in places such as human skin, water, dust, and soil. Micrococcus are
generally thought of as harmless bacterium. But, people with compromised
immune systems, such as HIV+ positive patients, have been known to get
skin infections caused by this bacterium. M. luteus on human skin breaks
down compounds in sweat into compounds with bad odor. It grows well in
environments with little water or high salt concentrations.
4-1Bacillus aryabhattai
It has extra-terrestrial background. Gram-Positive Cocci, non-endospore
forming (some strains do form). Used in cryotubes to collect air from
upper atmosphere. Found in India.
5-2 Staphylococcus cohnii
Gram-positive cocci, motile, and only occur singly or in pairs and display
facultative anaerobic metabolism; but, grow better in aerobic conditions. It
is isolated most frequently from human skin and infrequently from
primates. However, no known involvement in human disease has been
reported.
6-1, 6-2, 10-1 Staphylococcus epidermidis
Non-motile, gram positive Cocci, arranged in grape-like clusters. Most
common cause of nosocomial infections (create biofilms in catheters or
other surgical implants). Permanent ubiquitous colonizer of human skin.
7-1 failed
7-2, 9, 12 Staphylococcus hominis
Gram-positive spherical cells that commonly occur as a harmless
commensal on humans and animals. May occasionally cause infection in
immunocompromised pts.
8-2 Bacillus subtilis
Found in soil, air, plants, and compost. Gram positive, rod shaped and
resistant to high temperatures. It rarely colonizes on the human body and
generally remains dormant. It can be used as a fungicide for plants.
11-1 Staphylococcus caprae
Gram positive, coccus that is associated with goats. Typical bacteria on
human skin, but can cause infections of the bloodstream, urinary tract,
bones and joints.
11-2 Curtobacterium luteum
It is a gram-positive psychrotrophic bacterium, secreting an extracellular
protease that was isolated from the soil of Gangotri glacier, Western
Himalaya. It can survive in cold environments, so they are responsible for
spoiled food. They don’t usually cause human infection.
HOW DIRTY
IS YOUR
LIBRARY?
Noah Andone, Kacey
Christian, Danielle Day,
Stephanie Lombardo, Reshmi
Mathew, Rigen Saltivan

2. What materials were
used?
· Sterile swabs (16 total)
· Tubes to hold the swabs after taking
samples
· Streak for Isolation Plates (32 total)-
half to grow the samples, half to isolate
· 1X Sterile PBS buffer
· Microcentrifuge tubes
· Chemglass beads
· PowerClean™ DNA Solution 5
· Spin Filters
· PowerClean™ DNA Solution 6
· PowerClean™ DNA Solution 7
· BioTech Epoch Take 3 plate
· PCR Master Mix
· Buffer PB
· Buffer PE
· QIAquick columns
· Buffer EB
Procedures:
1. Gather swabs at Library West (two swabs per place at least)
a. Table
b. Chair
i. Couch type
ii. Wooden type
c. Keyboard
d. Books
e. Starbucks Counter
2. Grow the samples
3. Run a streakfor isolation for each swab
4. Run a PCR of each sample to amplify the DNA
DNA Extraction: Procedure
•Label everything.
•Take a micro-centrifuge tube with 1 mL 1x Sterile PBS buffer.
•Harvest one whole plate of cells (scrape off without getting agar pieces): Use
ALL cells and colonies, scrape off the agar plate using a sterile inoculating loop
and suspend them in the microtube with 1mL 1X Sterile PBS buffer (without
the glass beads) (2 people)
•Vortex at max speed for 5 sec.
•Centrifuge at 10,000 x g for 1 min. Lookfor a cell pellet. The DNA is in the
pellet inside the cells.
•Discard supernatant (suckoff with P1000, without touching the pellet) Add
0.5 mL 1x sterile PBS buffer to the pellet
•Resuspend the pellet by vortexing at max speed until the whole pellet is
dissolved.
•Place in hot water bath at 85°C for 5min. Transfer the content to a new
microcentrifuge tube containing 0.1 mL Chemglass beads.
•Vortex for 5 min at highest speed.
• Centrifuge for 1 min at 10,000 x g.
•Your DNA is now in the supernatant. Transfer supernatant to new tube.
Discard the pellet. (need help here)
DNA Purification
•Purpose?
◦To remove sample impurities (i.e.proteins and other cellular debris)
DNA Purification: Procedure Pt. 1
•Take 150uL of the DNA extraction supernatant.
•Add 400 μl of PowerClean™ DNA Solution 5 to the supernatant (be careful
that solution doesn’t exceed rim of Collection Tube) and vortex for 5 seconds.
◦PowerClean ™ DNA Solution 5 is a high salt concentration solution. Since
DNA binds tightly to silica at high salt concentrations, this solution will adjust
the salt concentrations to a5
•Carefully place the Spin Filter in a new Collection Tube (provided). Avoid
splashing any PowerClean™ DNA Solution 6 onto the Spin Filter.
◦Note: It is important to avoid any traces of the ethanol based wash solution.
•Add 50μl of PowerClean™ DNA Solution 7 to the center of the white filter
membrane.
◦Note: Placing this Solution (sterile elution buffer) in the center of the small
white membrane will make sure the entire membrane is wetted. This will result
in a more efficient release of the DNA from the silica Spin Filter membrane. As
PowerClean™DNA Solution 7 (sterile elution buffer) passes through the silica
membrane,
•DNA is released because it only stays bound to the silica Spin Filter
membrane in the presence of high concentration of salt. PowerClean™ DNA
Solution 7 is 10 mM Tris pH 8 and does not contain EDTA. Alternatively,
sterile DNA-Free PCR Grade Water (MO BIOLaboratories Catalog No. 17000-
10) may be used for elution from the silica Spin Filter membrane at this step.
•Centrifuge the Spin Filter at 10,000 x g for 30 seconds at room temperature.
•Discard the Spin Filter. The DNA in the Collection Tube is now ready. No
further steps are required. We recommend storing DNA frozen (-20° to -80°C).
PowerClean™ DNA Solution 7 does not contain EDTA.
DNA Quantification
•You will be pipetting 2ul of your purified DNA product onto the BioTech
Epoch Take 3 plate to quantify your DNA samples
5. Send for sequencing
Conclusions:
Most of the bacteria isolatedweren’t necessarily harmful to
healthyindividuals
Still recommendedyouwash your hands often/ keepyour
hands out of your faceandmouth, andlimit the surfaces
youtouch
Influx of people forexams means more bacteria
brought in
Cold/Flu Season means more infectious bacteria
brought in
Possible that we swabbedafter the library was
cleaned, so the data we havemay not be representative of
all the bacteria in thelibrary onanygivenday
References:
http://www.sciencedirect.com/science/article/pii/S1872207509600121
http://onlinelibrary.wiley.com/doi/10.1111/1469-0691.12743/abstract
http://web.mst.edu/~microbio/BIO221_2009/B_subtilis. html
http://www.globalrph.com/Staph-hominis.htm
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2807625/
http://www.vumicro.com/vumie/help/VUMICRO/Staphylococcus_cohnii_
ssp_cohnii.htm
https://microbewiki.kenyon.edu/index.php/Micrococcus
http://ijs.sgmjournals.org/content/59/12/2977.full
http://www.microbeworld.org/component/jlibrary/?view=article&id=80
54
http://www.tgw1916.net/Bacillus/megaterium.html
http://jcm.asm.org/content/43/3/1032.full
http://www.ncbi.nlm.nih.gov/pubmed/19966000
http://www.ncbi.nlm.nih.gov/bioproject/252898
http://www.ncbi.nlm.nih.gov/pubmed/22642843
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC262321/
https://www.healthtap.com/topics/what-disease-is-caused-by-bacillus-
megaterium
http://biosciencediscovery.com/Vol3No1/Semantairay138-145.pdf
http://ijs.sgmjournals.org/content/59/12/2977.full
http://www.bacterio.net/bacillus.html
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2807625/http://www.edlab.org/
glossary.html