Materials and methods Describe the staph st

Materials and methods
Describe the staph strip materials and methods
Look here:
http://microgenbioproducts.com/wp-
content/uploads/sites/8/2016/08/Microgen-Bioproducts-Fli
er-Staph-ID-LOW-REZ.pdf
http://www.tpm-
tpm.com.tw/downfile.php?file=products/145023165835.pdf
Gram stain (positive), catalase (positive) and latex
agglutination/coagulase tests are
performed as pre-tests on the isolate. Colony pigmentation is
recorded and then 1-2 colonies
are inoculated into the suspending medium. The suspension
is inoculated into the 12 well test
strip and incubated at 37°C. After 24 hours incubation the
strips are read and the Nitrate and
PYR reagents added. The resulting 5 digit numerical code is
entered in to the software and an
identi cation returned.
Blast search
The sequence data returned from the sequencing lab (add
trace file at the end - do not
include in page count) was trimmed of garbled ends and
compared by means of a BLAST

search to the database of prokaryotic 16S rRNA sequences
maintained by the National
Center for Biotechnology Information at the NIH.
Results
MSA plates
Pictures of the plates, each pic captioned with explanation of
which sample it is
List and describe the result of the staph strip
Electrophoresis
Photo of gel
Describe result
Blast search
The result ID links are for sequences 1 and 2 are,
respectively:
https://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Get&RID=0FDB
YFZ7014

https://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Get&RID=0FP3U
W8P014
The BLAST search returned 100% sequence identity over the
longest fragment to two
sequences from Staphylococcus epidermis. Other sequences
with 100% identity included S.
caprae, S. capitis, S. saccharolyticus. It returned a 99%
identity with S. aureus for fragment 1,
and 100% with fragment 2, both over shorter lengths. The hit
tables are available in
spreadsheet format (attached)
Discussion
While S. epidermis scores at the top, and 16S rRNA is highly
conserved, there is perhaps not
a very high level of confidence in the difference between
100% and 99% identical.
Nevertheless, the data is most consistent with the
identification of S. epidermis.
References
BLAST PROGRAMS. Altschul, S.F., Gish, W., Miller, W.,
Myers, E.W. & Lipman, D.J. (1990) "Basic
local alignment search tool." J. Mol. Biol. 215:403-410.
PubMed.
Proper citation for these if end up using them:

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content/uploads/sites/8/2016/08/Microgen-Bioproducts-Fli
er-Staph-ID-LOW-REZ.pdf
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Introduction
Staphylococci are gram-positive cocci (bacteria) typically about
1µm in size and grow in clumps, pairs, or occasionally short
chains. The staphylococci genus is home to around 40 different
strains of the bacteria, some being extremely common and
harmless relative to humans while others are much less present
and can cause serious infections when penetrating the barriers
of the human body(Staphylococci,2017). Staphylococci
epidermidis is one of these common types of staphylococci that
is part of the normal human flora and is typically found on the
skin and slightly less commonly in mucosal tissue due to it’s
ability to withstand higher salt concentrations. It typically
forms colonies 1-2mm in diameter and will test positive in the
catalase test, but negative in a coagulase one. Another
characteristic of this strain is that it is a facultative anaerobe,
meaning that it can grow utilizing both aerobic respiration and
fermentation. Being that S. epidermidis is typically found on
humans, it does not normally pose a threat. However, out of all
coagulase-negative staphylococci, s. epidermidis cause the most
amount of infections, typically through the use of medical
instruments, prosthetics, and catheters. This is due to the fact
that there is a vast amount of s. epidermidis on the skin and can
be moved into the organism through the instruments, posing
threat for infection(Otto, 2009). . Another common coagulase
negative staph is S saprophyticus. It is normally found in the
flora of the female genital region and inside the gastrointestinal

tract. S saprophyticus does not normally pose a threat but is
contributed to causing around 10-20% of urinary tract infections
in females. S. Aureus is a strain less common but still found on
the skin, in the nose, and the respiratory tract as it is also
tolerant to higher salt concetrations. It is a coagulase positive
strain, the only other coagulase positive staphylococci being s.
intermedius. Although common and can work commensally in
the body, S aureus is the main cause of staph infections and
poses many virulent factors. A main threat of s. aureus is that it
has gained the ability to become resistant to methicillin
antibiotics and is the bacteria responsible for MRSA
(methicillin-resistant S aureus). S aureus is responsible for
superficial skin lesions, deep-seated infections, food poisoning
due to the release of enterotoxins, and toxic shock syndrome by
releasing of super antigens into the bloodstream. Other common
staphylococci include S lugdunensis, S haemolyticus, S warneri,
S schleiferi, S intermedius but they are rare pathogens
(Kobayashi, 2015).
In order to identify and classify bacteria, physiological
characteristics of the bacteria must be determined. Through the
use of multi-test media, different physiological characteristics
can be observed. MSA plates were used to determine that the
strains taken from the nose and bottom of one show were not S
aureus, as they did not ferment the mannitol and would not turn
yellow.
16s rRNA is approximately 1500 base pairs and is used as the
main method for phylogentic purposes when it comes to
identifying bacteria. This method is used due to the fact that the
16s rRNA sequence is highly conserved between different
species of bacteria. The 16s rRNA subunit is a main component
in the 30s ribosomal complex in prokaryotes. The structural
importance of this gene is what has slowed the evolution of it
and led to it’s conservation. Through the method of PCR, the
16s rRNA sequence can be replicated and sequenced to
determine the genus of staphylococci present. There are
different PCR methods that can be used to extract and replicate

the 16s rRNA sequence, the one used in this lab consisted of
using an 8F primer and 1492R primer to result in a rRNA
sequence of approximately 1484 base pairs, which can be
sequenced and compared to identify the genus of staphylococci
present (Janda,2007).
Our experiment attempted to determine the species of two
unknown staphylococci that were not S aureus using the method
of 16S rRNA sequencing and analyzing the sequence compared
to other bacterial species called BLAST® by NCBI . One strain
was taken from the nose and the other from the bottom of one
shoe. After evaluation, we concluded that both the strain from
the nose and the strain from the bottom of the shoe was S
epidermidis.
Materials and Methods
Staph Lab Part I-Obtaining Specimens
The two Staphylococcus specimens were obtained from our nose
and the bottom of our shoe. We obtained the samples by first
sterilely pipeting 5ml of m-staphylococcus broth into 3 x 15 ml
screw top cap tubes. The tubs were then inoculated separately
by using sterile swabs to first swab the inside of one of our
nose’s and inoculating the tube and then the bottomof one our
shoes. The swab was gently moved about the area of concern to
pick up bacteria, then placed in the tube with the m-
staphylococcus broth and moved around for a short period of
time to transfer the microbes. They were then incubated at 37°C
for one week. The m-staphylococcus broth consisted of using 20
grams of powder and mixing it with 80ml of water and vacuum
filtrating it using .2µm filter to sterilize.
Staph Lab Part II-Streaking MSA Plates
After the m-staphylococcus broths had been incubating at 37°C
for a week, Mannitol Salt Agar plates were streaked using each
specimen. The bacteria growing in the tubes were transferred to
its own MSA plate through the use of a sterilized swab. The
plates were then incubated at 37°C. After a week of incubation,
the bacteria growing on the plates were first analyzed to

conclude that they were not S aureus by noting that the
specimens did not ferment the mannitol and no color change to
yellow agar was observed. Then, one isolated colony from each
plate was transferred from the MSA plate to its own MSA plate
using an inoculating loop. These were then also incubated at
37°C.
Staph Lab Part III-Gram Stain and 16s rRNA PCR
The bacterium on the MSA plates were Gram –stained and
observed under a microscope to securely indentify that they
were Gram-positive Cocci. The procedure of the Gram-Stain and
result were comparable to other research of such strains, in that
the bacteria resulted in clustering, purple, cocci.( link
1,Link2).Bacterium from each plate were then used to inoculate
a Microgen Staph ID test strip using an inoculating needle.
These were then incubated at 37°C and examined the next week
to gain an ID to match with the sequencing performed in the
next step.
16s rRNA sequencing was performed by first putting
.5ml of sterile saline in two separate centrifuge tubes and
labeling them. An inoculating loop was then used to transfer a
miniscule amount of each bacterium into each of the centrifuge
tubes, using separate tubes for each of the different samples. A
50ml PCR reaction was then set up using the following
concentrations of the additives:The components, once in the
tubes, were mixed by pipeting up and down. The tubes were
placed in a thermocycler that ran the denaturing, annealing, and
extension phases using the following cycle:This cycle was
repeated 40 times with a 5 minute 94°C step prior to the cycles
for first denaturation and a 7 minute 72°C step post cycles to
secure any other needed elongation. This provided us with a
theoretical yield of 240 copies of DNA.
Staph Lab Part IV
Cleaning Product
Since the PCR was run in part III, the mixture needs to be
cleaned up in order to be used for DNA sequencing. The
reaction contains unused primers and nucleotide, and a buffer

for PCR. The mixture is run through a small column, BioRad
Quantum Prep in this lab, which separates the molecules of he
contents by size. Resin beads inside the columns will trap the
smaller molecules such as the primers, nucleotides, and buffer
while allowing the large PCR product molecules to flow
through. This was done following the 7 step procedure
provided by BioRad; Step 1 was vortexing the column to
resuspend the resin making sure none was settled, Step 2 was
snapping off the tip and placing the whole column in a 2.0 ml
wash tube, we then spun the column for 1 minute in a
microcentrifuge at 735 x g. The column was then place in a
clean 1.5 ml collection tube to collect the cleaned sample as we
then added 50µl of the sample to the top of the column and spun
it for 2 minutes at 735 x g. The purified sample had then passed
through the resin beads and remained in the bottom of the 1.5ml
collection which was sent out to Macrogen,Inc in Maryland for
sequencing after it was tested for sound results through gel
electropherosis.
Gel Electropherosis
After the product was cleaned and was in the correct state to be
sequenced, we first ran the product on a gel electropherosis to
see if the PCR had been run successfully. It was expected that
all PCR products would be 1484 base pairs long, as the reverse
primer used was 1492R and the forward was 8F resulting in
1484 base pairs. Each cleaned DNA segment was run in 1%
agarose gel running in 1xTBE buffer and 10µL of the DNA was
used. The 1x TBE buffer consisted of 89 mM Tris base, 89 mM
Boric acid, and 2 mM EDTA. The stock was then diluted 10x to
make 500ml total volume. This was used with a 50ml gel that
was made using the following guidelines; .5g agarose was
weighed and put in a 25ml flask, 50ml 1xTBE buffer was added
and the agarose was then microwaved approximately 1 minute
until it melted. The hot agarose was then poured into a 50ml
blue cap tube and water was added to bring he volume back up
to 50ml. We then added 5µl of 10mg/ml ethidium bromide and
this was poured into the gel tray with an 8-well comb where it

was left to harden.
After the gel was made, the DNA had to be prepped to run on
the gel. This was done by placing 10 µl of the DNA in a 1.5ml
tube and had 2µl 6x gel loading buffer added to it. This then
allowed us to place it in the gel box and fill the box with 1x
TBE buffer until it was submerged , once the gel had hardened.
The gel was then loaded with our samples with a combined 5µl
of DNA size markers. The size markers used were BioRad EZ
Load 100 bp PCR Markers. These markers provide bands at
every 100bp between 100-3000bp, with thick bands at 1000 bp
and 3000 bp that contain triple the concentration. These markers
allow for a template to gauge the size of the DNA fragments in
which our products resulted in. The gel was hen run at 140V for
approximately 30 minutes to allow the fragments to move
through the gel. Once complete, the gel was viewed using an
ultraviolet transilluminator to see that our DNA fragments had
moved through the gel and using the markers we were able to
determine that the size of the segments were approximately
1484 base pairs. This indicated that our PCR had been run
successfully and could be sequenced. We prepared the sample
for sequencing by placing 15µl of the cleaned PCR DNA in a
tube labeled by specimen (shoe, nose) and sent to Macrogen,Inc.
in Rockville, Maryland to be sequenced.
Staph Lab Part I-Obtaining Specimens
The two Staphylococcus specimens were obtained from our nose
and the bottom of our shoe. We obtained the samples by first
sterilely pipeting 5ml of m-staphylococcus broth into 3 x 15 ml
screw top cap tubes. The tubs were then inoculated separately
by using sterile swabs to first swab the inside of one of our
nose’s and inoculating the tube and then the bottomof one our
shoes. The swab was gently moved about the area of concern to
pick up bacteria, then placed in the tube with the m-
staphylococcus broth and moved around for a short period of

time to transfer the microbes. They were then incubated at 37°C
for one week. The m-staphylococcus broth consisted of using 20
grams of powder and mixing it with 80ml of water and vacuum
filtrating it using .2µm filter to sterilize.
Staph Lab Part II-Streaking MSA Plates
After the m-staphylococcus broths had been incubating at 37°C
for a week, Mannitol Salt Agar plates were streaked using each
specimen. The bacteria growing in the tubes were transferred to
its own MSA plate through the use of a sterilized swab. The
plates were then incubated at 37°C. After a week of incubation,
the bacteria growing on the plates were first analyzed to
conclude that they were not S aureus by noting that the
specimens did not ferment the mannitol and no color change to
yellow agar was observed. Then, one isolated colony from each
plate was transferred from the MSA plate to its own MSA plate
using an inoculating loop. These were then also incubated at
37°C.
Staph Lab Part III-Gram Stain and 16s rRNA PCR
The bacterium on the MSA plates were Gram –stained and
observed under a microscope to securely indentify that they
were Gram-positive Cocci. The procedure of the Gram-Stain and
result were comparable to other research of such strains, in that
the bacteria resulted in clustering, purple, cocci.( (Namvar,
2014. Uconn,2017).Bacterium from each plate were then used to
inoculate a Microgen Staph ID test strip using an inoculating
needle. These were then incubated at 37°C and examined the
next week to gain an ID to match with the sequencing
performed in the next step.
16s rRNA sequencing was performed by first putting .5ml
of sterile saline in two separate centrifuge tubes and labeling
them. An inoculating loop was then used to transfer a miniscule
amount of each bacterium into each of the centrifuge tubes,
using separate tubes for each of the different samples. A 50ml
PCR reaction was then set up using the following concentrations
of the additives:

Reaction Component
Concentration in µl
2x SSO SYBR supermix
25
10µM 8F Primer
2
10µM 1492R primer
2
Water
17.5
DMSO
2.5
Bacteria colony in Saline
1
The components, once in the tubes, were mixed by pipeting up
and down. The tubes were placed in a thermocycler that ran the
denaturing, annealing, and extension phases using the following
cycle:
Cycle
Temperature(°C)°C)
Time(minutes)
Denature
94
0.5
Anneal
55
0.5
Elongation
72
2
This cycle was repeated 40 times with a 5 minute 94°C step
prior to the cycles for first denaturation and a 7 minute 72°C
step post cycles to secure any other needed elongation. This
provided us with a theoretical yield of 240 copies of DNA.
Staph Lab Part IV
Cleaning Product

Since the PCR was run in part III, the mixture needs to be
cleaned up in order to be used for DNA sequencing. The
reaction contains unused primers and nucleotide, and a buffer
for PCR. The mixture is run through a small column, BioRad
Quantum Prep in this lab, which separates the molecules of he
contents by size. Resin beads inside the columns will trap the
smaller molecules such as the primers, nucleotides, and buffer
while allowing the large PCR product molecules to flow
through. This was done following the 7 step procedure
provided by BioRad; Step 1 was vortexing the column to
resuspend the resin making sure none was settled, Step 2 was
snapping off the tip and placing the whole column in a 2.0 ml
wash tube, we then spun the column for 1 minute in a
microcentrifuge at 735 x g. The column was then place in a
clean 1.5 ml collection tube to collect the cleaned sample as we
then added 50µl of the sample to the top of the column and spun
it for 2 minutes at 735 x g. The purified sample had then passed
through the resin beads and remained in the bottom of the 1.5ml
collection which was sent out to Macrogen,Inc in Maryland for
sequencing after it was tested for sound results through gel
electropherosis.
Gel Electropherosis
After the product was cleaned and was in the correct state to be
sequenced, we first ran the product on a gel electropherosis to
see if the PCR had been run successfully. It was expected that
all PCR products would be 1484 base pairs long, as the reverse
primer used was 1492R and the forward was 8F resulting in
1484 base pairs. Each cleaned DNA segment was run in 1%
agarose gel running in 1xTBE buffer and 10µL of the DNA was
used. The 1x TBE buffer consisted of 89 mM Tris base, 89 mM
Boric acid, and 2 mM EDTA. The stock was then diluted 10x to
make 500ml total volume. This was used with a 50ml gel that
was made using the following guidelines; .5g agarose was
weighed and put in a 25ml flask, 50ml 1xTBE buffer was added
and the agarose was then microwaved approximately 1 minute
until it melted. The hot agarose was then poured into a 50ml

blue cap tube and water was added to bring he volume back up
to 50ml. We then added 5µl of 10mg/ml ethidium bromide and
this was poured into the gel tray with an 8-well comb where it
was left to harden.
After the gel was made, the DNA had to be prepped to run on
the gel. This was done by placing 10 µl of the DNA in a 1.5ml
tube and had 2µl 6x gel loading buffer added to it. This then
allowed us to place it in the gel box and fill the box with 1x
TBE buffer until it was submerged , once the gel had hardened.
The gel was then loaded with our samples with a combined 5µl
of DNA size markers. The size markers used were BioRad EZ
Load 100 bp PCR Markers. These markers provide bands at
every 100bp between 100-3000bp, with thick bands at 1000 bp
and 3000 bp that contain triple the concentration. These markers
allow for a template to gauge the size of the DNA fragments in
which our products resulted in. The gel was hen run at 140V for
approximately 30 minutes to allow the fragments to move
through the gel. Once complete, the gel was viewed using an
ultraviolet transilluminator to see that our DNA fragments had
moved through the gel and using the markers we were able to
determine that the size of the segments were approximately
1484 base pairs. This indicated that our PCR had been run
successfully and could be sequenced. We prepared the sample
for sequencing by placing 15µl of the cleaned PCR DNA in a
tube labeled by specimen (shoe, nose) and sent to Macrogen,Inc.
in Rockville, Maryland to be sequenced.
Kobayashi, S. D., Malachowa, N., & DeLeo, F. R. (2015).
Pathogenesis of Staphylococcus aureus Abscesses. The
American Journal of Pathology, 185(6), 1518–1527.
http://doi.org/10.1016/j.ajpath.2014.11.030
Janda, J. M., & Abbott, S. L. (2007). 16S rRNA Gene
Sequencing for Bacterial Identification in the Diagnostic
Laboratory: Pluses, Perils, and Pitfalls . Journal of Clinical
Microbiology, 45(9), 2761–2764.
http://doi.org/10.1128/JCM.01228-07

Namvar, A. E., Bastarahang, S., Abbasi, N., Ghehi, G. S.,
Farhadbakhtiarian, S., Arezi, P., … Chermahin, S. G. (2014).
Clinical characteristics of Staphylococcus epidermidis: a
systematic review. GMS Hygiene and Infection Control, 9(3),
Doc23. http://doi.org/10.3205/dgkh000243
(n.d.). Retrieved November 11, 2017, from
http://web.uconn.edu/mcbstaff/graf/Student%20presentations/S
%20epidermidis/sepidermidis.html
Otto, M. (2009). Staphylococcus epidermidis – the “accidental”
pathogen. Nature Reviews. Microbiology, 7(8), 555–567.
http://doi.org/10.1038/nrmicro2182
Staphylococcal Infections: MedlinePlus. (2017). Retrieved
November 9, 2017, from
https://medlineplus.gov/staphylococcalinfections.html
Introduction
Staphylococci are gram
-
positive cocci (bacteria) typically about 1µm in size and grow
in
clumps, pairs, or occasionally short chains. The staphylococci
genus is home to around 40
different strains of the bacteria, some being extremely common
and harmless r
elative to
humans while others are much less present and can cause
serious infections when penetrating
the b
arriers of the human body(Staphylococci,2017
).
Staphylococci epidermidis
is one of these

common types of staphylococci that is part of the normal human
flora an
d is typically found on
the skin and slightly less commonly in mucosal tissue due to it’s
ability to withstand higher salt
concentrations. It typically forms colonies 1
-
2mm in diameter and will test positive in the
catalase test, but negative in a coagulas
e one. Another characteristic of this strain is that it is a
facultative anaerobe, meaning that it can grow utilizing both
aerobic respiration and
fermentation. Being that
S. epidermidis
is typically found on humans, it does not normally pose
a threat. How
ever, out of all coagulase
-
negative staphylococci,
s. epidermidis
cause the most
amount of infections, typically through the use of medical
instruments, prosthetics, and
catheters. This is due to the fact that there is a vast amount of
s. epidermidis
on th
e skin and
can be moved into the organism through the instruments, posing
threat for infection
(Otto,
2009)
. . Another common coagulase negative staph is
S saprophyticus
. It is normally found in
the flora of the female genital region and inside the
gastrointestinal tra

ct.
S saprophyticus
does
not normally pose a threat but is contributed to causing around
10
-
20% of urinary tract
infections in females.
S. Aureus
is a strain less common but still found on the skin, in the nose,
and the respiratory tract as it is also tol
erant to higher salt concetrations. It is a coagulase
positive strain, the only other coagulase positive staphylococci
being
s. intermedius.
Although
common and can work commensally in the body,
S aureus
is the main cause of staph infections
and poses many
virulent factors. A main threat of
s. aureus
is that it has gained the ability to
become resistant to methicillin antibiotics and is the bacteria
responsible for MRSA (methicillin
-
resistant
S aureus
).
S aureus
is responsible for superficial skin lesions,
deep
-
seated infections,

food poisoning due to the release of enterotoxins, and toxic
shock syndrome by releasing of
super antigens into the bloodstream. Other common
staphylococci include
S lugdunensis, S
haemolyticus, S warneri, S schleiferi, S intermediu
s
but they are rare pathogens
(
Kobayashi,
201
5)
.
In order to identify and classify bacteria, physiological
characteristics of the bacteria
must be determined. Through the use of multi
-
test media, different physiological
characteristics can be observed. MSA plates were used
to determine that the strains taken from
the nose and bottom of one show were not
S aureus
, as they did not ferment the mannitol and
would not turn yellow.
16s rRNA is approximately 1500 base pairs and is used as the
main method for phylogentic
purposes wh
en it comes to identifying bacteria. This method is used due to
the fact that the 16s
rRNA sequence is highly conserved between different species of
bacteria. The 16s rRNA subunit
is a main component in the 30s ribosomal complex in

prokaryotes. The structu
ral importance of
this gene is what has slowed the evolution of it and led to it’s
conservation. Through the
method of PCR, the 16s rRNA sequence can be replicated and
sequenced to determine the
genus of staphylococci present. There are different PCR metho
ds that can be used to extract
and replicate the 16s rRNA sequence, the one used in this lab
consisted of using an 8F primer
STAPH LAB REPORTS
Microbiology 311L – Fall 2017
first draft due in lab 7 & 9 November
final copy due Monday 20 November
You and your lab partner are producing and handing in a JOINT
lab report for the Staph lab. You either both swim or you both
sink. The format and grading scheme are described below.
Realistically, you’re probably looking at 7-10 pages – maybe
more.
Remember that you’re telling a story to a reader: what you did,
why you did it and what you found. You’re telling the story in
a way so that readers can interpret your data, come to their own
conclusions, and if they want, have enough information to try
and reproduce your experiments.
Please DO NOT INCLUDE the mixed Gram (+) and Gram (-)
culture from the lab exercise or the PDFs of the DNA
sequencing chromatograms.
TITLE and AUTHORS
· do this on a separate page – each of the other sections don’t
have to start on separate pages

· come up with a descriptive title telling the reader what you did
in your project
· list the names of the people who did the work – you decide on
the order of the names
· Abstract: none needed for this report
INTRODUCTION
· Write some background of what is known about the
Staphylococci which live on / infect humans and exist otherwise
in the environment. This doesn’t need to be an exhaustive list
with what they all are or do. Which ones are the big players in
our normal flora / normal environment and which ones cause
some of the major problems?
· Talk briefly about the idea of multi-test media and about 16S
rRNA sequencing for identifying bacteria. Google 16S and
you’ll hit some good information.
· Typically the last paragraph of an introduction says something
like “In the present study, we attempted to…..” or “The goals of
these studies were to…..” and then something like “In our
studies, we found that…..”
MATERIALS AND METHODS
· Divide this section up into subheadings for each of your major
techniques. Information you want to have in here includes:

- how/where you collected samples and how you plated them
- how you did Gram staining or tests like coagulase (reference
these – not to my labs
but to real publications!)
- which sample was inoculated into the Staph strip and how you
finished the tests for the strip
- which sample was put into the PCR, how you set up the PCR
reactions and how were they cycled
- how you cleaned up your DNA with the spin columns
- how you did a gel (here’s how to say it – modify it a bit for
reactio
bromide in 1x Tris-borate-EDTA buffer (TBE). DNA size
markers (whatever we actually use) were run as standards. The
gel was run at 125V in 1x TBE for approximately 45 minutes.
PCR products were visualized and photographed on a UV
transilluminator). “Hyperladder” is the brand name of a
particular set of DNA gel size markers. We don’t use it so
please just refer to them as markers
- how DNA sequence analysis was done (see lab 8)
- you can include a flow chart if it will help to explain things
(call it a Figure)
- all the info for tests and molecular stuff is in your labs!
FIGURES / TABLES

· Make your figures and tables FIRST. They will be the basis
for writing the rest of your paper.
· You could include any pictures (plates / Staph strips); a table
of Staph strip results; a screen shot of your DNA sequence
analysis; further sequence analysis (% identity etc)
· Keep the figures simple. Massive tables of numbers are a
pretty sure way to loose the attention of a reader. You can use
color to some extent, but remember that most journals are still
in paper format and are published in black and white.
· If you show tables, make sure to provide column and row
headings.
· Make sure to have a figure legend for each figure / table.
Give a brief description of what the reader is seeing for each
and briefly tell what was done. You can embed them in the text
right next to the figure or you can put the figures and their
legends in a separate section of the document.
RESULTS
· Walk the reader through your figures and make sure to refer to
the figure/table numbers as you go. Assume they really don’t
know much about what you did and use the results to tell them
about it - think about some freshman bio major reading this, not
Dr.O.
· Stick to the facts in this section but don’t interpret the
findings here. That’s the purpose of your discussion. Lead the
reader from one figure to the next in some logical order with
some transitions between the figures in your text.
· If you didn’t get a PCR product from which to obtain DNA
sequence, still show your gel (markers with sizes indicated) and
just state what you were trying to do but that you didn’t get a
product and so no DNA sequence is available.
DISCUSSION
· This is where you can discuss your interpretations of your

results. Were your results in line with what is known at
Staphylococci in general and about the particular ones you
identified? Were your unknowns one that your would expect to
find in the source (nose, throat, cell phone etc) you got them
from? If not, why not???
· Here’s a good place to talk about some reasons why you might
not have gotten a PCR product but what information you could
have gotten from it (ie how does 16S rRNA sequence allow you
to determine genus/species?)
· Often, the first paragraph describes again what you were
trying to do and an overview of what you found.
REFERENCES / LITERATURE CITED
· You need to have at least 5 references IN ADDITION TO the
textbook.
· ONLY 2 WEBSITES CAN BE CITED as sources!
· WIKI is NOT an acceptable reference for a lab report. It is,
however, a good place to get started for some basic information.
Google Scholar and PubMed are great places to look for real
published information.
· Each scientific journal can have its own format for references.
Let’s all use this one from the Journal of Cellular Biochemistry
for citations from journals:
Owen TA, Aronow M, Shalhoub V, Barone LM, Wilming L,
Tassinari MS, Kennedy MB, Pockwinse S, Lian JB, Stein GS.
1990. Progressive development of the rat osteoblast phenotype
in vitro: reciprocal relationships in expression of genes
associated with osteoblast proliferation and differentiation
during formation of the bone extracellular matrix. J Cell Physiol
143:420-430.
or this one for citations from books:

Owen, T. A., J. Holthuis, V. Shalhoub, E. Markose, M. Aronow,
J. B. Lian, and G. S. Stein. 1990. Evidence for a functional
relationship between proliferation and initiation of osteoblast
phenotype development. in Calcium Regulation and Bone
Metabolism. D. V. Cohn, F. H. Glorieux, and T. J. Martin, eds.
Elsvier, Amsterdam, the Netherlands. pp. 371-376.
GRADING
· This report is worth 20% of your lab grade (ie 10% of course
grade)
-- 7% of your lab grade for the first draft
-- 13% of your lab grade for the final version
· Total points (100 possible) will be determined as follows for
EACH version:
(see the separate grading sheet for a more detailed break down
of points)
Possible Points
Appropriate title, names
10
Introduction
20
20
Figures and Results

20
Discussion
20
References (number, validity, format)
10
PRACTICAL STUFF
· I’m not counting pages, but am looking for you to do a nice
job telling your story.
· Please double space and use 11 or 12 point font. I’m old…….
· TAKE THE TIME TO PROOFREAD!!! In this age of spell
and grammar check, there’s absolutely not excuse for typos and
sentences that aren’t sentences. I will dock you points for this
stuff!
· If there are journal articles you find in PubMed that you would
like to use as sources but our library doesn’t have access to
them, please send me the PubMed ID number (PMID) and I’ll
try to get you the pdf file.
Micro 311L – Staph Lab format Fall 2017
Page 4

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