BS 244 Microbial Science Laboratory

Fall 2019

Module III Handbook

Table of Contents:

Lab 19 Overview of antimicrobial testing, set up Kirby Bauer, and synergy experiments……….….3

Lab 20 Results of KB, Synergy, set up E strip.……………………...….….………………...….……..16

Lab 21 E strip results, set up MIC, Introduction to 16S rDNA, Sanger Sequencing, Bioinformatics
and BLAST.………………...……………..……………………..……………………………………….19

Lab 22 MIC results, discuss MBS, set up MBC, set up multiplex PCR w isolates and known
resistant strains………………………………………………………………………………………….26

Lab 23 MBC results, Run gel of multiplex PCR, and BLAST…………………….………………….32

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 Lab 19
Overview of antimicrobial testing
Set up Kirby Bauer
D test and Synergy experiments
Overview of antimicrobial testing, Kirby Bauer AST, D Test and Synergy Experiments
Is there an abundance of antimicrobial resistant bacteria colonizing our restrooms? We will use the
bacteria isolated in the previous module to investigate this question by identifying if they exhibit resistant
phenotypes or encode known resistance genes in their DNA.

REMEMBER ANTIMICROBIALS AND RESISTANCE WERE ALSO DISCUSSED DURING

MODULE I

Antimicrobial resistant organisms can be spread through the environment in clinical settings (termed
nosocomial or hospital-acquired/associated infections) or non-clinical settings (community acquired
infections). There are a number of methods for testing bacteria for antimicrobial resistance in bacteria,
both phenotypically and genotypically. Phenotypic antimicrobial assays include qualitative and
quantitative or semi-quantitative methods whose results can be applied clinically for patient treatment.

ANTIMICROBIAL SENSITIVITY TESTING (AST)

The spread of antimicrobial resistance is a major clinical problem. The emergence of multiple drug
resistant (MDR) microbes requires constant vigilance through AST and the continual development of new
antimicrobials.
In the clinical laboratory, antimicrobial sensitivity may be determined by:
• Agar Diffusion Methods such as the Kirby-Bauer Technique or E-Test
● Kirby-Bauer (K-B) Technique is a semi-quantitative method via disk diffusion
● E-Test (Ellipse Test) is a quantitative method via strip diffusion

• Tube Dilution Tests - the determination of Minimum Inhibitory Concentration (MIC)

• Special Tests may sometimes be performed to assess mechanisms of resistance to antimicrobials, such
as PCR

All of these methodologies return a qualitative result, such as susceptible, resistant or intermediate
susceptible to a specific antibiotic.

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KIRBY-BAUER (K-B) TECHNIQUE AND INTERPRETATION
In the Kirby-Bauer Disk-Agar Diffusion Method, Mueller Hinton Agar (MHA) in a Petri dish is the
universally used medium. The MHA medium supports relatively good and reproducible growth by many
microbial species, and the MHA medium’s formula is relatively simple. To begin the (K-B) Method the
laboratorian inoculates a sample from a pure culture of the microorganism in question onto the surface of
the MHA plate. Ultimately, this inoculum should produce a pattern of heavy growth known as a
“Bacterial Lawn”.
Before incubation of the MHA plate, the laboratorian places commercially available paper disks, each
impregnated with a measured amount of a specific antimicrobial, on to the inoculated-plate surface. The
commercial antimicrobial disks are color coded and / or lettered for identification. Then the inoculated
MHA plate containing the antimicrobial disks incubates for a standard time at a standard temperature.
After the plate’s incubation, zones of inhibition around the disks in the growth of the “Bacterial Lawn” of
the microbe must be carefully examined. If an antimicrobial is able to inhibit microbial growth, a zone of
clearing (no growth - known as a Zone of Inhibition) appears around the disk. See Figure 1.
Any AST must only be performed on a pure culture. Remember, a pure culture is a plate or broth
growing only one organism.

Figure 1 Measuring the zone of inhibitions

The diameters of the observed zones of inhibition are measured in millimeters. Comparisons of inhibition
zone values to standard values in clinical correlation tables determine if the microbe is susceptible or
resistant to the action of a drug. The size of the zone cannot be used on its own, different antimicrobials
will have different sized zones which correlate to susceptibility or resistance.
The (K-B) Method is a Standardized Procedure. This means that any laboratory that uses this
procedure can compare results with other laboratory facilities. This is an important concept when
tracking the susceptibility pattern of an infectious bacteria by public health authorities such as the
Philadelphia Health Department, the Pennsylvania Public Health Department Bureau of Laboratories, or
the Centers for Disease Control and Prevention.

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K-B is a semi-quantitative test. The starting concentration, in µg or U (units), of each antimicrobial is
stamped on the disk. The antimicrobial diffuses out of the disk and into the agar becoming more dilute.
The longer the radius is from the center of the disk, the more dilute the antimicrobial concentration is.
This dilute concentration of the antimicrobial in the agar is unknown and results the minimum inhibitory
concentration of antimicrobial cannot be directly interpreted. The measurement of the zone of inhibition,
in mm, is compared to a chart used to interpret the zones of inhibition as Resistant, Intermediate, or
Susceptible.

Factors Affecting the Size of the Zone of Inhibition on Kirby-Bauer MHA Plates:
1. Composition of the Medium: The Mg++ and Ca++ content of the medium can affect the diffusion
of antimicrobials (e.g. Gentamicin) and can lower the zonal concentrations of the antimicrobial.
2. Antimicrobial Disk Content: A greater antimicrobial content in a disk usually produces a larger
zone of inhibition. However, beyond a certain amount, adding to the chemical content of a disk
does not increase zone size.
3. Diffusibility of Antimicrobial: Some antimicrobials such as Vancomycin do not diffuse readily
from the disk and thus, zone diameters are extremely small even for susceptible organisms.
4. Stability of Antimicrobial: Ampicillin is subject to spontaneous hydrolysis during incubation at
37oC. If the concentration of the antimicrobial diminishes sufficiently during the early part of
incubation, then a few organisms may survive and multiply to produce colonies making the zone
size smaller.
5. Bacterial Enzymes: Some bacteria have enzymes that inactivate certain antimicrobials, e.g., β-
lactamase containing bacteria that cleave the β-Lactam Ring in Penicillin, thus affecting the zone
size.

The Kirby-Bauer is a semi-quantitative assessment of antimicrobial effect on the organism. The

size of zones between different antimicrobials should not be directly compared.
Example #1: There is a 20 mm zone around your penicillin disk. There is a 20 mm zone around
your neomycin disk. The bacteria is resistant to penicillin but susceptible to neomycin.

Example #2: There is a 14 mm zone for ampicillin and a 20 mm zone of inhibition around
tetracycline. This does NOT mean you should use tetracycline because the zone is larger and the
bacteria seems more susceptible to it.

Materials per group:

1. 1 Large Mueller Hinton Agar Plates
2. 4 Small Mueller Hinton Agar Plates
3. Sterile Swabs
4. 1 Broth Culture of known Bacteria (listed below)
5. Your TSA plate of unknown isolated bacteria

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6. 1 Sterile Mueller Hinton Broth in 15 mL conical tube

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 7. Ruler (for measuring zones in the next lab)
8. 95% Ethanol in beaker
9. Forceps
10. 8 Conical tube with the following antimicrobials:
a. Ceftriaxone
b. Ciprofloxacin
c. Rifampicin
d. Erythromycin
e. Oxacillin
f. Penicillin
g. Tetracycline
h. Vancomycin

Procedure for K-B Using Organism Growing in Broth:

1. Obtain a large MH plate. Label it as per lab protocol.
2. You will use one of the following known bacteria:
a. Proteus mirabilis (Groups 5 & 7)
b. Pseudomonas aeruginosa (Groups 6 & 8)
c. Escherichia coli (Groups 1 & 3)
d. Staphylococcus aureus (Groups 2 & 4)

2. Remove a sterile swab from its protective cover and swirl the tip in the broth culture of the
assigned, known, strain of bacteria (above).
3. Aseptically streak the surface of an MHA plate with this swab. Evenly coat the whole surface of
the plate and spread the bacteria in three different directions, as shown in the Figure 2 below and
as described in a-d.
a. First, inoculate plate by placing one streak down the center of the plate
b. Second, using the same swab, cross streak down the whole plate
c. Third, turn the plate 1/3 around, and streak from the top to the bottom
d. Last, turn the plated another 1/3 around, and streak from the bottom to the top

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 Figure 2 Plate Inoculation for Kirby Bauer Antimicrobial Testing

4. PLEASE DO NOT RETURN THE USED SWAB INTO ITS WRAPPER.

a. Dispose the swab into a red bench top sharps container.
b. The wrapper is discarded in regular trash.
5. Place the plate lid-down on top of the template, Figure 3 below. With a sharpie, mark a dot
where each dot is on the template (where each disk will be placed) on the bottom of the plate.
6. Flip your plate back in the lid-up position. Using sterile forceps, apply one disk of each
antimicrobial to the media on top of each dot.
Note: cover plate with lid between disk application.
7. After disks are placed, using sterile forceps, gently tap disks to ensure adherence to the surface of
the MHA plate. You need good contact between the disk and the agar surface.
8. Incubate the MHA plate at 35oC for 18-24 hours.

Procedure for K-B Using Organism Growing on a Plate:

1. Obtain a large MH plate and label it as per lab protocol
2. Obtain a MH broth tube
3. Remove a sterile swab from its protective cover and sweep a small sample of your organism off
of a TSA plate
4. Swish your swab in the broth, making sure all of the organism comes off the swab and is well
mixed into your MH broth. Discard your swab in the sharps bin.

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5. Using a new swab, aseptically streak the surface of an MHA plate with this swab. Evenly coat
the whole surface of the plate and spread the bacteria in three different directions, as shown in the
Figure 2 and as described in a-d.
a. First, inoculate plate by placing one streak down the center of the plate
b. Second, using the same swab, cross streak down the whole plate
c. Third, turn the plate 1/3 around, and streak from the top to the bottom
d. Last, turn the plated another 1/3 around, and streak from the bottom to the top
6. PLEASE DO NOT RETURN THE USED SWAB INTO ITS WRAPPER.
a. Dispose the swab into a red bench top sharps container.
b. The wrapper is discarded in regular trash.
7. Place the plate lid-down on top of the template, Figure 3. With a sharpie, mark a dot where each
dot is on the template (where each disk will be placed) on the bottom of the plate.
8. Flip your plate back in the lid-up position. Using sterile forceps, apply one disk of each
antimicrobial to the media on top of each dot.
Note: cover plate with lid between disk application.
9. After disks are placed, using sterile forceps, gently tap disks to ensure adherence to the surface of
the MHA plate. You need good contact between the disk and the agar surface.
10. Incubate of the MHA plate at 35oC for 18-24 hours.

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 Figure 3 K-B Disk Placement Template
Record your KB results below
Using the mm side of a ruler, measure the diameter of the zone of inhibition for each antimicrobial in
millimeters (refer to Figure 1). These zones are measured to include the disk diameter itself, which is 7
mm. For example, if you have growth of bacteria all the way up to your disk, you would record “7mm”
as your zone measurement. Compare your zone diameters measured to Table 1 below, “Antimicrobial
Interpretation Chart”, for each antimicrobial and indicate if your organisms (known and unknown) are
Resistant (R), Intermediate Susceptibility (I), or Susceptible (S). Record all of the results in the tables
provided. Use Table 2 to record the class’ known bacteria and Table 3 to record your isolated organism
zones and interpretations.

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Table 1 Antimicrobial Interpretation Chart

Final evaluation of zone size involves comparison with the interpretation chart that accompanies the disk.
The zones are interpreted as exhibiting the Resistant (R), Intermediate Susceptibility (I), or Susceptible
(S) quality.
A representative chart that lists eleven antimicrobials and their zone size interpretations is presented in
Table 1. Notice that an organism with a zone size of 14 mm when tested against Ampicillin is susceptible
(sensitive on the chart) but an organism with a zone size of 20 mm for Penicillin is resistant.
This type of clinical correlation data as shown in Table 1 is produced by organizations such as the
American Society for Microbiology - http://www.ascls.org/), (ASM - http://www.asm.org/), and the
Clinical Laboratory Standards Institute (CLSI - https://clsi.org/).
For an example of Zone Diameter and MIC Breakpoints from CLSI, click on the hyperlink below. Click
on Guest User, M100, and search for Table 2C (Staphylococcus spp.):
http://em100.edaptivedocs.info/GetDoc.aspx?doc=CLSI%20M100%20ED28:2018&sbssok=CLSI%20M
100%20ED28:2018%20TABLE%202C&format=HTML#CLSI%20M100%20ED28:2018%20TABLE%
202C

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 Resistant (R) ↔ Intermediate (I) ↔ Sensitivity (S)

Table 2 Results of Known Organisms; Use Table 1 above to interpret the susceptibility pattern for your
known organism

Unknown Organism
Antimicrobial Zone Diameter (mm) R/I/S
Ceftriaxone
Ciprofloxacin
Rifampicin
Erythromycin
Oxacillin
Penicillin
Tetracycline
Vancomycin
Table 3 Results of Unknown Organism; Use Table 1 above to interpret the susceptibility pattern for your
unknown organism:

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ANTIMICROBIAL SYNERGISM:
In some instances, physicians prescribe combinations of more than one antimicrobial for treatment of an
infectious disease. Some rationales for using Combination Therapy are:
• Effective combinations may reduce the incidence of bacterial resistance
• Effective combinations may demonstrate an enhanced bactericidal effect
• Effective combinations may exhibit reduced drug toxicity due to treatment with lower
individual doses of each drug
Synergism occurs when Combination Therapy produces enhanced antimicrobial activity. A synergistic
effect requires the following result be observed:
The sum of the effects of the drugs used in combination > The sum of their effects when used
individually
In this lab, we will examine the effect of Sulfisoxazole 250 μg and Trimethoprim 5 μg separately on the
growth of Staphylococcus aureus. We will compare these results to the synergistic effect of a combination
of lower doses of the same two drugs on the growth of Staphylococcus aureus. The lower dose
combination of the two drugs is a commonly used strategy in the treatment of infectious diseases.
Both of these antimicrobials are Competitive Inhibitors of different enzymes. Competitive inhibitors are
chemicals that compete with a substrate for binding to an enzyme’s active / catalytic site. When a
competitive inhibitor is bound by an enzyme, the native substrate cannot bind and the enzyme catalyzed
reaction does not occur. Competitive inhibitors do not alter the amino acid sequence of an enzyme, and
when the inhibitor releases from the enzyme, a molecule of substrate (or another inhibitor molecule) can
be bound. When a substrate molecule binds, the enzyme catalyzed reaction will occur. In order to be
effective antimicrobials that are competitive inhibitors, they must bind very tightly to their target enzyme
and thereby block the enzyme catalyzed reaction most of the time.
Each of these antimicrobials acts at a different step in the Tetrahydrofolic Acid Synthesis Pathway. Their
combined inhibitory effects deprive bacteria of reduced folates for use in anabolic metabolism (especially
in the biosynthesis of Thymidylate – a required precursor for DNA synthesis).

FIGURE 4 Sulfisoxazole & Trimethoprim Competitively Inhibit Successive Enzyme Catalyzed Steps for
Tetrahydrofolic Acid Biosynthesis

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Materials per group:
1. 8 Small Mueller-Hinton Agar Plates
2. 1 Tube of Staphylococcus aureus growing in MH broth
3. 1 MH broth in 15 mL conical tubes
4. Your unknown isolated bacteria
5. Sterile Swabs
6. Beaker with 95% Alcohol
7. Forceps
8. 4 Sulfisoxazole Disks 250 μg
9. 4 Trimethoprim Disks 5 μg
10. 2 Trimethoprim 1.25 μg / Sulfisoxazole 23.75 μg combination disk (SXT)
11. Ruler

Procedure #1 for Synergy Test Using S. aureus Growing in Broth:

1. Obtain four (4) small MH plates. Label them as per lab protocol and with the disks you will be
testing.
2. Remove a sterile swab from its protective cover and swirl the tip in the broth culture of
Staphylococcus aureus.
3. Inoculate a bacterial lawn onto each Mueller Hinton Agar (MHA) plate’s surface following K-B
protocol. (Note: Use a new swab between plates)
4. To one inoculated MHA plates using alcohol-dipped and dried tweezers, apply a Sulfisoxazole
paper disk to the center of the plate.
5. To a second inoculated MHA plate, using alcohol alcohol-dipped and dried tweezers, apply a
Trimethoprim paper disk to the center of the plate.
6. To a third MH plate, mark two dots 20 mm apart in the center of the media (back of plate) with a
sharpie. Using alcohol-dipped and dried tweezers, apply the inside edge of one Sulfisoxazole
paper disk plus inside edge of one Trimethoprim paper disk on to the media, using the dots as a
guide. See Figure 5.

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 Figure 5 Placement of Sulfisoxazole and Trimethoprim Disks

7. To a fourth inoculated MH plate, using alcohol-dipped and dried tweezers, apply a

Trimethoprim/Sulfisoxazole combination paper disk (SXT) to the center of the plate.
8. Incubate the plates overnight at 35oC.
9. In the next laboratory period, measure the zones of inhibition in mm around each disk using the
provided ruler.
10. Determine if the response of the Staphylococcus aureus strain to each antimicrobial (plates 1, 2,
and 4) indicates that the organism is susceptible or resistant.
11. Observe plate 3 for any signs of synergism between the Sulfisoxazole and Trimethoprim (see
Figure 6).

Procedure #2 for Synergy Test Using Unknown Organism Growing on a Plate:

1. Obtain four (4) small MH plates
2. Label your plates according to laboratory protocol, the disks you will be testing, and the organism
tested.
3. Using your sterile swab, sweep a small sample of your organism off of a TSA plate. Place the
swab into 2 mL of MH broth in a 15 mL conical tube and swirl around to get the organism off of
the swab.
4. Inoculate a bacterial lawn onto each Mueller Hinton Agar (MHA) plate’s surface following K-B
protocol. (Note: Use a new swab between plates)
5. To one inoculated MHA plate, using alcohol-dipped and dried tweezers, apply a Sulfisoxazole
paper disk to the center of the plate.
6. To a second inoculated MHA plate, using alcohol-dipped and dried tweezers, apply a
Trimethoprim paper disk to the center of the plate.

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 7. To a third MH plate, mark two dots 20 mm apart in the center of the media (back of plate) with a
sharpie. Using alcohol-dipped and dried tweezers, apply the inside edge of one Sulfisoxazole
paper disk plus inside edge of one Trimethoprim paper disk on to the media, using the dots as a
guide. See Figure 5 above.
8. To a fourth inoculated MH plate, place a Trimethoprim/Sulfisoxazole combination paper disk
(SXT) to the center of the plate
9. Incubate the plates overnight at 35oC.
10. In the next laboratory period, measure the zones of inhibition in mm around each disk using the
provided ruler.
11. Determine if the response of the unknown organism to each antimicrobial (plates 1, 2, and 4)
indicates that the organism is susceptible.
12. Observe plate 3 for any signs of synergism between the Sulfisoxazole and Trimethoprim (see
Figure 6).

Figure 6 Individual disks of Trimethoprim and Sulfasoxizole both produce zones of inhibition. Synergy
between the two antimicrobials is visualized via a bridging of the zones of inhibition
Observations:
ANTIMICROBIAL SUSCEPTIBLE RESPONSE IN mm FOR Staphylococcus aureus ZONE OF
INHIBITION

Table 4
Staphylococcus aureus breakpoints for Sulfisoxazole, Trimethoprim, Trimethoprim-Sulfasoxazole

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For S. aureus, what was the zone of inhibition for:
Sulfisoxazole (250 ug) ___________________
Trimethoprim (5 ug) ___________________
SXT (S-23.75 ug/T-1.25 ug) ___________________

Did you observe synergy on plate #3 (two disks)? What did it look like?

Unknown Organism

Table 5 Unknown Organism

Sulfisoxazole (250 ug) ___________________
Trimethoprim (5 ug) ___________________
SXT (S-23.75 ug/T-1.25 ug) ___________________

Did you observe synergy on the plate 3 (two disks)?

Sub your unknowns to a new TSA plate and TSB broth.

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 Lab 20
Results of K-B, Synergy and
Set up E-test strip
THE E-TEST:
The E-Test is another agar diffusion method for assaying a microbe’s antimicrobial susceptibility. Instead
of a disk, we use a strip (the E-Strip) that is impregnated with a gradient of an antimicrobial. This means
that one end of the strip has extremely low but precise amounts of the drug and the other end has higher
but precise amounts. When dissolved in the medium, these amounts generate a gradient of antimicrobial
concentrations, which are numerically marked on the strip.
An E- Test strip is placed on a plate just inoculated with a culture of the test bacterium. After incubation
and development of the bacterial lawn, the shape, in the form of an ellipse, of the resulting zone of
inhibition allows us to read the Minimum Inhibitory Concentration (MIC) directly from the plate. See
Figure 7 below. Thus the E-test is a quantitative test for antimicrobial susceptibility/resistance.
The lowest concentration of an antimicrobial that inhibits bacterial growth is the MIC.
The E-Test adapts the disk diffusion assay to rapidly and quantitatively assess the level of antimicrobial
resistance associated with a microbe. The E-Test also provides insight about a microbe’s evolving drug
resistance.

How does the E-Test allow clinicians to follow the emergence of drug resistant bacteria? Why is this
ability important? (Hint: answer is not listed in this Handbook. You must research this on your own.)

Figure 7
AB = Antimicrobial Code; White = Ellipse of Inhibition; Grey = Bacterial Growth;
Numbers on strip = Gradient Concentration Indication

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Materials:
2 small (100 mm) Mueller Hinton Agar Plates
Sterile Swab
E. coli Strain A or E. coli Strain B in TSB
Your unknown isolated bacteria
2 Ampicillin E-Strip
2 Ampicillin Disk (AM-10)
Beaker with 95% Alcohol
Forceps
Procedure:
1. Label your plates according to laboratory protocol, the E-test strips and disks you will be testing, and
the organisms tested.
2. Following K-B protocol, inoculate a lawn culture onto 1 Mueller Hinton Agar plate using your
assigned E. coli strain. Repeat with the other MHA plate using your isolated unknown bacteria.
3. Refer to Figure 8 below when following the remainder of this procedure.
4. Use alcohol-dipped and dried forceps to remove an E-test strip from the covering. Carefully transfer
the E-test strip to your dominant gloved hand, touching ONLY the end with the “E” written on it.
Carefully place each E-test strip on to the right side of the MH plate. Gently tap it into place with
alcohol-dipped and dried forceps.
a. Be sure to keep the number side of the strip facing up.
b. To avoid kinks or wrinkles in your strip, attempt to anchor the bottom end of the strip against
the media and roll the strip down into place.
c. DO NOT MOVE THE STRIPS AFTER INITIAL APPLICATION.
(Watch this video: https://www.youtube.com/watch?v=Ij7_ffiKJMY)
5. Use alcohol-dipped and dried tweezers to apply one Ampicillin (AM -10) paper disk on the left side
of the MH plates for each organism.
6. Incubate all plates overnight at 35oC.
7. In the next laboratory period, observe and record the MIC value indicated by the E-Test Strips. Using
Table 9, determine if each MIC indicates a susceptible, intermediate, or resistant response for the
tested antimicrobial.
8. Measure the zones of inhibition for the Ampicillin disk. Using Table 9, determine if the organism is
susceptible, intermediate, or resistant. Record results in table 10.

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 Table 9 Zone Diameter and MIC Breakpoints used to interpret E. coli and your organism
Source CLSI M100 2018

Figure 8 Place the E-test strip on the right side of the agar plate. Leave about 1 inch of space between
the strip and the right edge of the plate. Place a disk on the left side of the agar plate. Leave about 1 inch
of space between the disk and the left edge of the plate.

Results:
Measure the zone of inhibition (in mm) for each disk, the MIC for each strip, and record the following:

AM-10 zone Susceptible or AMP E Strip MIC Susceptible or

(mm) Resistant Resistant
E. coli A
E. coli B
Unknown
Organism
Table 10

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If you had two patients, one infected with E. coli strain A, and one with E. coli strain B, who will you be
able to treat with Ampicillin?

Is your isolated organism ampicillin resistant or susceptible?

Sub your unknowns to a new TSA plate

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 Lab 21
Results of E-Test Strip
Set Up Serial Dilution MIC
THE MINIMUM INHIBITORY CONCENTRATION TEST:
To perform this test, bacteria is inoculated in multiple serial dilutions of an antimicrobial in Tryptic Soy
Broth (TSB). After incubation, the tubes are observed for bacterial growth. The concentration of
antimicrobial in the first tube that shows no growth is the Minimum Inhibitory Concentration (MIC) for
that specific antimicrobial against that specific bacterium. The MIC provides a Quantitative Assessment
of antimicrobial susceptibilities. In the clinical laboratory, this procedure is automated.

Materials:
18 Empty Sterile Eppendorf Tubes
Colored Tape
2 Tubes of Sterile Mueller Hinton Broth (MHB)
2 Tubes Containing 5.0 mL Sterile Saline
2 Tubes Containing 9.9 mL Sterile Saline
2 Tubes Containing Ampicillin at a Concentration of 128 μg/mL
Several 1.0 mL Volumetric Pipettes
Blue Pi Pump
E. coli Strain A or E. coli Strain B in TSB
Your unknown isolated bacteria

Procedure: *Correctly sketch out this procedure for 0.25 extra point to your quiz points. Due at
the start of this lab. No late submissions will be accepted. Must include Name, Section# and
Procedure Name or assignment will not be accepted.
1. Line the tubes up into 2 sets of 9.
a. Set #1: E coli A or B
b. Set #2: Unknown Organism
2. Label the tubes 1-9
3. Add 0.5 mL of sterile MHB to each tube
4. Preparation of Antimicrobial
a. Add 0.5 mL of 128 μg/mL Ampicillin to the first tube only.
b. Mix the contents thoroughly
c. Transfer 0.5 mL from this tube into the second tube.
d. Mix the contents of the second tube and transfer 0.5 mL to the third tube.
e. Continue this dilution process through tube number 7.

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 f. Discard 0.5 mL of broth from tube 7 into the waste beaker so that the final volume in all
tubes is 0.5 mL.
5. Preparation of Bacteria
a. Using an overnight culture of either Escherichia coli Strain A, or Escherichia coli Strain
B (set #1), or your unknown (set #2), prepare a slightly turbid suspension of the bacteria
by transferring 0.1 mL of the culture into a tube containing 5 mL saline.
b. Transfer 0.1 mL of the bacterial suspension into another tube containing 9.9 mL saline
(now known as Dilute Inoculum).
c. Mix the contents thoroughly and transfer 0.1 mL of the Dilute Inoculum to tubes 1-8.
d. Mix all tubes thoroughly and incubate the tubes for 24 hours
e. After incubation, keep these tubes for MBC evaluation
Tubes 8 and 9 serve as our controls. Tube 8 has no antimicrobial added, only bacteria. Therefore, it
serves as a Growth Control. Tube 9 has neither antimicrobial, nor bacteria. Therefore, it serves as a
Sterility Control.
RESULT

Tube Tube Tube Tube Tube Tube Tube Tube Tube

1 2 3 4 5 6 7 8 9
Concentration 64 32 16 8 4 2 1 Growth Sterility
of AMP Control Control
(ug/mL)
E. coli A
E. coli B
Unk. Organism
Table 11 Record MIC here

Determination of the MIC of ampicillin against the bacterial strains:

E. coli Strain A MIC _________________
E. coli Strain B MIC _________________
Unknown Organism MIC _________________

Use Table 9 to determine the interpretation of the E coli and your unknown against Ampicillin
E. coli Strain A MIC Interpretation _________________
E. coli Strain B MIC Interpretation _________________
Unknown Organism MIC Interpretation _________________
Does the Serial Dilution Tube MIC Method result match the E-test MIC results?

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Thinking about both the serial dilution MIC and the E-test MIC, which test is more reliable and why?
Sub your unknowns to TSA.

Introduction to 16S rDNA, Sanger Sequencing, Bioinformatics and BLAST

In bacteriology, the 16S rDNA is sequenced to identify the unknown organism. The portion of DNA that
codes for 16S rRNA portion of the ribosomes is a very conserved region. This means that between
bacterial species, this portion of the DNA does not change all that much. Sequencing this portion of the
DNA is a reliable method of identification.

Adapted from https://scholarblogs.emory.edu/fankhauserlab/2017/06/28/soil-microbes-and-food/16s-

rdna/

Adapted from http://faculty.samford.edu/~djohnso2/44962w/405/_06translation.html

Sanger Sequencing, or chain-termination sequencing, is a cheap and reliable way to sequence 16S rDNA
in an attempt to identify an unknown bacterial organism. The goal of chain-termination sequencing is to
learn the proper sequence of nucleic acids in a portion of DNA. Chain-termination sequencing is not
meant to sequence whole genomes due to the limitations of the chemistry of the PCR reaction. A more
powerful technique, such as Next Generation Sequencing (NGS) is a more sophisticated way of
sequencing both small and large segments of DNA. We will not be covering NGS in this laboratory
portion of the course.
Chain-termination sequencing DNA extraction and PCR reactions uses the routine methods and chemical
mixture of DNA template, forward and reverse primers, buffer, water, Mg++, DNA Taq-polymerase, and

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4-dNTPs (dATP, dGTP, dCTP, and dTTP) (refer to Module I). What makes this PCR reaction mixture
special is the addition of fluorescently-labeled ddNTPs (ddATP, ddGTP, ddCTP, ddTTP). Each type of
nucleic acid ddNTP will possess a unique fluorescent color. The fluorescently-labeled ddNTPs do not
have an -OH group on the ribose that is needed for chain extension. The absence of the -OH is what
makes the extension of the growing chain terminate (see figure below).

https://www.quora.com/In-terms-of-biology-what-is-the-main-and-most-important-difference-between-ddNTP-and-dNTP

After ~30 cycles, the PCR process is complete. The amplicon needs to be cleaned up to get rid of the
excess fluorescently-labeled ddNTPs. The process uses a column of hollow beads (think about a bead that
has a hole in the center where you can thread a string through) that traps the fluorescently-labeled
ddNTPs, but allows for the strands of new amplicon to flow in between the beads and out of the column.
The liquid captured in the bottom of the column catch tube is used for sequencing analysis.

When the PCR reaction is allowed to run as usual, there will be several lengths of amplicon (DNA
copies), each with one fluorescently-labeled ddNTP on the end. The shortest length is considered to be the
first nucleic acid in the target sequence, the longest length is considered to be the last nucleic acid
sequence, and then there are lengths of every size in between.

23
 Adapted from http://oregonstate.edu/instruct/bb331/lecture05/FigG4.html

A genetic analyzer (https://www.thermofisher.com/order/catalog/product/4359571) that has a laser and an

“eye” to read the fluorescently-labeled DNA strands is used to “read” the sequence. The sequencing
machine is just like PAGE that we used to visualize bands (see Module I), except the acrylamide gel used
is not set, but rather it is in a liquid state and within capillary tubes. The fluorescently-labeled DNA
strands flow through the acrylamide gel within the capillary tubes using an electrical current (remember
which way DNA will flow to when exposed to (+) and (-) current?) A portion of the tubes are unsheathed
to allow the fluorescently-labeled DNA strands to be excited by the laser and then to be read by the “eye”.
A computer captures this information and processes it into a way in which humans can understand - a
electropherogram of DNA sequence of A, T, G, and C patterns.

https://bitesizebio.com/27985/sanger-sequencing-genome-won/
An electropherogram is the raw data captured from the genetic analyzer. The peaks are the strengths at which each fluorescently-
labeled nucleic acid registers. Reading an electropherogram is a useful way to ensure your sample is pure. If this sample were
mixed with a contaminating bacteria, you would be able to see more than one peak at each “location”. (ex. 120 - 130 nucleic acid
# in the sequence).

24
 Adapted from http://oregonstate.edu/instruct/bb331/lecture05/FigG4.html
This is a more simplistic way of thinking about chain-terminating sequencing. The shortest strand is seen at the bottom (the first
nucleic acid in the sequence), while you can see the progressive “building” of the DNA strand as each progressively longer
nucleic acid strand goes through the capillaries one by one and are read in that order.

Bioinformatics is an area of science that combines sequencing data, molecular data, computer databases,
and programming algorithms (such as R and MatLab) to solve complex biological problems at the
molecular level. There are many different uses for bioinformatics such as: drug design and development,
nucleic acid sequencing analysis, and amino acid (protein) sequencing analysis for viral, prokaryotic, and
eukaryotic research. There are a multitude of databases available that house bioinformatics data and
programing algorithms to process and make sense of the data stated above. BLAST is one of those
programs.
Basic Local Alignment Search Tool (BLAST) maintained by the National Center for Biotechnology
Information (NCBI, https://blast.ncbi.nlm.nih.gov/Blast.cgi) is a bioinformatics database that stores many
types of information related to nucleic acids and amino acids (protein). The database is populated using
many different sources, such as individual researchers and large research entities, and can be accessed
free of charge for the public’s use. Primary publications, annotations, and other tools and resources are
available in NCBI BlAST. One of the most notable uses of BLAST is in matching (searching) an
unknown nucleic acid sequence to a similar sequence in the database in the hopes to identify the unknown
organism.
During a future lab in this module, we will be “BLASTing” a nucleic acid sequence. We will also do
some further database mining to learn about navigating the NCBI BLAST tools.

25
 Lab 22
Results of Serial Dilution MIC
Set UP MBC and Multiplex PCR

THE MINIMUM BACTERICIDAL CONCENTRATION TEST:

The MIC test can be further extended to determine if an antimicrobial is Bactericidal (kills the microbe)
or Bacteriostatic (arrests the microbe’s growth and multiplication).
From the MIC tubes that showed no growth, sub-cultures are made on to MHA plates. These plates can
be evaluated for bacterial growth after 18-24 hours of incubation at 35oC.
Materials:
Mueller Hinton Agar Plates (equal to the number of no growth tubes)
Sterile Swabs
Procedure:
1. Obtain and label your MHA plates as per lab protocol. Include the tube # and the concentration
of ampicillin.
2. Mix the NG tubes thoroughly.
3. Dip a sterile swab into the no growth tube and inoculate plate as per K-B protocol.
4. Discard the swab in the bench-top sharps.
5. Discard the paper wrapper in the regular trash.
6. Incubate plates at 350C for 24 to 48 hours.
7. Record results in Table 12 below. Note “G” for growing and “NG” for no growth. Cross out the
concentrations what were not plated.
Results:
Tube 1 Tube 2 Tube 3 Tube 4 Tube 5 Tube 6 Tube 7
Concentration 64 32 16 8 4 2 1
(ug/mL)
E. coli A
E. coli B
Unknown
Organism
Table 12 Record MBC here

What was the MBC for E coli A?

26
What was the MBC for E coli B?
What was the MBC for your unknown organism?

THE β-LACTAMASE ENZYME ASSAY (revisited):

PCR AMPLIFICATION OF ANTIMICROBIAL RESISTANCE GENES
We can test for antimicrobial resistance genes as well. Remember the technique we practiced in Module
I, polymerase chain reaction (PCR). PCR uses sequence specific primers to pair with bacterial DNA and
then amplifies the sequence so it can be visualized and detected. We can use multiple primers for
multiple antimicrobial resistance genes in a single-tube multiplex polymerase chain reaction (MP-PCR).
MP-PCR is a technique that allows the simultaneous production of millions of copies of several genes in a
test tube without the use of all the cellular enzymes normally needed to copy DNA. It relies on several
sets of gene-specific primers, buffer + dNTPs, the gene templates (DNA sequences), and the presence of,
plus the thermostability of, an enzyme called Taq I DNA polymerase. This enzyme was originally
isolated from a bacterium Thermophilus aquaticus that lives in hot springs. Unlike most mammalian
proteins, Taq I does not denature at high temperatures. If all the essential components of each MP-PCR
reaction are present in a test tube, gene-specific DNA may be synthesized for each MP-PCR reaction
cycle.
The three stages of any PCR cycle are:
● Denaturation/Melting: the two strands of DNA separate when the sample heats to between
90oC and 98oC
● Annealing/Hybridization: the hybridization of DNA primers to their complementary target
DNA regions takes place between 55oC and 68oC
● Synthesis/Extension: involves the binding of a primer and template-dependent DNA
polymerase to the DNA-primer pair followed by incorporation of complementary nucleotides to
extend the primer from its 3’ end (hence copying the primed-DNA template strand) (occurs
between 70oC and 75oC – best at ~ 72oC)
By sequentially repeating these stages, the quantity of DNA increases exponentially with each PCR cycle.
At the end of approximately 30 PCR cycles (~ 2 hours), each sequence of targeted DNA increases to
about a billion copies, μg to mg quantities of DNA product! PCR cycles are performed in an instrument
known as a Thermal Cycler that controls the specific temperature and time for each cycle stage. Thermal
cyclers heat and cool rapidly through the preprogrammed temperatures and times for the required number
of cycles.

27
For this exercise, the thermal cycler settings for the temperatures and times for the three stages of each
PCR cycle are:
● Denaturation/melting at 95oC for 40 seconds
● Annealing/hybridization at 59oC for 50 seconds
● Synthesis/extension at 72oC for 60 seconds

For the PCR reaction to amplify the ampicillin resistance gene, two DNA primers that specifically bind,
or hybridize, to opposite ends of the β-Lactamase (bla) gene sequence are used (Refer to Figure 11):
● The primer hybridizing at the start, or forward, of the bla gene, ampFor, has the sequence: (5’)
ATGAGTATT CAACATTTCCGTGTCG (3’) (25 mer)
● The primer hybridizing at the end, or reverse, of the bla gene, ampRev, has the sequence: (5’)
GTTACCAAT GCTTAATCAGTGAGGC (3’) (25 mer)

Figure 11 ampForward primer and ampRevers primer (RED); Bolded and Underlined portion is the bla
gene of interest
For the PCR reaction to amplify the tetracycline (tetA) resistance gene, two DNA primers that specifically
bind, or hybridize, to opposite ends of the tetA gene sequence are used (Refer to Figure 12):
● The primer hybridizing at the start, or forward, of the tetA gene, Tet-F, has the sequence: (5’)
GCG CGT TCA ATC GGA CCA GCG (3’) (21 mer)
● The primer hybridizing at the end, or reverse, of the tetA gene, Tet-R, has the sequence: (5’)
CCG GAA GTC GCC TTG ACC CGC (3’) (21 mer)

28
Figure 12 Tet-Forward primer and Tet-Revers primer (RED); Bolded and Underlined portion is the tetA
gene of interest

For the PCR reaction to amplify the neomycin / kanamycin resistance gene, two DNA primers that
specifically bind, or hybridize, to opposite ends of the aphA gene sequence are used (Refer to Figure 13):
● The primer hybridizing at the start, or forward, of the aphA gene, Kan-F (aphAF – V04), has
the sequence: (5’) GCGGCTCACGTGATGGG (3’) (17 mer)
● The primer hybridizing at the end, or reverse, of the aphA gene, Kan-R (aphAR – V05), has the
sequence: (5’) CGATGCAGCCGACTACC (3’) (17 mer)

29
Figure 13 Kan-Forward primer and Kan-Revers primer (RED); Bolded and Underlined portion is the
aphA gene of interest

Each pair of primer sequences are complementary to flanking regions (next to the gene of interest) of the
targeted gene sequence. If the target (flanking regions of the gene) is present, the primers will hybridize
(by Watson / Crick base pairing) to those regions during the annealing cycle. This will allow the DNA
polymerase to extend the fully hybridized primer sequences from the 3’ end of the primer at the synthesis
temperature of 72oC, and creating a copy of the template gene. The copy now become the template for
the next cycle.

Preparation of DNA for the Multiplex Polymerase Chain Reaction (MP-PCR)

Materials:
2-1 mL microcentrifuge tubes
Sterile distilled water in a microcentrifuge tube
P1000 pipette
Pipette tips
Inoculating loop or needle
Bacti-cinerator
Centrifuge

30
Heat block set to 95oC to 100oC
Vat of ice chips
TSA plate of E coli A or E coli B
TSA plate of unknown isolated organism

Procedure for Isolation and Purification of DNA:

1. Label two microcentrifuge tubes as E coli and Unknown. Include your group #.
2. Transfer 100 uL of bacterial culture E. coli (A or B) microcentrifuge tube labeled E coli.
3. Centrifuge the sample at 10,000 rpm for two (2) minutes to obtain a pellet of the cells.
4. Discard the supernatant in the provided waste beaker.
5. Resuspend the cell pellet in 500 μL of sterile distilled water.
6. To a second microcentrifuge tube labeled “unknown”, add 500 uL of water. Then inoculate the
water with 4 -5 well isolated colonies from unknown growing on TSA. Mix well. Continue with
step 7 for both E. coli and unknown organism.
7. To promote cell lysis and plasmid DNA isolation, incubate the cell suspension at 95oC to 100oC
for eight (8) minutes followed by incubation on ice for three (3) minutes.
8. Centrifuge the lysate at 10,000 rpm for three (3) minutes to pellet the cellular debris.
9. Stop here and provide your tube to your instructor.
10. The instructor will add 1 uL of your prepared, extracted DNA to a tube containing
a. dNTPs, buffer, in a Master mix
b. Fwd and reverse primers
c. taq polymerase
(Total volume = 25 μL)

Note: Typically, the bacterial chromosome is attached to the cell membrane and will be present in the
pellet along with other cellular components. Plasmids, which are relatively small DNA
molecules, will end up in the supernatant. Therefore, to determine the presence of the bla, tet, and
aphA genes, we will collect the step 7 supernatant to Perform the MP-PCR as described below.

Note: E. coli A and B can also be used as positive and negative controls. Which organism represent the
positive control and which represents the negative control? Why did you choose those organisms?

Set up MP-PCR Reaction Mix

Materials:
1. Template DNA (isolated in the procedure above)
2. Taq DNA polymerase (the enzyme required for primer and template-dependent DNA synthesis)
3. 2’deoxy-Ribo-Nucleotide Triphosphates (dNTPs) (A,C,G,T, the building blocks of DNA)
4. Primers [18-30 base DNA polymers that are complementary (capable of forming Watson /Crick
base pairs) to each of the targeted DNA sequences on the RK2 DNA template. To successfully
copy a gene, two primers are needed, one for each strand of targeted DNA sequence. Each primer

31
 5. should ideally form 100% Watson / Crick base pairs (100% Complementarity) only with a region
flanking, or within, the gene of interest.]
6. Buffer plus metal ions (provides conditions for optimal enzyme activity)

Procedure for Multiplex Polymerase Chain Reaction (MP-PCR):

A TA will add the following components to two (2) PCR tubes for your group:
● Go Taq Green: TaqI DNA Polymerase/dNTPs/Buffer/Mg++/BSA ……………..12.50 μL
● ampFor Primer ……………………………………………………………………..1.00 μL
● ampRev Primer……………………………………………………………………..1.00 uL
● Tet-F Primer .............................................................................................................1.00 uL
● Tet-R Primer………………………………………………………………………..1.00 uL
● Kan-F Primer……………………………………………………………………….1.00 μL
● Kan-R Primer……………………………………………………………………….1.00 uL
● Sterile Distilled Water ………………………………………………………………5.50 uL
To the FIRST mixture, your group will add 1 μL of supernatant contacting the bacterial plasmid (if
any) from E coli A or B. To the SECOND mixture, your group will add 1 μL of supernatant
contacting the bacterial plasmid (if any) from your unknown organism. Your group will be called up one
at a time to add your samples.
Total PCR reaction mixture volume is 25 μL.
These tubes will be placed in a thermal cycler preset to the conditions optimal for amplifying the MP-
PCR products. The expected sizes of the three PCR products are:
● bla gene – 862 bp
● tet gene – 1279 bp
● aphA gene – 513 bp

32
 Lab 23
Run Gel for Multiplex PCR and BLAST Nucleic Acid Sequence

Analysis of PCR DNA Products by Gel Electrophoresis:

This is a simple but powerful technique used to analyze the size, quality and quantity of DNA fragments
that were generated by our MP-PCR procedure. In this procedure DNA is forced to travel through a
“sieve” of agarose pores. Agarose by itself is an inert material and does not affect the mobility or
properties of DNA. An electrical charge is placed onto the gel. DNA loaded into the gel migrates towards
the cathode (+) when the electrical current is applied. The current is active for 30 minutes.
֎Why is the DNA migration in this direction?

Because of the sieving effect of the agarose gel, smaller fragments will move faster (and further) than
larger fragments. In this lab, a chemical called Gel Red is added to the agarose gel mixture before it is
poured into the molds. DNA binds to Gel Red and the bands fluoresce when exposed to UV light. A UV
light box is used to visualize the bands. There are multiple ways to visualize bands of DNA. Gel Red is
one of the safer methods and does not pose any carcinogenic or mutagenic risk.

Materials:
● Agarose Gel w/ Gel Red
● Gel Box
● Buffer
● DNA Molecular Weight Marker (1KB)
● MP-PCR Products
o Your organism, E coli A or E coli B
● P10 Micropipettors and sterile Micropipettor Tips
● UV Box
Procedure for Loading and Running an Agarose Gel:
1. Gently mix and load 12 μL of PCR Product into the designated well on the gel.
2. The instructor will load a DNA molecular weight marker into one the lanes on either side of the
loaded samples and controls.
֎Why is a molecular weight marker needed?

3. Close the gel box and apply electric field of 100V. Run for 30-45 minutes, usually until you see the
dye about 2/3-3/4 of the way. Go Taq Green is a mixture of blue and yellow dye. The two colors
will separate out during the electrophoresis process.

33
4. The instructor will move the gel on to the UV light box. Look for the presence of fluorescent DNA
bands at the correct positions in the various lanes. Compare the bands to the DNA ladder loaded onto
either side of the gel to “measure” the approximate base pairs of your bands.

Results Observed on the Agarose Electropherogram:

Record which of the following MP-PCR samples produced the expected DNA bands of 862, 1279 and
513 bp:
● E. coli B:
Bands (sizes) (circle all that apply): 862 1279 513

Products suggested by these bands (circle all that apply): bla gene tet gene aphA genes

Is this sample the “Positive” or the “Negative” control?

● E. coli A
Bands (sizes) (circle all that apply): 862 1279 513

Products suggested by these bands (circle all that apply): bla gene tet gene aphA genes

Is this sample the “Positive” or the “Negative” control?

● Your isolated bacteria

Bands (sizes) (circle all that apply): 862 1279 513

Products suggested by these bands (circle all that apply): bla gene tet gene aphA genes

34
 BLAST Nucleic Acid Sequence
1 - Log onto google chrome. Open the NCBI BLAST website: https://blast.ncbi.nlm.nih.gov/Blast.cgi.

Click on Nucleotide Blast.

2 - Copy and paste the nucleotide sequence (available in lab) into the yellow box. Assign the Job Title as
your group #.
The sequence you are using is a FASTA sequence. In bioinformatics and biochemistry, the FASTA
format is a text-based format for representing either nucleotide sequencesor amino acid (protein)
sequences, in which nucleotides or amino acids are represented using single-letter codes. The format also
allows for sequence names and comments to precede the sequences. The format originates from the
FASTA software package, but has now become a near universal standard in the field of bioinformatics.
(https://en.wikipedia.org/wiki/FASTA_format)
A sequence in FASTA format begins with a single-line description, followed by lines of sequence data.
The description line (defline) is distinguished from the sequence data by a greater-than (">") symbol at
the beginning. It is recommended that all lines of text be shorter than 80 characters in length. An example
sequence in FASTA format is:
>P01013 GENE X PROTEIN (OVALBUMIN-RELATED)

QIKDLLVSSSTDLDTTLVLVNAIYFKGMWKTAFNAEDTREMPFHVTKQESKPVQMMCMNNSFNVATLPAE
KMKILELPFASGDLSMLVLLPDEVSDLERIEKTINFEKLTEWTNPNTMEKRRVKVYLPQMKIEEKYNLTS
VLMALGMTDLFIPSANLTGISSAESLKISQAVHGAFMELSEDGIEMAGSTGVIEDIKHSPESEQFRADHP
FLFLIKHNPTNTIVYFGRYWSP

Blank lines are not allowed in the middle of FASTA input.

(https://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&PAGE_TYPE=BlastDocs&DOC_TYPE=BlastHel
p)

35
36
3 - Leave the settings in default mode and press BLAST at the bottom.

4 - The search will update with the time lapsed to perform a BLAST search.

37
5 - Job Summary
BLAST Results Summary: A job title which defaults to the search type if not assigned in step 2.
Your job title should be your group #.

6 - Graphic Summary
A graphic summary will be displayed. The colored bars represent the alignment score. The sequence you
will be searching has complete alignment so all of you will have alignment of >200 in your searches. A
graph with a mix of colors alerts the user that the alignment of the query to the database was not
significant.

38
7 - Descriptions
The sequences producing significant alignment or “hits” will be shown below the graph. The query cover
with score of 100% is a very significant match and one can surmise with great confidence that their query
sequence has been correctly identified. Each institution that uses NCBI BLAST to identify thier organism
may set a query cover score that they may wish to accept identifications. You must be aware, however,
that the lower the query score, the lower the probability that the query sequence has been correctly
identified. In the example below, the query score is 100% for Streptococcus pneumoniae strain ATCC
33400 16S ribosomal RNA, partial sequence.
First: Query Cover ___________ % Organism ID: _________________________________
Accession #: _____________

Middle: Query Cover ___________ % Organism ID: _________________________________

Accession #: _____________

Last: Query Cover ___________ % Organism ID: _________________________________

Accession #: _____________

39
8 - Alignments
The alignments section shows each “hit” with the query sequence aligned with the significant alignment
sequence listed in “Descriptions”. This allows the user to manually screen over the alignment so see if
there are any mismatches or gaps. Since this particular alignment as a query cover of 100%, there is only
one match, and all 1515 out of 1515 nucleotides search match this sequence exactly.

First: Identities __________/__________ ( %) Gaps __________/__________ ( %)

Middle: Identities __________/__________ ( %) Gaps __________/__________ ( %)

Last: Identities __________/__________ ( %) Gaps __________/__________ ( %)

First:
# Gaps ____
Query Nucleotide Position _______ Nucleotide ____
Subject Nucleotide Position _______ Nucleotide ____

40
# Gaps ____
Query Nucleotide Position _______ Nucleotide ____
Subject Nucleotide Position _______ Nucleotide ____
# Gaps ____
Query Nucleotide Position _______ Nucleotide ____
Subject Nucleotide Position _______ Nucleotide ____

# Mismatches ____
Query Nucleotide Position _______ Nucleotide ____
Subject Nucleotide Position _______ Nucleotide ____

Query Nucleotide Position _______ Nucleotide ____

Subject Nucleotide Position _______ Nucleotide ____

Query Nucleotide Position _______ Nucleotide ____

Subject Nucleotide Position _______ Nucleotide ____

Middle:
# Gaps ____
Query Nucleotide Position _______ Nucleotide ____
Subject Nucleotide Position _______ Nucleotide ____

# Gaps ____
Query Nucleotide Position _______ Nucleotide ____
Subject Nucleotide Position _______ Nucleotide ____

# Gaps ____
Query Nucleotide Position _______ Nucleotide ____
Subject Nucleotide Position _______ Nucleotide ____

# Mismatches ____
Query Nucleotide Position _______ Nucleotide ____

41
Subject Nucleotide Position _______ Nucleotide ____

Query Nucleotide Position _______ Nucleotide ____

Subject Nucleotide Position _______ Nucleotide ____

Query Nucleotide Position _______ Nucleotide ____

Subject Nucleotide Position _______ Nucleotide ____

Last:
# Gaps ____
Query Nucleotide Position _______ Nucleotide ____
Subject Nucleotide Position _______ Nucleotide ____

# Gaps ____
Query Nucleotide Position _______ Nucleotide ____
Subject Nucleotide Position _______ Nucleotide ____

# Gaps ____
Query Nucleotide Position _______ Nucleotide ____
Subject Nucleotide Position _______ Nucleotide ____

# Mismatches ____
Query Nucleotide Position _______ Nucleotide ____
Subject Nucleotide Position _______ Nucleotide ____

Query Nucleotide Position _______ Nucleotide ____

Subject Nucleotide Position _______ Nucleotide ____

Query Nucleotide Position _______ Nucleotide ____

Subject Nucleotide Position _______ Nucleotide ____

42
In the alignment below, in the 1506 nucleotides on the subject sequence, only 1501 matched. There is 1
gap out of 1506. Can you find the 1 gap and the 4 mismatches?

43
44
9 - Sequence ID
Going back to the first alignment (query cover of 100%) and click on the NR_#### link next to sequence
ID.

A new window will/may open to the Nucleotide database within NCBI. This is where you will find
detailed information on the organism.

Highlight the name of the organism next to definition and paste it into the search bar above

45
Change the drop down search menu to PubMed and press search or hit enter.

Scroll through the articles listed. Click on the article link of your choice and read the abstract. Did you
learn anything (new) about this organism from reading this abstract?
Article Name: ________________________________________________________________
What did you learn from the abstract?

46
47