Will an S. aureus culture exhibit cell death if silver, copper, or zinc
ions are bound to autoinducing peptide (AIP) and inserted into the
If silver, copper, or zinc ions are bound to AIP and inserted into an S.
aureus culture, then cell death will occur.
Quorum sensing is the method
by which bacteria within a
biofilm express genes
In pathogenic bacteria, such
as S. aureus, these genes
regulate the production of
Autoinducing peptide, or AIP-
1, is the autoinducer of the
agr (accessory gene regulator)
quorum sensing system (Kong
et al., 2006) of S. aureus.
Figure 1. Structure of AIP-1. The top image shows the
atomic structure of AIP-1 while the bottom image shows
the amino acid structure. (Kjaerulff et al., 2013)
Materials and Methods
Table 1. List of materials used in the project and their sources
Silver nitrate Biological
Centrifuge Flasks Staphyloccoc
Zinc acetate Balance LS 55
Pipet gun Culture tubes Double-
SYTO 9 dye
Refrigerator Pipettes Micropipette PI (propidium
Metal Ion Solution Creation 96-Well Plate Preparation
Figure 2. Flowchart showing how metal ion solutions were prepared
Figure 3. Flowchart depicting how the metal ion plate was prepared
Metal Ion Exposure Tests
Figure 4. Flowchart depicting how the timed metal ion exposure experiments were conducted
Definition of RatioG/R
The live/dead assay involved two dyes.
SYTO 9 (abbreviated to G, because of its ability to stain live cells
green) and propidium iodide (abbreviated R, because of its ability to
stain dead cells red) were used to determine the overall viability of
Forming RatioG/R by dividing the intensity of the green fluorescence
(510 – 540 nm) by the intensity of the red fluorescence (620 – 650 nm)
allows the viability of the bacterial cells to be assessed.
96-Well Plate Test
Figure 5. The RatioG/R of the control group consisting of 15 wells of S.
aureus, no metal ions, and no AIP in the 96-well plate experiment
Figure 6. The RatioG/R of the silver group consisting of 15 wells of S.
aureus, silver ions, and no AIP in the 96-well plate experiment
Figure 7. The RatioG/R of the zinc group consisting of 15 wells of S.
aureus, zinc (II) ions, and no AIP in the 96-well plate experiment
Figure 8. The RatioG/R of the copper group consisting of 15 wells of
S. aureus, copper (II) ions, and no AIP in the 96-well plate
First Metal Ion Exposure Test
Figure 9. A comparison of the three groups (the control group, the copper group, and the zinc group) in the first fluorescence
Figure 10. A comparison of the two groups (the control group and the copper group) in the second fluorescence
Second Metal Ion Exposure Test
96-Well Plate Data Analysis
The positive slopes for the silver and zinc groups (0.0287 and 0.0433
respectively) were unexpected because both silver and zinc have been
proven to be antimicrobials (see Figures 6 and 7).
One possible cause of these results could have been that the plate
was not left to incubate for an extensive period of time.
The slope of the copper group (see Figure 8), -0.0244, indicates that
copper ions could react with bacterial cells more quickly than zinc
ions or silver ions can.
Metal Ion Exposure Tests Analysis
In the first timed exposure experiment, both the zinc group and the
copper group showed a negative trend in RatioG/R as time elapsed (see
For the copper group, the steepest decrease in RatioG/R occurred from
30 minutes to 90 minutes, suggesting that most of copper’s
antimicrobial activity occurs early on when exposed to bacteria.
The zinc group appeared to have its steepest decrease in RatioG/R
occur from 30 to 120 minutes.
RatioG/R at 90 minutes for zinc showed an unusually sharp increase.
The conditions under which the zinc group was incubating should not
have been conducive for bacterial growth.
The copper group in the second timed experiment showed a negative
trend from 0 minutes to 116 minutes (see Figure 10), while the copper
group in the first experiment showed a steeper negative trend from 30
to 90 minutes and a less steep negative trend onwards.
These results still suggest that copper has an increased period of
antibacterial activity early on.
The copper group of the second fluorescence spectroscopy experiment
showed a period of inactivity from 129 to 160 minutes, which is
similar to the first spectroscopy experiment in which the copper group
showed a period of inactivity after 100 minutes.
Determine a procedure to purify AIP.
Conduct the same experiments but with added AIP.
Determine a procedure to conduct the experiments using silver.
Conduct experimentation using different metal ions.
Preparation of the Metal Ion Plate and Modelling of Copper Binding to AIP-1
Figure 11. Photograph of Achilles Gatsonis
preparing the 96-well plate for the metal ion test
Figure 12. Model of Cu2+ binding to AIP-1 (John Cvitkovic)