Pseudomonas TRUE FALSEPositive 99.57% 0.22%Negative 99.78% 0.43%

A New Method of Sample Preparation for Laser-Based Bacterial IdentificationDerek Gillies

University of Windsor, Department of Physics

A: Scanning electron images verified complete

coverage of the deposited bacterial lawn.

A: By creating serial dilutions, a calibration

curve for E. coli cell number was obtained in an

attempt to solve for the limit of detection. Due

to cellular attraction and clumping, lower

concentrations caused non-uniform bacterial

lawns.

I would like to thank Dr. Rehse, my supervisor, for his

continued guidance on these projects as well as

Russell Putnam, Dylan Malenfant and Anthony Piazza

for their help. I would also like to thank Dr. Eugene Kim,

the co-op faculty advisor and the University of Windsor.

Derek Gillies: gilliesd@uwindsor.ca

Physics and High Technology (Medical Physics) Co−op

3rd Year Undergraduate, Summer 2014

Employer: Dr. Steven J. Rehse

Department of Physics, University of Windsor

[1] - "Healthcare-Associated Infections - —Due Diligence." Public

Health Agency of Canada. <http://www.phac-

aspc.gc.ca/cphorsphc-respcacsp/2013/infections-eng.php>

[2] – R.A. Putnam, Q.I. Mohaidat, A. Daabous, S.J. Rehse, A

comparison of multivariate analysis techniques and variable

selection strategies in a laser-induced breakdown spectroscopy

bacterial classification, Spectrochim. Acta Part B 87 (2003)

161-167.

Escherichia TRUE FALSEPositive 98.28% 0.77%Negative 99.23% 1.72%

Staphylococcus TRUE FALSEPositive 97.75% 1.44%Negative 98.56% 2.25%

Mycobacterium TRUE FALSEPositive 95.36% 0.33%Negative 99.67% 4.64%

Sensitivity: 98 ± 2% Specificity: 99 ± 1%

Escherichia TRUE FALSE

Positive 96.55% 1.12%Negative 98.88% 3.45%

Staphylococcus TRUE FALSE

Positive 96.75% 1.53%Negative 98.47% 3.25%

Mycobacterium TRUE FALSEPositive 97.02% 0.41%Negative 99.59% 2.98%

Pseudomonas TRUE FALSEPositive 98.92% 0.33%Negative 99.67% 1.08%

Sensitivity: 97 ± 3% Specificity: 99 ± 2%

Current Clinical Method of Bacteria Classification

Blood is drawn from

patient.

Sample is place in an

indicating jar to test if

bacteria is present.

If the sample tests

positive, bacteria is

grown and cultured.

A Gram stain test is completed

on bacteria to reduce possible

species.

Bacteria is placed in a

multiwell assay to

classify species.

Average time to complete bacterial classification = 24 to 72 hours

0.01 0.1 1

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tal In

ten

sity (

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.)

Relative Concentration

Concentration Curve of E. Coli

• Escherichia coli

• Staphylococcus aureus

• Mycobacterium smegmatis

• Pseudomonas aeruginosa

Q: Can bacteria be deposited in a controlled

manner?

A: A steel disk was designed in order to create a

reproducible area for bacteria to be placed on.

13 mm

4.3 mm

A: Once the appropriate laser focal distance was

found on a steel sample, LIBS intensities were

reproducible and controlled.

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sit

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Relative Height of Stage (mm)

Height Analysis of Fe Wavelengths

Sum FeISum Fe II

172.78 μm140.02 μm

Q: Can cell number be determined?

With a sensitivity of 97% and specificity of 99%, the nitrocellulose filters proved to be an easier and more

effective method for discriminating bacteria using LIBS. With the introduction of a systematic testing

procedure, more serious issues can be investigated in the future such as urinary tract infections, meningitis

and MRSA infections.

Due to a lack of uniformity (through agglutination) in the cell lawn causing relatively high standard

deviations at low concentrations, a limit of detection was not obtained for the bacterial specimen. In order to

avoid elemental additions to the samples via chemical separation techniques, future work involves the use of

sonication to disrupt cell clumps. However, since the cellulose filters contribute a non-zero background to the

LIBS spectra, low cell counts may continue to be an issue for this method.

Q: Can the laser ablate in a controlled fashion?

A: With crater diameters of about 150 μm, the

quantity of cells ablated for each spectrum was

estimated at 106 (verified using optical

densitometry) including a non-zero filter signal.

Our Method of Bacteria Classification

Bacteria is cultured

using trypticase soy

agar (TSA).

Colonies are

removed and

placed in 1.5 mL

distilled water.

30 μL of vortexed

sample are deposited

on a standard 0.22

μm cellulose filter in

contained wells.

Colloidal suspension

is dried forming a

bacterial lawn on the

clinician-friendly

disposable filter.

Filter is placed in an

argon environment and

ablated using a pulsed

1064 nm Nd: YAG laser.

Échelle diffraction grating

spectrometer is used to obtain

the atomic spectrum and

composition of sample.

Atomic composition is

used to discriminate

bacteria against pre-

existing library.

Average time to complete bacterial classification = 1 hour

There is an urgent need to develop faster ways to identify pathogenic bacteria. Improved patient outcomes and lower medical costs would result. Specific

diseases/infections that our LIBS-based bacterial identification technology could address are MRSA, meninigitis, and urinary tract infections. In 2013, the Public

Health Agency of Canada reported “the healthcare-associated methicillin-resistant Staphylococcus aureus (MRSA) infection rate increased more than 1,000%

from 1995 to 2009.”1 In a previous study, BactoTM Agar was used as an element-free background for mounting bacteria to obtain spectra via laser-induced

breakdown spectroscopy (LIBS), determining their accurate atomic composition, and allowing objective classification of the bacteria.2

The purpose of this experiment was to increase accuracy for discriminating bacterial species using a controlled method for mounting cells that is familiar to

clinical microbiologists and faster than mounting on agar. Controlled mounting offers the possibility for accurately measuring the absolute cell count. Since

bacterial titer has the potential to double in 15 minutes, rapidly discriminating between samples with a cell count different by a factor of 2 could be useful for an

antibiotic resistance test.

Q: Can the new method still classify bacteria?

A: Bacterial identities were externally validated

using a discriminant function analysis (DFA) and a

partial least squares discriminant analysis (PLS-DA)

performed with SPSS v.22 (IBM, Inc.) and

PLS_toolbox v6.7.1 operating with Matlab v7.6

(Eigenvector Research, Inc.) respectively. A new

model was created by increasing the number of

independent variables from 80 to 164. After 1513

measurements, DFA classified best (see below).

Autoclaved bacteria samples were also tested; after

inactivation (sterility

verified), they were

not distinguishable

when compared to

standard procedures

(see graph at right).

Pathogenic Organisms Used

in this Project

Acknowledgements

References

Conclusions and Future Work

DFA Classification Grouped by Species

PLS-DA Classification Grouped by Species

Motivation

SEM image of

three ablation

craters in the

fractured

bacterial lawn.

Contact