1 of 27

PHYSICAL PROPERTIES OF BACTERIA CELLS GROWN UNDER AMPICILLIN

Presented by: Amy Carranza-Parras

Faculty Mentor: Dr. Keenan E. Dungey

Summer 2021 URP

Figure I. Undiluted Escherichia coli (Static Force, 780ms, Stat0.2LAuD).

Figure I. Undiluted Escherichia coli (Force Curve).

2 of 27

OVERVIEW

2

Conclusion

Trends

Future Plans

Results and Discussion

Inhibition zone measurements

Bacterial Cell measurements

Force Curves

Methodology

AFM technique

Culturing bacteria

Introduction

Cells response to stress

Purpose

3 of 27

INTRODUCTION

Cells response to stress

Project Purpose

​

3

4 of 27

STRESS RESPONSE

​

  • Living organisms respond to stress in order to increase their survivability.
    • Humans have the well-known “flight or fight” response to stress.
      • Remove ourselves from stressful situations
      • Choose to take on the battle

​

Cells respond to stress as well.

  • Living cells use cell surface proteins, such as mechanosensors, to constantly sense and respond to their environment.

​

4

5 of 27

ANTIBIOTIC-INDUCED MODIFICATIONS OF THE STIFFNESS OF BACTERIAL MEMBRANES

  • Infections due to multidrug-resistant bacteria are a major public health problem, causing increasing mortality.

​

  • Two stiffer areas could be identified (red and yellow ovals).

​

  • Cells became stiffer when exposed to ampicillin.

5

Longo, G., Rio, L. M., Trampuz, A., Dietler, G., Bizzini, A., & Kasas, S. Antibiotic-induced modifications of the stiffness of bacterial membranes. Journal of Microbiological Methods, 93(2), 80-84. 2013. https://doi.org/10.1016/j.mimet.2013.01.022

​

Figure III. Topography spectroscopy of E. coli in PBS environment (5×5 μm)

​

6 of 27

PROJECT PURPOSE

  • To analyze the physical properties of E. coli cells under ampicillin using the Atomic Force Microscope (AFM).

​

Do bacterial cells become more rigid and lose their elasticity when under stress?

  • Our hypothesis is that, when confronted with ampicillin, the E.coli cells will progressively become more rigid.
  • Collaboration with Dr. Sanchez Diaz and his research students who are using other tools to analyze E. coli .

​

6

7 of 27

RESEARCH GOALS

7

Imaging in air and liquid series of E. coli generations grown to resist ampicillin with the Atomic Force Microscope.

Measure the dimension of E. coli with the Gwyddion Software.

Further analyze bacteria cells by measuring the physical properties such as cell stiffness, viscoelasticity, hardness and adhesion through Force Spectroscopy.

Data Analysis for possible trends in order to test our hypothesis: when confronted with antimicrobial chemicals, the cells will become more rigid.

Figure II. 1:10 Diluted Escherichia coli (Dynamic Force, 680ms, Dyn190Al).

8 of 27

METHODOLOGY

AFM Technique

Culturing Bacteria

8

9 of 27

AFM TECHNIQUE – IMAGING CELLS

9

 

10 of 27

AFM TECHNIQUE – FORCE SPECTROSCOPY

10

  • Slope= measure for sample stiffness
  • Fadh = adhesive force properties of the sample
  • Wadh = measure for the irreversible work performed
  • Indentation into sample is indirect measurement of sample stiffness.

​

https://www.nanosurf.com/en/support/afm-modes-overview/force-spectroscopy

11 of 27

FORCE DISTANCE CURVES– CALIBRATION OF CANTILEVER

11

Deflection Sensitivity

Spring Constant

12 of 27

CALIBRATION – DEFLECTION SENSITIVITY

  • Deflection Sensitivity is the relationship between deflection and displacement.
  • Max Deflection must be found by measuring length of force curve slope on hard surface in Static Mode.

​

​

12

Laser

Detector

Deflection: Volts

Surface

cantilever bends

Displacement: Meters

13 of 27

CALIBRATION– SPRING CONSTANT

  •  

13

Figure IV. Frequency Sweep of Dyn190Al

 

14 of 27

CALIBRATION– SPRING CONSTANT

14

http://www.ampc.ms.unimelb.edu.au/afm/webapp.html

15 of 27

CULTURING E.COLI

15

16 of 27

CULTURING E.COLI STEPS (PART 1)

16

17 of 27

CULTURING E.COLI STEPS PART 2

17

18 of 27

RESULTS AND DISCUSSION

18

Inhibition Zone measurements

Bacteria cell measurements

Force Curve

19 of 27

INHIBITION ZONE OF PLATED CULTURES

19

Figure V. Generation 1A-F Plates with Ampicillin (6/2/21)

G2A

G2B

G2C

G2D

G2E

G2F

20 of 27

MEASURING E.COLI CELLS USING GWYDDION

20

  • Average Height was recorded for each cell

21 of 27

BACTERIAL CELL MEASUREMENTS 

21

Figure II. Height (nm) of Generation 1-5

Decrease in Height from Generation 2—5!

22 of 27

FORCE CURVES 

Hertz model was used to fit data.

22

23 of 27

COMPARISON OF FORCE CURVES

23

24 of 27

GENERATION 3 FORCE SPECTROSCOPY 

24

A1

A2

A3

A4

A5

Figure VII. Generation 3 E. coli Cell (Dyn190Al,780ms)

25 of 27

CONCLUSIONS

  • Cell diameter decreased after Generation 2
  • Cell stiffness of Generation 3 larger than Generation 1

​

​

25

IMAGING E. COLI GENERATIONS GROWN TO RESIST AMPICILLIN IN AIR USING AFM.

MEASURE THE DIMENSION OF E. COLI WITH THE GWYDDION SOFTWARE.

FURTHER ANALYZE BACTERIA CELLS BY MEASURING THE PHYSICAL PROPERTIES SUCH AS CELL STIFFNESS, VISCOELASTICITY, HARDNESS AND ADHESION THROUGH FORCE SPECTROSCOPY.

DATA ANALYSIS FOR POSSIBLE TRENDS IN ORDER TO TEST OUR HYPOTHESIS: WHEN CONFRONTED WITH ANTIMICROBIAL CHEMICALS, THE CELLS WILL BECOME MORE RIGID.

26 of 27

FUTURE PLANS

  • Continue research Fall 2021
  • Present at SERMACS
  • Continue measuring bacteria cells for Epsilon (least growth) data set
  • Calculate the elasticity for all cells
  • Repeat experiment with gram-positive bacteria

26

27 of 27

ACKNOWLEDGEMENTS

  • Dr. Keenan E. Dungey
  • Dr. Luis Sanchez Diaz
  • Mackenzie Smith
  • Patrick Kenney
  • Grote Endowment Fund
  • Westbrook Scholarship

​

27