Bacteria distribution on thermal gradients Avances tesis Presentación.ppt

Thesis work of bacteria distribution over thermal gradients

1. Temperature gradients generation by
deposition of graphene inks:
Bacteria distribution evaluation
(e.coli)
Masters Thesis of Daniel Alberto González
Aradillas
Thesis Advisors: Dra. Mildred Quintana Ruiz
Dra. Vanesa Olivares Illana
1

2. Abstract:
Bacteria responds to changing thermal environment by moving towards or
away from a particular location. In this report, we looked into thermal
gradient generation and response of E. Coli to thermal gradient with different
temperatures, optical density of starting colonies, and incubation times.
Thermal gradients are generated by using a graphene based ink that
generates heat when an electrical current pass through the material. We
analyzed then, bacteria distribution according to temperature to observe the
effect of temperature gradient over bacteria.
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Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)

3. • Temperature gradient
• Thermal Camera
• Importance of thermal gradients over growth and
distribution of e. coli bacteria
3
Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)
Introduction

4. Temperature gradient
"A temperature gradient is a physical quantity that describes in which
direction and at what rate the temperature changes the most rapidly
around a particular location. The temperature gradient is
a dimensional quantity expressed in units of degrees (on a particular
temperature scale) per unit length. The SI unit is Kelvin per meter
(K/m)."
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Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)
Introduction

5. Thermal Camera
To measure temperature
gradient we use a thermal
camera, which is a device that
uses infrared light to calculate
temperature distribution and
shows it in a form of colors or
so. It helps (with the correct
software) to see temperature
distribution in a surface and
to know emissive
temperature.
We used ®FLIR E6 Infrared
Camera.
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Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)
Introduction

6. Importance of thermal gradients over growth and distribution
of e. coli bacteria
Microorganisms often have to navigate through their surroundings in
search of nutrients and a better environment for survival. One of the
important environmental cues microorganisms follow is temperature.
Bacteria sense temperature differences and follow thermal gradients
towards their favourable temperature which could help bacteria
colonize certain regions, form biofilms, and infect host cells or
tissues. 
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Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)
Introduction

7. •Design and construction of TGGCs
•Design and construction of Incubator
•Evaluation of growth and distribution of bacteria through
temperature gradient
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Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)
Special Objectives

8. •Culture/Growth Medium
•Preparation of Bacteria
•Characterization of Bacteria Colonies
•Optic Microscopy
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Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)
Methodology

9. Culture/Growth Medium
With specific concentrations (2.5 g) of this medium in water (100 ml),
a standard growth medium solution with a pH of 7.2 has to be
obtained to later on, add a bacteria colony (as seed) and then
incubate at 37 ºC in a shaking incubator at 250-300 rpm. As note: In
order to have a sterilized medium, it is heated above 100 ºC and
procedure of adding bacteria into the LB medium is made near the
flame of a burner (less than 10 cm away from the flame) with
previously sterilized material, as micropipette tips, tubes used as
containers, etc.
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Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)
Methodology

10. Preparation of Bacteria
To understand bacteria distribution at any kind of growth technique,
and to ensure about the same quantity of bacteria on each
experiment, it is needed to know the density of bacteria disperse in a
LB medium (liquid). Achieving this is possible with an absorption
spectrometer used at 600 nm of wave length (which is the wave-
length needed to detect e. coli bacteria), and starting from the LB-
bacteria medium, some dilutions are measured; primal solution, 1:10,
1:100 and 1:1000 solutions. So we know we have same density of
bacteria each time (for each dilution and experiment).
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Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)
Methodology

11. Characterization of Bacteria Colonies
Bacteria Colonies are studied by growing them under specific
conditions . First of all a well known method is implemented, it is a
standard growth method which consists on the addition of LB-
bacteria solutions over an Agar-LB based medium and settled on a
traditional incubator (which is an isolated box with adjustable heater)
at 37 ºC for 24 h. This allows us to observe the normal growth
behaviour of bacteria for each dilution so we can compare that with
temperature gradient growth and it helps to select which density of
bacteria suits better for our experiments.
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Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)
Methodology

12. Optic Microscopy
Once we run an experiment and some bacteria are growth on our
GCs, we observe under the microscope (10-40x of magnification) how
much bacteria do we have. First of all we observe the growth- cells
after letting bacteria divide for 24 h at 37 ºC in a conventional
incubator and then we observe the temperature-gradient growth-
cells to see its effect on bacteria division for different temperatures
and different time periods.
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Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)
Methodology

13. •GCs and TGGCs fabrication
•Incubator
•GCs and TGGCs characterization
•Bacteria optical densities
•Bacteria distribution over temperature gradients
13
Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)
Results

14. GCs and TGGCs fabrication
GCs are basically films of 2mm of thickness with a surface of 22x22
mm made of Agar placed over a glass cover slip, but a mold was
needed to achieve that shape and size.
To fabricate them a glass slip is placed at the bottom of each of four
cavities of a mold and then 1 ml of liquid agar (it is liquid at around
70 ºC) is poured inside each of them too over the glass slip. Two
molds were used each time so we got eight 1 ml jellies with the
desired shape.
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Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)
Results

15. GCs and TGGCs fabrication
15
Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)
Results
Fig. 1 - A: glass coverslip is placed on the mold; B: 1 ml of liquid Agar is poured over the glass slip,
it is left over there for about 20 min to let it cold and solidify.

16. GCs and TGGCs fabrication
GTGGCs are just a glass cover slip with a printed lane of
resistive ink and their respective AuNPs based conductive ink
contacts as shown in Fig. 2
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Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)
Results
Fig. 2 - A: glass coverslip; B: glass slip with the resistive ing printed on it; C: conductive contacts
placed over the resistive ink.

17. Incubator
The incubator was made
of a collection of pieces
cut from black acrylic of
3mm of thickness,
assembled with CHCl3 as
shown in the Fig. 3
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Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)
Results
Fig. 3 - A: Surface of cavities; B: Electric circuit layer; C: Space layer; D:
Base; E: Side walls of the covering; F: Frontal wall of the covering; G:
Rear wall of the covering; H: First ceiling; I: Second ceiling; J:
Asembled Incuvator.

18. 18
Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)
Results

19. 19
Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)
Results

20. 20
Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)
Results

21. 21
Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)
Results

22. 22
Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)
Results

23. GCs and TGGCs
characterization
To characterize GCs we
poured 100 µl of LB-
bacteria solution of
different bacteria densities
to observe bacteria growth
under standard conditions,
this is to place the squared
Agar films into a Petri box
and then placed inside a
traditional incubator for 24
h. Then we observed results
shown in Pic. 1 and Img. 1.
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Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)
Results
Pic. 1 - Picture of four of
the Agar cells after
traditional growht for
densities of 1:100 and
1:1000
Img. 1 - Image of some
colonies taken from an
optical microscope
camera at 10x
magnification.

24. GCs and TGGCs characterization
24
Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)
Results

25. GCs and TGGCs characterization
To characterize TGGCs different measurements (with a fixed
bacteria density each time) were made taking a rank of
temperatures going from 32 ºC to 60 ºC to know which density
of bacteria was going to be used and to select a temperature
for the TGGCs (shown in Pic. 2 and Img. 2)
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Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)
Results

26. GCs and TGGCs characterization
26
Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)
Results
Pic. 2 - Picture of eigth TGGCs placed over the
Incubator.
Img. 2 - Image taken from FLIR Tools software, aquired with a E6 Infrared Camera.
Temperature of (a) was 60 ºC and it was decreased in steps of 4 ºC down to 32 ºC
at (h).

27. GCs and TGGCs characterization
27
Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)
Results
Img. 3 - Image taken from FLIR Tools software, aquired with a E6
Infrared Camera. Temperature of all cells is 38 ºC.

28. Bacteria optical densities
Optical densities of bacteria were measured by absorbance
spectrometry and values obtained are shown in Tab. 1
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Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)
Results
Dilutions 1:1 1:10 1:100 1:1000
Optical Densities0.7714 - 0.91020.1524 - 0.20110.0283 - 0.03240.0008 - 0.0010
Tab. 3 - Optical densities are measured in absorbance units, and it is shown as a rank from the lowest to the
highest measured densities.

29. Bacteria distribution over temperature gradients
First of all, length of image given a 40x magnification on the
optical microscope was made, to ensure a correct
measurement of growth cells and temperature location. This is
shown on Img. 4.
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Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)
Results
540 µm
Img. 4.- Shows a sample measured by a calibrated ruler from side
to side of the image taken from the optical microscope at 40x
magnification..

30. Bacteria distribution over
temperature gradients
Second stage was a collection
of all images taken from the
optical microscope from 2500
to 19500 µm. This is shown on
Graph. 1 (as temperature
distribution along the sample)
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Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)
Results
Graph. 1.- Shows the temperature distribution along the sample.

31. Bacteria distribution over temperature gradients
Bacteria was analyzed in two stages, the first one was a
collection of specific spots in which bacteria colonies were
observed taking on count location (distance from the heating
edge) and temperature given each spot. This results are shown
in Graph.1 and Imgs.5-6.
31
Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)
Results

32. Bacteria distribution over temperature gradients
32
Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)
Results
Img. 5.- Shows the bacteria distribution observed by optic microscopy, in some positions along the sample for a 1:100 dilution.
a: 2160-2700 µm / 36.80 ºC; b: 3780-4320 µm / 37.86 ºC; c: 5400-5940 µm / 31.95 ºC; d: 7020-7560 µm / 27.93 ºC; e: 8640-9180 µm / 26.05 ºC; f: 10260-
10800 µm / 25.08 ºC; g: 11880-12420 µm / 24.64 ºC; h: 13500-14040 µm / 24.28 ºC; i: 14580-15120 µm / 24.11 ºC; j: 15660-16200 µm / 23.99 ºC; k: 16740-
17280 µm / 23.87 ºC; l: 17820-18360 µm / 23.82 ºC

33. Bacteria distribution over temperature gradients
33
Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)
Results
Img. 6.- Shows the bacteria distribution observed by optic microscopy, in some positions along the sample for a 1:1000 dilution.
a: 3780-4320 µm / 37.86 ºC; b: 5940-6480 µm / 30.21 ºC; c: 8100-8640 µm / 26.58 ºC; d: 10260-10800 µm / 25.08 ºC; e: 12420-12960 µm /
24.48 ºC; f: 14580-15120 µm / 24.11 ºC; g: 16740-17280 µm / 23.87ºC; h: 18900-19440 µm / 23.89 ºC.

34. Bacteria distribution over temperature gradients
Imgs. 7-8 (where bacteria distribution is shown for 8 and 12 h
of culture with a dilution rate of 1:100)
34
Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)
Results

35. Bacteria distribution over temperature gradients
35
Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)
Results
Img. 7 - 32 pictures from 2700 µm at 36.80 ºC to 19440 µm at 23.89 ºC (see Graph. 1). For a dilution rate of 1:100 on a growht time of 8 h.

36. Bacteria distribution over temperature gradients
36
Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)
Results
Img. 7 - 32 pictures from 2700 µm at 36.80 ºC to 19440 µm at 23.89 ºC (see Graph. 1). For a dilution rate of 1:100 on a growht time of 8 h.

37. Bacteria distribution over temperature gradients
37
Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)
Results
Img. 7 - 32 pictures from 2700 µm at 36.80 ºC to 19440 µm at 23.89 ºC (see Graph. 1). For a dilution rate of 1:100 on a growht time of 8 h.

38. Bacteria distribution over temperature gradients
38
Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)
Results
Img. 7 - 32 pictures from 2700 µm at 36.80 ºC to 19440 µm at 23.89 ºC (see Graph. 1). For a dilution rate of 1:100 on a growht time of 8 h.

39. Bacteria distribution over temperature gradients
39
Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)
Results
Img. 7 - 32 pictures from 2700 µm at 36.80 ºC to 19440 µm at 23.89 ºC (see Graph. 1). For a dilution rate of 1:100 on a growht time of 8 h.

40. Bacteria distribution over temperature gradients
40
Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)
Results
Img. 8 - 32 pictures from 2700 µm at 36.80 ºC to 19440 µm at 23.89 ºC (see Graph. 1). For a dilution rate of 1:100 on a growht time of 12 h.

41. Bacteria distribution over temperature gradients
41
Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)
Results
Img. 8 - 32 pictures from 2700 µm at 36.80 ºC to 19440 µm at 23.89 ºC (see Graph. 1). For a dilution rate of 1:100 on a growht time of 12 h.

42. Bacteria distribution over temperature gradients
42
Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)
Results
Img. 8 - 32 pictures from 2700 µm at 36.80 ºC to 19440 µm at 23.89 ºC (see Graph. 1). For a dilution rate of 1:100 on a growht time of 12 h.

43. Bacteria distribution over temperature gradients
43
Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)
Results
Img. 8 - 32 pictures from 2700 µm at 36.80 ºC to 19440 µm at 23.89 ºC (see Graph. 1). For a dilution rate of 1:100 on a growht time of 12 h.

44. Bacteria distribution over temperature gradients
44
Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)
Results
Img. 8 - 32 pictures from 2700 µm at 36.80 ºC to 19440 µm at 23.89 ºC (see Graph. 1). For a dilution rate of 1:100 on a growht time of 12 h.

45. Bacteria distribution over temperature gradients
45
Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)
Results
Img. 8 - 32 pictures from 2700 µm at 36.80 ºC to 19440 µm at 23.89 ºC (see Graph. 1). For a dilution rate of 1:100 on a growht time of 12 h.

46. At the beginning of the project we expected to have different
results than obtained, because logic told us bacteria was going
to grow better on its optimal temperature which is 37 ºC, but we
observed that bacteria has a different behaviour than expected.
46
Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)
Conclutions

47. Ratkowsky showed that there is a behaviour dependent of
temperature for speed of growing and the life time of bacteria,
it is not only the temperature the important topic but the time
in which division occurs. Even if bacteria has its most efficient
development at 37 ºC, when temperature is higher division
occurs faster but life time of individuals decay, and in the other
hand, when temperature is lower than optimal division occurs
slower but lifetime is extended.
47
Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)
Conclutions

48. This is what we observed on this work, because even if bacteria
had optimal conditions most of individuals never divided due to
a short life time and far from optimal conditions populations of
bacteria trended to be more extensive. We concluded that
bacteria can reproduce better at 37 ºC but they die so fast that
colonies have not the same opportunity to develop a huge
population.
48
Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)
Conclutions

49. Referencias:
[1] Variation in cell volume and community composition of bacteria in response to
temperature; Johanna Sjöstedt, Ake Hagström, Ulla Li Zweifel, Department of
Natural Sciences, Linnaeus University, 39182, Kalmar, Sweden, Department of Cell
and Molecular Biology, University of Gothenburg, 405 30 Göteborg, Sweden.
[2] A Mechanism for Precision-Sensing via a Gradient-Sensing Pathway:A Model of
Escherichia coli Thermotaxis; Lili Jiang, Qi Ouyang, Yuhai Tu, Center for Theoretical
Biology and School of Physics, Peking University, Beijing 100871, China; and ‡IBM T.
J. Watson Research Center, Yorktown Heights, New York 10598.
[3] Transient Rates of Synthesis of Individual Polypeptides in E. coli Following
Temperature Shifts; Peggy G. Lemaux, Sherrie L. Herendeen, Phillip L. Bloch,
Frederick C. Neidhardt, Department of Microbiology The University of Michigan Ann
Arbor, Michigan 48109.
[4] Effect of gold nanoparticles on thermal gradient generation and thermotaxis of
E. coli cells in microfluidic device; Nithya Murugesan, Tapobrata Panda, Sarit K. Das,
Springer Science&Business Media New York 2016 - Biomed Microdevices (2016).
[5] The thermal impulse response of Escherichia coli; Eli Paster, William S. Ryu,
Lewis–Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ
08544.
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Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)

50. Referencias:
[6] Effect of temperature on the composition of fatty acids in escherichia coli; Allen
G. Marr, Jhon L. Ingraham, Department of Bacteriology, University of California,
Davis, California.
[7] Levels of Major Proteins of Escherichia coli During Growth at Different
Temperatures; Sherrie L. Herendeen, Ruth A. Vanbogelen, Frederick C. Neidhardt,
Department ofMicrobiology, The University ofMichigan, Ann Arbor, Michigan 48109.
[8] Relationship Between Temperature and Growth Rate of Bacterial Cultures; D. A.
Ratkowsky, June Olley, T. A. Mcmeekin, A. Ball, Division of Mathematics and Statistics
and Division of Food Research, Commonwealth Scientific and Industrial Research
Organization; and Department ofAgricultural Science, University of Tasmania;
Hobart, Tasmania, Australia 7000.
[9] Model for Bacterial Culture Growth Rate Throughout the Entire Biokinetic
Temperature Range; D. A. Ratkowsky, R. K. Lowry, T. A. Mcmeekin, A. N. Stokes, R. E.
Chandler, Department of Agricultural Science, University of Tasmania, and Division
of Mathematics and Statistics, Commonwealth Scientific and Industrial Research
Organization, Hobart, Tasmania, Australia 7000, and Division of Mathematics and
Statistics, Commonwealth Scientific and Industrial Research Organization,
Melbourne, Victoria, Australia 3168.
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Temperature gradients generation by deposition of
graphene inks:
Bacteria distribution evaluation (e.coli)

51. Point-of-care nano-biosensor to measure
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Article in Science119(3095):551-552 · April 1954
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Callister, W. D. Wiley: New York, 2007
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J. G. Grabmaier and C. B. Watson
Article first published online: 31 MAR 2006 OI: 10.1002/pssb.19690320155
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Shintaro Miyazawa, Yasunobu Ishii, Satoru Ishida and Yasushi Nanishi
 Appl. Phys. Lett. 43, 853 (1983)
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