Pablo Bravo
PhD Candidate in Quantitative Biosciences at GeorgiaTech. I am interested in biofilms, and how their topography evolves and what we could learn about their development by looking at them
growth and in biofilms
Pablo Bravo
YunkerLab, Georgia Institute of Technology
QBioS 4th year seminar
Vertical
topographies
Outline
I. Biofilms
- What are they, and why we should care
II. Vertical growth dynamics
III.Biofilm topographies
- How to measure vertical growth
- Behavior and clues
- Heuristic model
- Why topographies
- Characterization
- Apples to oranges?
Extracellular matrix formed of polysaccharides, DNA, and proteins
Surface
Interface
Cells
Biofilms are 3D structures
complex surface-attached
cycle
The (expanded) biofilm
- How do aggregates grow?
- What are the underlying processes?
- Simple quantitative model?
Horizontal Growth
Vertical
Growth
Growth and accumulation
Outline
I. Biofilms
II. Vertical growth dynamics
III.Biofilm topographies
- What are they, and why we should care
- How to measure vertical growth
- Behavior and clues
- Heuristic model
- Why topographies
- Characterization
- Apples to oranges?
- Multiple light sources in the instrument
- Super-resolution measurements
- Non-invasive
- No preparation needed
\( 0.5 mm\)
0
2
4
6
8
10
\(\Delta z\) (\( \mu m\))
Central region of a vibrio cholerae biofilm
Surface topography + intensity!
White-light interferometry
Using to measure
interferometry
biofilm growth
Homeland
Agar
Coffee Ring
Things didn't go quite well the first ~10 attempts
Hours
0
48
during biofilm growth
Two regimes
Two regimes in which vertical growth depends with the height of the colony
linearly
in the agar?
Nutrient depletion
Agar is not running out of nutrients!
Colonies must be slowing down for a different reason
inside the colony
Nutrient dynamics
\(z\)
\frac{\partial c}{\partial t} = D \cdot \frac{\partial^2 c}{\partial z^2} - \lambda \cdot \frac{c}{k+c}
Diffusion constant
Consumption rate
Monod constant
c(0, t) = c_0 \quad \quad \quad \dot{c}(h, t) = 0
Concentration in the substrate
No flux in the top
Region where cells can grow is finite
\int_0^h \frac{c(z)}{k+c(z)}dz \approx \text{min}(h, L)
of a colony saturates once they reach a critical length \(L\)
Total growth
for vertical
biofilm growth
Heuristic model
Empirical data + biophysical insight:
\frac{\partial h}{\partial t} = \alpha \cdot \text{min}(h, L) - \beta \cdot h\\
Growth rate
Decay rate
Diffusion length
- Fits the dynamics across timescales
- Captures two growth regimes
- \(D, \lambda, k \rightarrow L\)
behavior
Long-time
- Measure colonies every 2 days
- Equilibrium in maximum height
-
Model height prediction:
\(h_{\text{max}} = \frac{\alpha L}{\beta}\)
-
Same behavior, different parameters
-
Good agreement even early!
in vertical growth
Universality
Experiments across a large cohort of microbes
- Bacteria and fungi
- Gram + and -
- Different shapes
- Different EPS production
Cool way of getting
numbers from growth in solid media
biologically relevant
Do the make sense?
parameters
\frac{\partial c}{\partial t} = D \cdot \frac{\partial^2 c}{\partial z^2} - \lambda \cdot \frac{c}{k+c}
\(800 \mu m^2 \cdot s^{-1}\)
\(38 \mu M\)
\(1.3\cdot10^3 \mu M \cdot s ^{-1}\)
Eschericia coli growing in agar -> limited by L-serine
Using literature parameters we obtain
\(L = 14.8 \mu m\)
And using the interface model
\(L = 14.3 \pm 1 \mu m\)
Outline
I. Biofilms
- What are they, and why we should care
II. Vertical growth dynamics
III.Biofilm topographies
- How to measure vertical growth
- Behavior and clues
- Heuristic model
- Why topographies
- Characterization
- Apples to oranges?
What is ?
Profiles are flat. A few cells in amplitude, over thousands of micrometers!
\(500 \mu m\)
biofilm topography
Using white-light interferometry, we can capture the profiles of a growing colonies for extended periods of time
are different between strains
Staphylococcus aureus
Bacillus cereus
Eschericia coli
- What is leading to the differences between profiles?
- How do we characterize the dynamics?
\(2 mm\)
\(8 \mu m\)
Time since inoculation [hours]
\(0\)
\(24\)
\(48\)
Fluctuation dynamics
Fluctuations can
Aeromonas veronii
Eschericia coli
or
relax
increase
- Width of perpendicular fluctuations in the height profile:
- With a roughness/Hurst scaling exponent \(H\):
- The width saturates at \(l_\text{sat}\), and across the whole sample the width is \(w_\text{sat}\)
w_l(\mathbf{r}, t) = \langle (h (\mathbf{r}, t) - \langle h(\mathbf{r}, t) \rangle_l )^2 \rangle_l^{1/2}
\(w_l(t) \propto l^{H} \)
fluctuations
Dervaux, et al. 2014
Martinez-Calvo and Bhattacharjee, et al. 2022
\(H\)
\(l_{\text{sat}}\)
\(w_{\text{sat}}\)
Quantifying
across strains
Roughness \(H\), after a period of time, stabilizes at \(H_{\text{steady}} \sim 0.8\)
Roughening
and
There is high variability between microbes
\(w_{sat}\)
vertical growth
Colonies reach nutrient depletion length \(L\)
And an apparent correlation between vertical growth dynamics and the topography!
Knowing the moment when the colony is growing the fastest, just by looking at
fluctuations
Are fluctuations ?
\(S(k) [\mu m^4]\)
\(k [\mu m^{-1}]\)
\(10^4\)
\(10^3\)
\(10^2\)
\(10^1\)
Dynamic scaling, a test for self-affinity requires:
\(\nu = 1+2H\)
We do not see observe dynamic scaling.
It is close but not the same!
scale-free
Testing again
self-affinity
We can test self-affinity using the fractal dimension \(D\)
\(D + H = 2\)
\(D + H = n +1\), where \(n\) is the base dimension of the system
Topography dynamics as a consequence of growth through a viscoelastic material:
- Reach SA while growth is fast
- Relax when the colony is tall, and fluctuations get damped
Acknowledgements
NIH-NIMS
NSF BMAT
Biolocity
Dr. Peter Yunker
Dr. Brian Hammer
Dr. Siu Ling Ng
Dr. Thomas Day
Aawaz Pokhrel
Emma Bingham
Funding
Thanks!
Adam Krueger
Raymond Copeland
Maryam Hejri
Lin Zhao
Chris Zhang
Lots to learn about , both from and
vertical growth
biofilm development
biotopographies
4x speed
10 \( \mu L \) inoculation
11 days
2x speed
Some stretch for really long before breaking
Two error messages
1. The deviation in controller X is too large
2. The controller target window has not been reached in target monitoring time
Petite yeast (this time for real)
- Is this because of the stitching? (~20 stitches shown)
- Could a setting in the stitching software help?
| Strain | Media | Species | Date | Comment |
|---|---|---|---|---|
| JT1080 | LB 1.5% | Vibrio cholerae | 2020-11-10 | EPS- |
| SN503 | LB 1.5% | Vibrio cholerae | 2021-01-11 | EPS+ |
| BH1543 | LB 1.5% | Vibrio cholerae | 2021-04-12 | EPS++ |
| BGT127 | LB 1.5% | Aeromonas | 2021-06-25 | |
| bacillus* | LB 1.5% | Bacillus subtilis | 2021-07-30 | Gram + |
| JT305 | LB 1.5% | Eschericia Coli | 2021-08-27 | |
| pyeast* | YPD | Saccharomyces cerevisiae | 2021-09-03 | Aerotolerant anaerobe |
| CC151 (~JT305?) | YPD | Eschericia Coli | 2022-01-21 | wt ecoli on different media |
| pyeast* | YPD | Saccharomyces cerevisiae | 2022-01-28 | Aerobic |
Growth timelapses
Do we need more/specific combination?
QBioS 4th year seminar
By Pablo Bravo
QBioS 4th year seminar
Discover how white-light interferometry can measure biofilm growth, their 3D structures, dynamics, and topographies. Presentation made for the QBioS 4th year Seminar at GeorgiaTech.
