This readme.txt file was generated on 2022-02-25 by Francesco Giorgi

GENERAL INFORMATION

1. Title of Dataset: Supplementary material: "Real-time monitoring of dynamics and interactions of bacteria and the early-stage formation of biofilms"

2. Author Information
	A. Principal Investigator Contact Information
		Name: Francesco Giorgi
		Institution: University of Liverpool
		Email: francesco.giorgi@liverpool.ac.uk

3. Date of data collection: from 2021-03 to 2021-09.

4. Geographic location of data collection: Liverpool, UK.

5. Information about funding sources that supported the collection of the data: The Author was founded by the EPSRC.


DATA & FILE OVERVIEW

1. File List: 

Figure S1: Population of E.coli bacteria dispersed in PBS and exposed to a glass surface for 30 minutes. The black arrows highlight the bacteria experiencing lateral adhesion.

Figure S2: Magnification of the middle-right E.coli bacterium in figure S1 which is experiencing lateral adhesion on the control glass surface.

Figure S3: Population of E.coli Escherichia coli bacteria dispersed in a solution of 10% LB in PBS and exposed to a glass surface for 30 minutes. The black arrows point at the bacteria experiencing lateral adhesion while the white arrows point at bacteria experiencing rotary adhesion.

Figure S4: Magnification of the bottom-right E.coli bacteriim in figure S3 experiencing lateral adhesion.

Figure S5: Transition of the dynamics of E.coli bacteria from lateral to rotary adhesion.

Figure S6: Transition of the dynamics of E.coli bacteria from rotary adhesion to random motion over the surface.

Figure S7: Detail of E.coli bacteria cell division.

Figure S8: Transition of the dynamics of E.coli bacteria from lateral to rotary adhesion.

Figure S9: Transition of the dynamics of  E.coli bacteria from rotary motion to random motion over the surface and back to rotary adhesion and motion. 

Figure S10: Transition of the dynamics of  E.coli bacteria from random motion over the surface to rotary adhesion.

Figure S11: E.coli bacteria statically adhere to glass surface coated with BKC in PBS. The white arrow points at a bacteria already statically adhered to the surface. The bacteria highlighted by the arrow at the beginning of the video is out of focus, because the focus was set to the bacteria further from the surface which was appoaching the surface..

Figure S12: E.coli bacteria statically adhere to glass surface coated with BKC in PBS. The white arrow points at a bacterium already statically adhered to the surface. The bacteria highlighted by the arrow at the beginning of the video is out of focus, because the focus was set to the bacteria further from the surface which was appoaching the surface.

Figure S13: Population of statically adhered E.coli bacteria dispersed in PBS and exposed to a glass surface treated with BKC for 1h. It is possible to recognise that the only bacterium not static is the one not in contact with the surface (out of focus).

Figure S14: Population of statically adhered E.coli bacteria dispersed in a solution of 10% LB in PBS and exposed to a glass surface treated with BKC for 1h. 

Figure S15: P.Aureginosa bacterium exhibiting a rotary dynamics once attached to the glass control surface. 

Figure S16: P.Aureginosa bacterium exhibiting a lateral dynamics once attached to the glass control surface.

Figure S17: Population of statically adhered P. Aureginosa bacteria dispersed in PBS and exposed to a glass surface treated with BKC for 1h. It is possible to recognise that the only bacteria not static are the ones not in contact with the surface (out of focus).

Figure S18: Detaila of two P. Aureginosa bacteria statically attached to a surface trated with BKC.

Figure S19: E.coli Bacteria clustering to start biofilms.

Figure S20: E.coli Bacteria clustering to start biofilms.

Figure S21: Detail of the dynamics of a multilayered E.coli biofilm. 

Figure S22: Population of E.coli bacteria dispersed in PBS and exposed to a glass surface for 24h

Figure S23: Population of E.coli bacteria dispersed in a solution of  10% LB in PBS and exposed to a glass surface for 24h.

Figure S24: Population of statically adhered E.coli bacteria dispersed in a PBS and exposed to a glass surface treated with BKC for 24h.




METHODOLOGICAL INFORMATION

1. Description of methods used for collection/generation/processing of data: 

Caustic signatures of the bacteria investigated in this work were generated in a standard inverted optical microscope (Axio Observer.Z1 m, Carl Zeiss, DE) used in transmission mode and mounted on antivibration feet (VIBe, Newport, US) to isolate the sample from the environment. Some simple adjustments were made to the original setup of the microscope to increase the coherence of the illuminating light. In brief, the microscope was adjusted for Köhler illumination and equipped with a 100W halogen lamp, a condenser lens-assembly, and a green interference filter (Olympus, JP, centred on 550 nm, 45 nm bandwidth). The condenser aperture was closed down to its minimum (1 mm in diameter), so that the sample was illuminated by a coherent and focused ray of light. Images and videos were acquired using a CCD monochrome camera (AxioCam ICm1, Carl Zeiss, DE) coupled to the camera port on the microscope. A stage-top incubation system (Incubator PM S1, Heating Insert P S1, Temp and CO2 module S1, Carl Zeiss, DE) was used to mantain a constant temperature of 37°C during the experiments. 
Overnight cultures of Escherichia coli were diluted to McFarland Standard 0.5 in Luria-Bertani (LB) broth or phosphate buffered saline solution (PBS) as appropriate to obtain a working concentration of approximately 108 CFU/mL. Bacteria were diluted in pure PBS or in solution of 10% LB in PBS, to reduce the noise generated by the medium nutrients and to limit their growth and ability to proliferate, thus allowing for longer monitoring of the interaction between single bacterium and the target surface, and the subsequent formation of the biofilm. Solution of bacteria were analysed using 60 µl of solution in a deep cavity (250 ± 10 ?m in depth) in sterile microscopy slides.

The efficiency of benzalkonium chloride (BKC) as antimicrobial agent and its effect on the bacterial-surface interaction was tested by spreading a 0.1% solution of BKC in ultrapure-deionised water on same glass surfaces. Then solutions of bacteria were exposed to the surfaces treated with BKC after 3 h, to allow for the complete evaporation of the BKC solution and for the deposition of the BKC film.

The dynamics of the bacteria was evaluated using the ImageJ plugin Trackmate.