Team:Tsinghua/Hardware
Hardware
Description
In order to imitate the flowing condition of the drain-pipe used in our lives, we designed a hardware in which we could cultivate biofilm of the P. aeruginosa and observe it in real time. The main element of the hardware was designed in a suitable size and a transparent material so that it could be placed on an inverted microscope. Silicone tubes connected the main element and an infusion bag to form an airtight container for broth and P. aeruginosa, aiming to keep the culture uncontaminated by other microorganisms. The silicone tubes also connected the element to the pump so that a flowing condition would be formed. After the assembly, this hardware could be put on the microscope, then the cultivation and observation of the biofilm could be conducted in real time. The schematic picture of the hardware is shown in Figure A.
Figure A. The schematic diagram of the hardware
The hardware used in our program is shown in Figure B and C.
Figure B and C. The real product of the hardware
Materials and equipment
1.Main element
The main element was custom made from Eastsea Chuangkai Quartz Limited. After several days of communication with the engineers in the company, every parameter of the element was qualified precisely in order to fit the size of the microscope. The final product could work properly and all the parameters were shown below.
Material: quartz
Thickness of the material: 2mm
Main body:
Shape: Cuboid shape
Length: 60mm
Width: 24mm
Height: 10mm
Connecting tubes:
Shape: Circular
Length: 15mm
Figure D. The schematic picture of the main element.
2.Silicone tubes
Outer diameter: 4mm
Inner diameter: 2mm
3.Disposable sterile infusion bag
250ml sterile infusion bag from Hongda Company
4.Flowing pump:
Brand: Lead fluid
Model number: 348
5.Inverted Microscope:
Olympus IX73
Methods and results
Bacterial strains
P. aeruginosa biofilm culture experiments were performed with a single typical P. aeruginosa biofilm-forming Strain PAO1. To observe the biofilm under the fluorescence microscope, we select a PAO1 strain with chromosomally expressed green fluorescent protein (GFP).
Stock cultures of PAO1-GFP were streaked onto Luria-Bertani (LB) culture plates, and incubated for 24 hours at 37°C 220rpm. Single colonies of each strain were transferred into separate tubes containing 2 mL of sterile m9 broth, and grown overnight in a shaker at 37 °C for injection into the hardware.
Hardware preparation
First, we connected all the tubes and element, closed the valve of the infusion bag, and made sure all the hardware was airtight. Then, 100ml 75% ethyl alcohol was added to the infusion bag in the asepsis work table, along with the pump being turned up for 1h to circulate the ethyl alcohol and disinfect the hardware. Next, we opened valve of the infusion bag and drain out the ethyl alcohol. To make sure the left ethyl alcohol was too little to influence the PAO1-GFP which we planned to add next, we used 100ml m9 broth to wash the hardware twice, each for 20 minutes. Finally, 100ml m9 broth along with 1ml PAO1-GFP solution were added into the hardware through the valve, and the cultivation could start.
Cultivation and observation
The whole hardware was moved to an inverted microscope and the cultivation was conducted there. Before the cultivation, the main element was put on the console of the microscope, so that all the cultivation process could be recorded by the microscope in a real time pattern. During the cultivation, the pump was working all the time and the flowing velocity parameter was controlled at 13, a rather suitable flowing velocity to imitate most drain tubes in our daily lives.
Figure E. P. aeruginosa biofilm observed from instrucment we built
Extra system used in our study: Microfluidic system
At first, we considered two systems to measure the biofilm in the flowing condition: one was the original hardware mentioned above, and another was microfluidic system which had a high accuracy and was often used by biofilm scientists. We discussed the ideas with the Anti-Biofilm Community, Jingtao Liu’s lab and Guoqiang Chen’s lab during the integrated HP process, confirming and improving our hardware designs. Then we developed our microfluidic software and installed it into the microfluidic system. However, due to the limited time and human resources, we haven’t cultivated the biofilm of PAO1-GFP in the microfluidic system. It becomes a work which needs our future exploration.
Figure F. CellAASIC ONIX2 Microfluidic System
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