Real-Time Monitoring of Bacterial Growth Using a Microfluidic Microdroplet Culture System

Abstract

Source: Jian, X. et. al., Automated Microbial Cultivation and Adaptive Evolution using Microbial Microdroplet Culture System (MMC). J. Vis. Exp. (2022)

This video demonstrates the use of a microfluidic droplet-based platform for real-time monitoring of bacterial growth. By generating uniform droplets containing bacterial suspensions and guiding them through a detection pathway, the system enables continuous optical measurement of cell density to construct a growth curve.

Protocol

1. Preparations

1. Connect the syringe needle (inner diameter is 0.41 mm and outer diameter is 0.71 mm), quick connector A, and reagent bottle (Figure 1C), and autoclave them at 121 °C for 15 min.
   NOTE: Unscrew the cap of the reagent bottle slightly during sterilization. A few more reagent bottles can be prepared each time for use.
2. Use a 0.22 µm polyvinylidene fluoride (PVDF) filter to filter the microdroplet culture system (MMC) oil. Put the microfluidic chip (Figure 1B) and MMC oil into the clean bench in advance and sterilize them by ultraviolet irradiation for 30 min before use.
   NOTE: For the details of Quick connector A, reagent bottle, MMC oil and microfluidic chip refer to the Table of Materials.
3. Install the microfluidic chip
   1. Open the door of the operation chamber (Figure 1A) and lift the optical fiber probe.
   2. Align the electric field holes with the electric field needles and gently place the chip on the chip pedestal. Then insert the two positioning columns into the positioning holes and put down the optical fiber probe (Figure 1D).
   3. Connect the quick connector A on the chip to the corresponding port of the MMC according to the position number (C5-O5, C4-O4, C6-O6, C2-O2, CF-OF, C1-O1, C3-O3). Then close the door of the operation chamber.
4. Replenish the MMC oil (to about 80 mL) in the oil bottle and empty the waste liquid in the waste bottle before use.
   NOTE: The waste liquid is usually organic waste. Please refer to regional law and regulation upon disposal, subject to change based on experimental setup.

2. Growth curve measurement in MMC

1. Preparation for the initial bacterial solution
   1. Follow the related standard regulations to prepare Luria-Bertani (LB) medium and autoclave at 121°C for 15 min.
      NOTE: Components of LB medium: NaCl (10 g/L), yeast extract (5 g/L) and tryptone (10 g/L).
   2. Take out the Escherichia coli (E. coli) MG1655 strain from glycerol stock and cultivate it in a 50 mL shake flask with 10 mL of LB medium in a shaking incubator (200 rpm) at 37 °C for 5-8 h.
      NOTE: The cultivation time depends on the specific strains. It is optimal to cultivate the strain in the logarithmic period/phase.
   3. Dilute the cultured E. coli MG1655 solution with fresh medium to an optical density at 600 nanometers (OD₆₀₀) of 0.05-0.1 to obtain an initial bacterial solution (prepare about 10 mL).
2. Click on Initialization to initialize the MMC. After the initialization interface appears, set the cultivation temperature as 37 °C and the photoelectric signal value as 0.6 (Figure 2A). Initialization will take about 20 minutes.
3. Turn on the ultraviolet (UV) lamp (wavelength 254 nm) during initialization.
4. Inject the initial bacteria solution and MMC oil into the reagent bottle.
   1. Take out a sterilized reagent bottle on the clean bench and tighten the cap.
   2. Use a 10 mL sterile syringe to inject 3-5 mL of MMC oil from the syringe needle of the side tube. Tilt and rotate the reagent bottle slowly to make the oil fully infiltrate the inner wall.
   3. Inject about 5 mL of initial bacteria solution, and then fill the reagent bottle by injecting 5-7 mL of the oil again.
   4. Pull out the independent quick connector A, and insert the quick connector A of the reagent bottle into its quick connector B to complete the sample injection operation (Figure 3A).
5. Wait for the initialization to end and then turn off the UV lamp (wavelength 254 nm).
6. Open the door of the operation chamber, and put the reagent bottle into the metal bath.
7. Pull out the C2 connector of the chip and the quick connector A of the reagent bottle. Connect the side tube connector of the reagent bottle to the C2 connector and the top tube connector to the O2 connector. Then close the door of the operation chamber.
8. Click on Growth Curve to choose the function of growth curve measurement (Figure 2A). In the parameter setting interface, input the Number as 15, turn on the OD detection switch, and set the Wavelength as 600 nm. Click on Start to start droplet generation. It will take about 10 minutes.NOTE: Here, Number refers to the number of droplets to be generated. Wavelength refers to the wavelength of the OD to be detected. Set the Number (maximum 200) and Wavelength (350-800 nm) according to the experiment requirements.
9. When a pop-up window appears on the main interface prompting "Remove the reagent bottle between C2 and O2, then please click the OK button after completion", open the door of the operation chamber to take out the reagent bottle and connect the C2 and O2 connectors.
10. Close the door, and click the OK button in the pop-up window to automatically cultivate the droplets and detect the OD values.
    NOTE: The MMC detects the OD value when the droplet passes the optical fiber probe. Therefore, the detection period depends on the number of droplets generated.
11. When the growth curve reaches the stationary phase, click the Data Export button to export the OD data. Select the data save path and export the OD value recorded during the cultivation period in the .csv format, which can be opened by appropriate software (e.g., Microsoft Excel). Then use a mapping software (e.g., EXCEL and Origin 9.0) to plot the growth curve.
    NOTE: During the cultivation process, it is feasible to click on the Data Export at any time to export the OD data of all the current droplets.

Results

Figure 1: Structure and essential tools of MMC. (A) External and operational chamber of MMC. (B) The microfluidic chip of...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.22 μm PVDF filter membrane	Merck Millipore Ltd.	SLGPR33RB	Sterilize the MMC oil
4 °C refrigerator	Haier	BCD-289BSW	For reagent storage
Agar	Becton, Dickinson and Company	214010	For solid plate preparation
Clean bench	Beijing Donglian Har Instrument Manufacture Co., Ltd.	DL-CJ-INDII	For aseptic operation and UV sterilization
Computer	Lenovo	E450	Software installation and MMC control
Constant temperature incubator	Shanghai qixin scientific instrument co., LTD	LRH 250	For the microbial cultivation using solid medium
Electronic balance	OHAUS	AR 3130	For reagent weighing
EP tube	Thermo Fisher	1.5 mL	For droplet collection
Freezing tube	Thermo Fisher	2.0 mL	For strain preservation
Glycerol	GENERAL-REAGENT	G66258A	For strain preservation
High-pressure steam sterilization pot	SANYO Electric	MLS3020	For autoclaved sterilization
Microbial microdroplet culture system (MMC)	Luoyang TMAXTREE Biotechnology Co., Ltd.	MMC-I	Performing growth curve determination and adaptive evolution. Please refer to http://www.tmaxtree.com/en/index.php?v=news&id=110
Microfluidic chip	Luoyang TMAXTREE Biotechnology Co., Ltd.	MMC-ALE-OD	For various droplet operations. Please refer to http://www.tmaxtree.com/en/
MMC oil	Luoyang TMAXTREE Biotechnology Co., Ltd.	MMC-M/S-OD	The oil phase for droplet microfluidics. Please refer to http://www.tmaxtree.com/en/
NaCl	GENERAL-REAGENT	G81793J	Component of the LB medium
Petri dish	Corning Incorporated	90 mm	For the preparation of solid medium
Pipette	eppendorf	2.5 μL, 10 μL, 100μL, 1000μL	For liquid handling
Quick connector A	Luoyang TMAXTREE Biotechnology Co., Ltd.	—	For the connection of each joint. Please refer to http://www.tmaxtree.com/en/
Reagent bottle	Luoyang TMAXTREE Biotechnology Co., Ltd.	MMC-PCB	Sampling and storage of bacteria solution and reagents. Please refer to http://www.tmaxtree.com/en/
Shake flask	Union-Biotech	50 mL	For microbial cultivation
Shaking incubator	Shanghai Sukun Industrial Co., Ltd.	SKY-210 2B	For the microbial cultivation in shake flask
Syringe	JIANGSU ZHIYU MEDICAL INSTRUCTMENT CO., LTD	10 mL	Draw liquid and inject it into the reagent bottle
Syringe needle	OUBEL Hardware Store	22G	Inner diameter is 0.41 mm and outer diameter is 0.71 mm.
Tryptone	Oxoid Ltd.	LP0042	Component of the LB medium
Ultra low temperature refrigerator	SANYO Ultra-low	MDF-U4086S	For strain preservation (-80 °C)
UV–Vis spectrophotometer	General Electric Company	Ultrospec 3100 pro	For the measurement of OD values
Yeast extract	Oxoid Ltd.	LP0021	Component of the LB medium