The dialogue between protozoa and bacteria in a microfluidic device

Abstract

In nature, protozoa play a major role in controlling bacterial populations. This paper proposes a microfluidic device for the study of protozoa behaviors change due to their chemotactic response in the presence of bacterial cells. A three-channel microfluidic device was designed using a nitrocellulose membrane into which channels were cut using a laser cutter. The membrane was sandwiched between two glass slides; a Euglena suspension was then allowed to flow through the central channel. The two side channels were filled with either, 0.1% peptone as a negative control, or a Listeria suspension respectively. The membrane design prevented direct interaction but allowed Euglena cells to detect Listeria cells as secretions diffused through the nitrocellulose membrane. A significant number of Euglena cells migrated toward the chambers near the bacterial cells, indicating a positive chemotactic response of Euglena toward chemical cues released from Listeria cells. Filtrates collected from Listeria suspension with a series of molecular weight cutoffs (3k, 10k and 100k) were examined in Euglena chemotaxis tests. Euglena cells were attracted to all filtrates collected from the membrane filtration with different molecular weight cutoffs, suggesting small molecules from Listeria might be the chemical cues to attract protozoa. Headspace volatile organic compounds (VOC) released from Listeria were collected, spiked to 0.1% peptone and tested as the chemotactic effectors. It was discovered that the Euglena cells responded quickly to Listeria VOCs including decanal, 3,5- dimethylbenzaldehyde, ethyl acetate, indicating bacterial VOCs were used by Euglena to track the location of bacteria.

Fig 1. Conceptual design of the microfluidic device used in this study.

The sample was introduced from the right end of the center channel. The chambers are named as 1^st, 2^nd, and 3^rd (from right to left) set of chambers to represent the sequences of a sample to reach the entrances of the chambers.

Fig 2. Microfluidic assembly on the stage of the microscope.

A syringe pump was used to control the flow rate of the Euglena suspension. The Euglena sample was added in the reservoir on the right end of the central channel and pulled through the microfluidic system using a syringe pump connected on the left.

Fig 3. Euglena cell distribution inside the microfluidic device with two side channel filled with 0.1% peptone during the experimental duration.

The flow rate of Euglena suspension was 3 μL/h. Pictures were taken at the end of each set of chambers at 0 min, 10 min, 20 min, and 30 mins after the chemmoeffector (0.1% peptone) was introduced in the microfluidic system.

Fig 4. A suspension of 6 μm polystyrene beads was pulled into the center channel at a flow rate of 3 μl/h by a syringe pump.

The pictures were taken at the both ends of the first chamber and parts of center channel between the entrances of the first set of chambers. It was observed that the beads did not reach the bottom of chambers and they had a tendency to accumulate in the central channel.

Fig 5. Euglena distribution (percentage of observed cells in top chambers over time) inside the microfluidic chambers without a chemoeffector.

Uneven distribution of the illumination light from the microscope caused more cells to move to the top of the chambers.

Fig 6

Euglena cell distribution inside the microfluidic device with one side channel (top) filled with 0.1% peptone and the other side channel (bottom) filled with Listeria suspension during the experimental duration. The flow rate of Euglena suspension was 3 μL/h. Pictures were taken at the both ends of the chambers and used for cell distribution analysis.

Fig 7. Euglena distribution (percent of total observed cells over time from initiation of experiment) inside the microfluidic chambers with the bottom side channel filled with Listeria suspension.

Fig 8

Total ion GCXGC chromatographs of a peptone buffer control (top) and a Listeria VOC sample (bottom) showed different VOC features present in VOC sample compared to the buffer. Peaks can be located with two retention times as x and y axis and peak intensity is in z axis.

Fig 9. Population shift of Euglena cells inside the microfluidic system with different VOC molecules served as chemoattractant.

Significant population shift can be observed when ethylhexyl acetate and decanal were used as chemoattractants.

Fig 10

Euglena cell distribution inside the microfluidic device with one side channel (top) filled with 0.1% peptone and the other side channel (bottom) filled with decanal spiked 0.1% peptone during the experimental duration. The flow rate of euglena suspension was 3 μL/h.