Discussion of the mobility of Ps.fluroscens and E.coli

Comparing the data of the mobility of bacteria E. coli, it can be found that the speed of E. coli motions measured in three different patterns fluctuates little. The speed of E. coli in irregular blocks is within the range of speed measured in free motions in plaza zone. It decreased little when the bacteria are in parallel tracks. However, comparing the routines of E. coli in three patterns as well as the angles, it can be observed that it is becoming difficult for bacteria to move in a more complex track as the irregular-blocks part shows. In addition, both three parts of the E. coli indicates that it is difficult for E. coli cells to make a turn on a large angles, typically, larger than 90℃. Therefore, the movement of E. coli cells in confined channels is indeed affected by the patterns, and different patterns can influence the mobility in different aspects like the speed and the angles.

Comparable analysis of the mobility of another bacteria Ps. fluroscens can be obtained basing on the results as well. The causes to the variations in mobility of two bacteria in observations can be explained not only because of the different structure of patterns, but also the bacteria own behaviors. However, the variations of mobility of E. coli cells concern more about the aspect of patterns, while the changes of that of Ps. Fluroscens are associated more with the bacteria own behavior. As it mentions, narrower channels would result in a smaller speed of E. coli, and more complicated channels with different turning points would influence the routines of E. coli and even the marching process consequently. But the remarkable distinctions of the mobility of each Ps. fluroscens cell should be further explained by the bacteria behavior. During the experiment of Ps. Fluroscens, it can be found that a large proportion of bacteria were kept still or spinning at certain locations for a long time. Moreover, it is observed that the bacteria would be much easier to be jammed at each corner of channel or the turning points of a right angle. This phenomenon is related with the peritrichous flagella of the bacteria, as the flagella may be stuck to the channel or the surface of PDMS, which consequently leads to each individual cell to rotate or to be motionless. As there are a large number of Ps. Fluoroscens become jammed in the PDMS channel, the sample size of moving bacteria are too small, so that the analysis of mobility of Ps. Fluroscens may lack accuracy and ubiquity.

Other factors that may cause inaccurate final results are related with the bacteria physiology and the utilization of the software Image J for data processing. Both the E. coli and Ps. Fluroscens have the cell division process during the motions in micro channels, and it slowed down the march of some bacteria. The proper reason for this may be the surface area were growing larger because of the cell division which it is easier for the bacteria to be stuck to the wall channel, and also cell division will charge more energy of bacteria.In addition, the Ps. Fluroscens are may bleach to colorless when they are exposed to the UV lights for a very long time as Figure 1 and 2 indicates. This will result in the great difficulties in observing and recording the bacteria, and errors therefore may be generated in the analysis of the motions of Ps. Fluroscens. There are some errors created during the data analysis with image J, for example, the errors in tracking the trajectories of bacteria manually, and the time measurement. Apart from that, as it has been mentioned previously, errors of measurement are existed since the bacteria may go deeper in the channels instead of going straight forward.

Figure 1 Bacteria initial states

Figure 2 Bacteria bleach after exposing to the UV light for a long time

The mobility of Ps.fluroscens

Three patterns used in the experiment of E. coli, the plaza zone, the parallel tracks and the irregular blocks, have also been applied in the research of Ps. Fluroscens. As mentioned in introduction, the Ps. Fluroscens has a type of specific protein that enables the bacteria to present a bright green color under the UV lights. Then, it will be clearer to observing the Ps. Fluorescens under a dark background as all the figures present. Firstly, in the plaza zone, cells of Ps. Fluroscens in this area are generally in random movements as Figure 1 presented, and the trajectories of cell are similar to an irregular wave form. The average speed measured and calculated by the Image J due to the trajectories recorded is around the 14.01 um/s with an error of -6.57 um/s. The error is mainly resulted from the wave-liked routines of Ps. Fluroscens movements. Image J can only record and measure the projection distance of the wavy routines on the surface. So the vertical distance in microfluidics channel has not been involved in the calculations as the part of the movements of Ps. Fluroscens cells.

This phenomenon is also existed in the Ps. Fluroscens movements in parallel tracks. However, the measured mobility of bacteria in parallel tracks has not been influenced much by this phenomenon, since the channel is much narrower to the bacteria comparing to the plaza zone, and the Ps. Fluroscens cells have smaller free space to vary the directions. Velocities of bacteria in track six, seven and eight have been calculated. The speed of bacteria in these three tracks are in a range from 10 um/s to 29.14 um/s and the average speed is about 24.975 um/s. From the observation, the lowest speed of the bacterium can mainly be explained as the trajectory in microfluidics is going deeper inside of the channel rather than going forward as Figure 2 and 3 shows. As the Image J can only measure the displacement projected on the surface, the measured length of trace is smaller than the actual length, and the speed in calculation is smaller in consequence as well.

Figure 1 Trajectories in plaza zone

Figure 2 Ps. fluroscens enter the tracks first

Figure 3 Ps. Fluroscens going deeper in the tracks

Irregular block

Since the pseudomonas fluorescens can produce fluorescence when they are exposed by UV light, the observation of the mobility of pseudomonas fluorescens in the irregular block area become intuitionistic to see. It can be seen that the movement of them varied significantly from that of E. coli. Pseudomonas fluorescens did not resemble E. coli which preferred to swim straight in the microchannels or even turn at smaller angles. The majority of pseudomonas fluorescens just tended to stick together and remained their location with only small swing of their body. This phenomenon was generated by one of characteristics of ps. Fluorescens, which is the flagella of them prefers to adhere to the wall or other place for a long time. In that case, bacteria are not capable to leave although they dislike being stable. Hence, some bacteria who struggled to be free just could rotate themselves with fixed flagella. The figure 4 shows that two pseudomonas fluorescens were stable to keep their location in parallel at initial time and then one of bacteria spun to change its orientation. This phenomenon also indicated that some of them had the will to move in the microchannels. It can also be concluded that the bulk of them stayed at the corner of each irregular block without any shift in displacement and since the longtime of being irradiated by UV light, they became dim. The bacteria in the plaza-like space which is separated by the irregular block could be divided in several groups rather than distributed anywhere (figure 5). All the observations demonstrated that pseudomonas fluorescens processed the property to stick to the substance and the probability of moving was lower than others.

Figure 4 Ps. fluroscens stick together

Figure 5 Ps.fluroscens in groups

E. coli in Plaza zone and plaza tracks

E. coli cells in plaza zones normally are in random motions with no permanent routines. The trace of bacteria have been tracked and recorded by Image J. Hence, the routines of E. coli cells are visualized, and the speed of their movements can be measured as well. According to the statistics, the swimming speed of E. coli is in the range from 19.15 um/s to 35.39 um/s, and the average speed of E. coli cell in microfluidics are 23.37 um/s. In addition, it is found that a number of E. coli have the tendency to swim until they reach the wall of PDMS, and then the bacteria will continue moving along the planar as the Figure1 and Figure2 showed. This is related with the flagella of the E. coli cells. As Ramia et al. has concluded in the model of hydro-dynamism, the movement of bacteria in aqueous medium can be benefited from the propulsion generated during the process when it is closed to the surface and move along with planar.

Figure 1

Figure 2

The second pattern in study is the parallel track that has been mentioned in previous section. Eight parallel tracks with different width connecting with the plaza zone have been provided to the E. coli cells. The motions of E. coli in this pattern can be divided into three stages, entering the tracks, moving inside the tracks, and exiting the tracks. According to the observation results, the movements of entering the tracks from the plaza zone and exiting the tracks to the plaza zone are similar. The average speed of entering the channels from the plaza is 26.69 um/s, and the average speed of exiting the channels to the plaza is 25.51 um/s. Moreover, when considering the routines of entering the channel, as the Figure 3 has presented, it can be seen that three traces of three E. coli cells entering the channel from the plaza at the same time are generally in a line along with the same orientation that parallel to the channels. There is a little number of E. coli turning a right angle immediately after coming in or out of the channels from or to the plaza.

Figure 3

After bacteria enter the channel, their movements inside the parallel tracks are typically the linear motions along with the wall of channels.The average speeds of eight different channels have been calculated based on the measurements. It can be seen that the speed of E. coli in channel 1 with the largest width is similar to the speed in plaza zone. When the channel width decreases, but is still larger than the size of bacteria, the speed of bacteria has decreased to around 15 um/s. Reasonable explanations to the too small number of velocity may be related with the bacteria behaviors such as the cell division, which will be discussed later. The low velocity of channel eight indicates that the bacteria swim quite slower comparing to those of other channels. This is because the width of channel eight is very close to the size of E. coli cells, so that flagella of E. coli cells are difficult to be entirely stretched to assist the swimming.

                                                                                                   Wrote on 21/02/2012

The analysis of the bacteria in irregular blocks

In this area, the space is divided by several irregular blocks with different angles at the corner. Bacteria in the plaza-like space near this pattern would not like to swim into channels since during seven seconds, only three to eight bacteria move into it. Bacteria in the right plaza-like space have more probability to go into channels. According to the analysis of the movement of the bacteria; the majority of them tend to go straight through the channels and moving closely along the wall is a character of their movement. Compared with the bacteria which move into this area through the middle and nether channel, the probability of changing direction for bacteria which swim into the top channel is also lower. When there are about 3 bacteria in the whole area, generally only one bacterium choose to turn at any block and when the number of bacteria increase to 7 or 8, about three of them will change their direction of mobility. However, if the bacteria swim into the end of the channel, the majority of them do not know where to go and just stay or rotate at the origin location and several of them will go back and only few of them know to turn at the right angle. Therefore, it can be seen that the bacteria prefer to turn at the smaller angle which is easy to vary their own direction. This is the reason why those bacteria move into the middle channel like to turn right rather then left. The statistics also indicate that, large percentage of the bacteria choose to turn at the block 1 or 8 and then to turn at the block 2 or 7 since the angle fluctuation of block 1 is just 28.24°, block 8 is 26.68°and angles of other blocks generally increase. The average speed of the movement is about 22.83μm/s.

                                                                                        Wrote on 13/02/2012 

Observation of the mobility of bacteria

The second part of the project is observing the mobility of bacteria in microfluidic structures. We practiced this in the bioelectronics laboratory on Feb 3rd, 2012 with Dr. Andrew Libberton.
Two kinds of bacteria which are called E.coli and Pseudomonas fluorescens were used to be researched. Escherichia coli which is gram-negative and rod-shaped is often found in the lower intestine of warm-blooded organisms (Figure 1). Pseudomonas fluoresces is also a gram-negative and rod-shaped bacteria. They have multiple flagella and can be in the soil and in water (Figure 2).

Figure1.E.coli

Figure2. Pseudomonas fluorescens

Since the PDMS has been produced, the next step is to put the bacteria into the channels on the PDMS. Seen from our eyes, the shape of the channel is just a stripe (Figure 3). However, seen from microscope, there are several different kinds of patterns. Using the pipette to collect the bacteria and put them on both sides of the channel uniformly (Figure 4). When collecting the bacteria, the experimenter should keep the cover of the bacteria container closed so as to protect them from contamination. Lid the container of the PDMS and paste the cover with PARAFILM (Figure 5).

Figure 3 PDMS container

Figure 4 Pipette

Figure 5 PARAFILM

The mobility of the bacteria should be observed in the relatively dark room. Camera was used to record the image which is observed by the microscope. The temperature for the operation is minus 75 ℃. A kind of oil can be used to lubricate the lens of the microscope and the glass for better observation.

There are several patterns for recording the movement of the bacteria. Firstly, we observed the mobility of the bacteria in the plaza-like space without channels and recorded whether the most of them went into channels or just move in the space. Secondly, we saw the bacteria swim in the stripe structure. Some of them just went straight quickly through the channel. Some of them swam a few micrometres and went back. The third pattern has several channels and these channels were divided by the different kinds of polygon. We wanted to know which channel the bacteria like to move and what angle the bacteria tend to turn. The fourth pattern is diamond structure with several squares inside it so that bacteria have so many choices or channels to swim from one block to another. Other patterns such as comb structure, small maze structure or a structure like a duck were all used in the experiment to record the various kinds of movement of the bacteria.

All videos of the mobility of the bacteria have been downloaded and they will be the vital materials for us to analyse the characteristic of the movement with software Image J in the next week.

Thanks to the Dr. Andrew Libberton and good luck to us.

                                                                                                           Wrote on 05/02/2012

The fabrication of PDMS

We started our first project practice work on Jan 30th, 2012, Monday, this week. Thanks to the great help from Dr. Andrew Libberton, we recieved a valuable video of the microfabrication of PDMS by a research group in MIT. This provides us a basis on how to make the PDMS, which is the fundamental experiment required appliance as the part of microfluidics.

The video link: http://www.jove.com/video/203/chemotactic-response-of-marine-micro-organisms-to-micro-scale-nutrient-layers

Here is the video notes dictation records.

Stage 1:
Clean the wafer with specifically required solvents.
Use the shining side to code.
Dry the wafer with Nitrogen.
Bake, 130°C, 5-10 mins
Prepare two hot plates, 65°C and 95°C at the same time.

Stage 2:
Spin-coating the wafer:
Pouring the photoresist, low and no bubbles.
Let the photoresist stay on the wafer for 1 min before spin.
Spin time: total: 55 secs; 0-500 rpm, 5 secs; spin extra 10 secs; 500-3000 rpm, 7secs; spin extra 30 secs.
Place the wafer after spining on hot plates: 65°C, 5 mins; then 95°C, 20 mins.
Cooling down: 65°C 2-3 mins; room temperature, 5 mins or more.

Stage 3:
UV lights expose:
Exposed part: harder.
Unexposed part: wash away.
Mask used for exposition: the side with ink on it is adhere to the wafer.
Safty! Wearing Glasses!
Then expose the wafer.
Finally, put the wafer exposed on hot plates: 65°C 5mins, 95°C 20 mins (the time on hot plates varies from numbers of parameters involved.

Stage 4:
Develop:
Before the further operation, the photoresist on wafer was needed to be removed first.
Place the specific solvent (PMMA) into a beaker, then put the wafer in.
Fresh solvent to wash the extra photoresist on wafer, then, another solvent again.
At last, use the hydrogen to dry the wafer.
Stage 5:
PDMS:
Mix PDMS powder and curing agent (liquid) with ration 10:1;
Large amount of bubbles should be generated while mixing.
Fix the wafer with tape
Use the compressor to clean the dust on wafer.
Pour the PDMS on wafer.
Put the mixed PDMS and wafer in vacuum container to debubbled.
Finally, when there is no bubble, put them into the oven; 65 °C, 12hrs.

Stage 6:
Get the PDMS:
Use a knife to peel off the PDMS, and take them out. Knife should be careful with little pressure. The first cut stops when the knife hit the wafer.
Punch the holes, for inlet and outle.
Tap the channel to remove the dust.
Bond the PDMS to a glass slide for experiment.


On Monday afternoon, we went to the Bioelectronics lab in University of Liverpool to fabricate the PDMS in practise. We are not actual touch the equipment or wafer or solvents, because most of them are dangerous, and are required of operations by highly qualified experimenters. However, it is still fantastic to take part into this.

Photos of lab work were attached in blog.

First time to the Bioelectronics Lab

Vacumm container

Wafer on the heating pot.

This is for spinning the photoresist.

Part of the equipment used for fabricating the PDMS.

                                                                                                       Wrote on 02/02/2012

Preparation work 2

Today our project has a great progress! We had met with Dr. Ben Libberton to learn about his present project that is related with our project topic. I really appreciate him for all the information he provided! It is really important for us and has great help to us understand project well.

Here are records of what Dr. Ben Libberton provided. All information is under the property of Dr. Libberton.

Firstly, his project was about observing the mobility of bacteria in microfluidics to solve mathematical problems, which is quite similar with ours. Mathematical puzzles such as the maze can be solved by bacteria when the base number of the problem is so large that solving process by computer would take a long time. The essential part is the junction channel built for mathematical problems. I think we need to to focus this part in our project, and try to figure out the thesis before the lab work. There are two types junction, three folks and four folks.

The bottleneck at this stage basically is some types of bacteria have the phenomenon called swarming that they may block one particular channel consequently terribly influence the results. Solutions for this are two, changing the types of bacteria, and changing the media or taxis. Actually, I think this two are relevant, because you may need to change the nutritive subtanceof agar to obtain a different taxis when you switch the bacteria in use. One part of his current experiment is to modify the sort of bacteria.

Some other fundamental concepts includes the constructing process of the PDMS, which is applying the microlithography technology on the silicon vafer. Construction may contains the step the UV radiaction. Next stage we would figure out the exact steps of construction.

We also have learned a numbers of names of bacteria, for examples, pseudomonas, serratia, and flavobaterium. Some common bacteria already known are E.coli, cyanobacteria and spirochetes. These bacteria has differnet taxis, but generally most of them have the chemotaxis. Other taxis that can be utilized are phototaxis, geotaxis and magnetotaxis. The chemical and physical properties of the substratum will be modified for specific requirement I think.

I have borrowed some books about the MICROFLUIDICS to do more reference acknowledgment. Dr. Libberton was willing to provide us some reference about the researching problem like the mathematical puzzles or something like that. Knowledge of bacteria, biochemistry lab work process, and some other parts related to this project will be complemented in the following Chrismas break. Oh, hope the God would help me to accomplish all of this.

Here again express my appreciation to Dr. Libberton.

                                                                                                            Wrote on 13/ 12/ 2011