Introduction

Motility is one of the most readily demonstrated bacterial characters, and chemotaxis is one of the most studied bacterial behaviors. Motile organisms are attracted by certain chemicals and repelled by others (positive and negative chemotaxis). Quantification of chemotactic motion is necessary to identify chemoeffectors and to determine the structure of bacterial communities.

Current methods of quantifying chemotaxis use chemotactic bacteria such as Escherichia Coli, which is assayed by measuring the number of organisms attracted into a capillary tube containing a chemoattractant.

UPO-Sevilla team has carried out different experimental prototypes which try to have under control all the parameters involved in chemotaxis. The goal of the group was to design different assays that allow us to study this effect in both point of views, qualitative and quantitative. Also we had to test induction of sensing systems and chemoattractant production. Anyway, these behaviours are highly related with the chemotaxis process in Bacterial Crowding project. This is why we wanted to measure chemoattractant production by counting the range of attracted bacteria by using chemotaxis assays. The induction of sensing systems could be tested by using GFP measures when its promoter is PfecA or PprhJ; or also due to the levels of chemoattractant production.

Qualitative Assays

Agar Soft Plates

Our qualitative assays were made in soft agar plates thanks to the protocols we received from Ph.D Parkinson (University of Utah).

This kind of plates allows bacteria to swim through the agar freely and to show their chemotactic capacities. A colony, inserted in soft agar plate, starts to grow while running out of the environmental sources. For this reason chemotactic bacteria move to places where the sources are not limited. That phenomenon produces a number of halos which are spread within the plate and increase in volume as the sources are lowered. The number of halos give us information about the number of chemoattractants that are running out.

Fig 1. Soft agar plate assay. Different E. coli strains are shown. Every strain carries out a mutation that affects its chemotactic response, but the wild type. In the wt you can observe two halos produced by chemotactic responses to aspartate (bigger halo) and glutamate.

The assay protocol is simple; once the soft agar plates are prepared, a colony is inserted in the agar. Let it grow at 30ºC. The soft agar is a delicate element, so it is important to be careful when moving the plates.

In those plates it could appear different concentric circles which represent chemotaxis to certain attractants. For instance, when two amino acids, which act as chemoattractants, are running out from the medium, two circles will appear. The inner one will show the amino acids limit with low chemotactic response; while the outer one will mean that the amino acid which causes a higher response is running down.

Optical and Fluorescence Microscopy

The microscopy techniques allow us to see the development of the assay in situ without waiting. In this part we will see how we can carry out an experiment that we could see under the microscope.

Fig 2. Preparation of capillary assay to look under a microscope.

Two capillaries are put over a microscope slide which will hold up a cover slip. Then we insert the bacterial dilution between the slide and the cover slip. Two new capillaries are inserted between the slide and the cover slip, inside of the bacterial dilution. One of those capillaries would contain a chemoattractant while the other one would be the control. Under a microscope we could see the difference between both capillaries and we would definitely be able to observe if there is chemotaxis toward this chemoattractant.

Apart from that, we could detect fluorescence emitted by a fluorophore which should be presented in bacteria by using a fluorescent microscope.

This assay can become quantitative too if we spread on agar plates the content of capillaries and count the number of colonies that grew there. Also we could measure the fluorescence inside each capillary, taking to this way other quantitative results.

Fig 3.Results of a capillary assay using microscope techniques. We can see that the chemotatic response toward aspartate is increasing as time passes by. Also there are major differences between the control without aspartate and the control with aspartate.

Quantitative Assays

Capillary assays

The capillary assays are the most useful to quantify chemotaxis. This team has performed different kinds of capillary assays.

Fig 4. On the left, a capillary assay with 96-well PVC microplates and 1μl capillary pipettes; capillaries are inserted through a 2% agarose gel in order to hold it. On the right, a capillary assay using needles and a tip chamber. Both of them were being incubated at 30ºC when pictures was taken.

• Foundamental Points

  When a capillary with chemoattractant is put in a bacterial dilution a concentration gradient of chemoatractant is developed according to the Fick law. This gradient would be sensed by bacteria that are going from low to high concentration places, thus we should have someThis gradient is sensed by bacteria and they start going from low to high concentration places. This is how some of these bacteria finish into the capillary and so it is possible to demonstrate that capillaries with chemoattractant attract more bacteria than another without any substance (the control), just buffer. The control has to continue the same treatment than the other capillaries. In the same way it could be tested repellent’s efficacy by showing that in its capillary there are less bacteria than in the control.

  Bacterial dilution recipient or Chemotaxis Chambers	Attractant or Buffer recipient or Capillaries
  tip chambers	syringe’s needles (more or less thin)
  needle’s cups or heated closed 100ml tips	Needles
  tip chambers	10μl micropipette’s tips
  96-well PVC microplates	1μl capillary pipettes
  drop between microscope slide and cover slip	1μl capillary pipettes
  Flow-chamber	1μl capillary pipettes

  Table 1. Several ways to permorm chemotaxis assays we have carried out.

  This team has performed those assays on different scales, using differents chemotactic chambers where the bacterial dilution was put inside and with differents capillaries. Some types of these variants are presented in the Table 1. The attractant concentration in the capillary depends on the substance itself, but for aspartate the optimum is reached at 10mM.

  

  Fig 5. Flow-chamber Capillary Assay. This picture is shown a three channel flow-chamber, which acts as chemotaxis chamber, with 1μl capillaries fixed in each end of one channel. At that time it was observed with a fluorescence microscope.

  One assay that deserves to be explain is the Multi-capillary assay. It uses 96-well PVC microplates as chemotaxis chamber and 1μl glass capillaries. You can see a picture of it in figure 4, at the left. We designed this chemotaxis assay when we were looking for a good way to perform numerous and simultaneous capillary assays. The assembly of the assay required to make holes in a microplate lid to allow glass capillaries to go through it. The lid is put upside down over the microplate and it is filled with 2% agarose gel. We also used parafilm to avoid that the gel drip through the lid holes while it was still humid. The function of the gel is to hold capillaries, in its dry way. In orther to fill capillaries we closed one of its ends by heating and heated the glass; then it was introduce in a attractant or buffer suspension and automatically it is filled. It is important to introduce the capillaries in the lid and gel by its closed end to keep the sterility on the other end. Then it is only necessary to put the open ends of capillaries inside the wells of the microplate. Previously these wells should have been filled with a bacterial dilution. At this point the assay continues in the same way than others: 1h incubation, clean outside of capillaries with bidistilled water, dilution of the capillaries content and spread in agar plates.

  We got our best results in the last kind of capillary assay showed in the table. It uses flow-chambers as chemotaxis chambers and 1μl capillary pipettes as capillaries. This method allows us to quantify bacterial density in two different ways, visualization of flow-chambers by fluorescent microscope techniques and spreading bacteria inside capillaries in LB plates . Actually, we think that this assay leads to less errors than others due to some reasons: when we put a capillary into the flow-chamber the fixing is perfect and the capillary can not move, the exposed part of the capillay inside the chamber is minimal so bacteria can not attach to the outside of the capillary making errors in dilutions, also it is a small device easy to work with.

• Protocol

  

  Fig 6.Representation of capillary assay in a tip chambers.

  The assays can start in two different ways: setting up inocula from the strains we are going to work with into triptone broth or in minimal medium, in a low shaking at 30ºC overnight. High shaking might cause the loss of flagella, and also the production of flagella would not be possible in rich medium since bacteria would not need it.

  The following day the inocula should be diluted in the same medium a hundred of times and wait for the growing phase to be the appropriate. For Escherichia Coli it would be necessary to wait for the exponential middle phase, since it is in this phase when flagella develop the flagellar motor. For Pseudomonas instead it would de better to wait for the late exponential phase, as the flagellum is developed later in this organism.

  Once the culture is ready, it must be changed to an appropriate medium for mobility and chemotaxis. For that, it is necessary to wash the culture twice with chemotaxis buffer by centrifuging at low speed, since flagella may be lost if culture is treated abruptly.

  When the culture is in the right medium, number of bacteria should be adjusted roughly 10⁷ fcu/ml. This dilution has to be distributed in chemotaxis chambers where our capillaries will be introduced. The volume of capillaries has not been fully established and usually we used as standard volume 100 μl of diluted chemoattractant in chemotaxis buffer. It is important not to forget controls. Negative controls for chemotaxis assays are filled with chemotaxis buffer. Then chambers and capillaries have to be incubated at 30º during 60 minutes. After that, we have to quantify bacterial popularion contained into capillaries. In order to achieve that we could do it either with dilution and spread in plates or analyzing the fluorescence, supposing that bacteria have any kind of fluorophore.

• Advices

  One of the elements we bear in mind is the chemotaxis buffer: chemotaxis medium contains potassium phosphate buffer (pH 7), ethylenediaminetetraacetate (EDTA) and glycerol (energy source). Glycerol is only necessary in long incubations; meanwhile in short incubations the typical sources of bacteria are enough to maintain the chemotactic machinery. It is all-important to underline that the chemotaxis medium must be free of any other substance which may have chemotactic effects, since this could disturb the results. This is one of the reasons why the carbon source is not glucose, as you may expect. Another important detail to bear in mind is EDTA, this chelation causes the precipitation of magnesium which may dull the movement of bacteria and the flagellar machinery. It would be complicated to be successful in the chemotaxis assays without this chelation. Incubation of bacteria must be carried at 30ºC since it helps motility. Shaking must be low because flagella can be lost in high shaking.

  It is crucial to be careful when choosing the strains to be used in the chemotaxis assays, since it may not have motility. Strains used in laboratories have normally no motility, as at that point they have usually suffer different screening process in benefit of immobile bacteria. A bacterium which has no motility will not have to invest any source in motility or chemotaxis; this would encourage the creation of a bigger colony and more eye-catching than usual, so Scientifics would be probably leaded to select one of this kind. This problem happened to us and we were trying to attract a non mobile strain toward different attractants until we saw the light.

Buridan’s Donkey

At the beginning of the summer we wanted to test bacterial chemotaxis by using a three-channel device based on flow-chamber biofilm. It would be able to produce a linear gradient within narrow tubes that connect the chambers. The linear chemical gradient would be generated by diffusion of the chemoattractant through a dialysis membrane located on the limit of the chamber. This membrane also makes impossible the movement of the chemoattractant-producing bacteria through the tube.

The first assay would involve only chemoattractants, and the second producing bacterias. As result, it would be expected that the movement of the cells in the center chamber was directed to the chamber containing chemoattractant-producing bacterias. It is necessary to clear up that the chemoattractant production would be activated solely by the contact of bacteria with plant cell walls that reside in the same chamber. Bacteria have to “decide” between going toward control empty chamber or going toward chamber with chemoattractant-producing bacteria.

This device could provide some advantages in chemotaxis studies: rapid and easy implementation, parallel and simultaneous test, visual proofs, different assays possibilities. Also some experimental conditions could be changed easily: concentration of bacterial population, chambers distances, bacterial cultures, chemoattractans. Despite its advantages, it maybe requires more than an hour of incubation.

Although we had not enough time to perform this assay, an explaining diagram of this device is provided below.

Measuring the performance of the chemotaxis circuits (Buridan's donkey assay principle)

Buridan's donkey assays with three-channel flow cells

Special acknowledgements to Ph.D Parkinson (chemotaxis researcher in University of Utah) who gave us some advices, handed us over some protocols of him, even mobile and mutant E. Coli strains.

References

• J. Adler (1972) A Method for Measuring Chemotaxis and Use of the Method to Determine Optimum Conditions for Chemotaxis by Escherichia coli. - Journal of General Microbiology ( I 973), 74, 77-91
• Guocheng Han and Joseph J. Cooney (1993) A modified capillary assay for chemotaxis - Journal of Industrial Microbiology, 12 (1993) 396—398
• Hanbin Mao, Paul S. Cremer, and Michael D. Manson (2003) A sensitive, versatile microfluidic assay for bacterial chemotaxis - PNAS MICROBIOLOGY vol. 100 no. 9 5449–5454.
• Russell Bainer, Heungwon Park, Philippe Cluzel (2003) A high-throughput capillary assay for bacterial chemotaxis - Journal of Microbiological Methods 55 (2003) 315– 319.