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 an attractant.

UPO-Sevilla team has carried out different experimental prototypes that are better to achieve. The goal of the group was designing different assays that allow us to study this effect in both point of views, qualitative and quantitative.

Qualitative Assays

Agar Soft Plates

Our qualitative assays were made in soft agar thanks to the protocols that we had received from Mr. Parkinson (University of Utah).

This kind of plates allows bacteria to swim trough the agar freely and showing their chemotactic capacities. A colony, inserted in soft agar plate, start to grow while running out the environmental sources. For this reason bacteria would 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 assay protocol is simple; once the soft agar plates are prepared, a colony is inserted in the previous plate. Let it grow in 30ºC. The soft agar is a delicate element, so it is important to be careful when moving the plates.

In those plates it might appear different concentric circles which represent chemiotaxis in a certain attractant. For instance, when two amino acids are run out from the medium, two circles will appear. The inner one will show the amino acids limit with low chemotactic ability; while the outer one will mean that the amino acid with a bigger ability will be running down.

Optical and Fluorescence Microscopy

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

Over a microscope slide two capillaries are put 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 close to the bacterial dilution. One of those capillaries will contain a chemoattractant while the other one will be the control. Under the microscope we can see the different between both capillaries and we will definitely be able to observe if there is chemotaxis toward this chemoattractant.

Apart from that, we can detect the fluorescence emitted by the fluorophore which are present in bacteria using a fluorescen microcope.

Quantitative Assays

Capillary assay

The capillary assays are the most useful to quantify chemotaxis. Although with some problems, this team has performed a capillary assay.

• Foundations

  A capillary, which is put in a bacterial dilution, makes a concentration gradient of chemoatractant, produced by thr flow that goes from capillary to the medium according to the Fick law (see http://en.wikipedia.org/wiki/Fick's_laws_of_diffusion). This gradient would be sensed by bacteria that are going from low to high concentration places thus we should have some bacteria into the capillary. We can demonstrate that when we compare a capillary chemoattractant with another without any substance (the blank), just the buffer. The blank has to continue the same protocols than the others. In the same way could be tested the efficacy of a repellent since the capillary with the repellent will have less bacteria than the blank.

  This team has performed this assay using chemotactic chambers where the bacterial dilution was put inside. We used both syrnge’s needle (more or less thin) and micropipetes’ tips (10 l). The attractant concentration in the capillary depends on the substance itself.

• Protocol

  The experiment may start in two different ways; putting inocula from the strains we are going to work with into triptone broth or in minimal medium in a low shaking at 30ºC overnight. A high shaking might provoke the loss of flagella. The production of flagella wouldn’t be possible in a rich medium since bacteria wouldn’t need it.

  The following day the inoculla 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 this phase when flagella develop the “flager” motor. For Pseudomonas instead it would better wait for the late exponential phase, as the flagellum is developed later in this organism.

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

  When the cultive is in the right medium the number of bacteria is adjusted roughly to 107 fcu/ml. This dilution has been distributed in chemotactic chambers; our capillaries have been introduced in it. The volume of capillaries can be unsettled; we have used as a standard volume 100 ul of diluted chemoattractant in chemotactic buffer. Mind controls, they will be capillaries thanks to the chemotactic buffer.

  Incubate the experiment at 30º (información adicional!) during 60 minutes, after that we have to quantify bacteria that are contained into the 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.

• Advice

  One of the elements we bear in mind is the chemotaxis buffer: chemotaxis medium contain potassium potassium phospgate buffer (pH 7), ethylenediaminetetraacetate (EDTA) and glycerol (energy source). The 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 chemotactic 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, how you may expect. Other important detail to bear in mind is EDTA, this chelation provoke the precipitation of magnesium which may dulled the movement of bacteria and the flagellar machinery. It would be complicated to success in the chemotactic assays without this chelation. Incubation of bacteria must be carried at 30ºC since it helps motility. Shaking must be low as flagella can be lost in high shaking.

  It is crucial to be careful when choosing the strains to be used in the chemotactic 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 won’t have to invest in any source in order to create the flagelar motor; this would encourage the creation of a colony bigger and more eye-catching than usual, so Scientifics would be probably leaded to select one of this kind.

Buridan’s Donkey

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

The first assay involves only chemoattractants, and the second one includes producing bacterias. As result, it is expected that the movement of the cells in the center chamber was directed to the chamber containing chemoattractant-producing bacterias, for the cells chemotactic response, but not in the control chamber, in the opposite side. It is necessary to clear up that the chemoattractant production is 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 provides a lot of advantages in the study of chemotaxis: rapid and easy implementation, parallel and simultaneous test, visual proofs, different assays possibilities. This mechanism also let us changes experimental conditions, for instance: concentration of bacterial population, chambers distances, bacterial cultures, chemoattractans.

An explaining diagram of this device is provided below.

Special acknowledgements to Mr. Parkinson (University of Utah) who gave us some advices, handed us over some protocols of him, even an E. Coli mobile strain.

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.