Which methods did we use?

Qualitative Chemotaxis Analysis – Drop Plate Assay

The qualitative investigation of chemotaxis induction has been investigated by drop-plate-assays which give an insight into movement properties of bacterial colonies on bacto-agar/LMT-agarose-plates. When the chemotaxis specificity towards Cd2+ validated we further investigate migration speed and efficiency in different chip-based assays.

Equipment and Growth Media * 10x PBS recipe at end of document (1 L)
  • Chemotaxis-Buffer (CB1) = Phosphate Buffered Saline (PBS), pH 7.2 + 20 µM EDTA
  • Chemotaxis-Buffer (CB2) = Phosphate Buffered Saline (PBS), pH 7.2
  • Noble-Agar (Ultra-pure agar w/o any possible attractants)
  • MilliQ-Water
  • LB-medium
  • Chemoattractant = Asp, Glu, Ser, Thr, Asn, Gln, Tyr, (Cys, His), malonic acid, succinic acid, maltose, glucose, ribose, galactose, CoCl2, NiCl2, FeCl2, ZnCl2, MgCl2, CaCl2, MnCl2, CdCl2 (If no chloride is available any other salt resulting in a divalent cation is suitable too), Dipetides (Asp-Phe) – Create 500 µL stock-solutions of 1 M
  • Petri-dishes (96 mm)
Growing the Bacterial Inoculum for Chemotactic Test
  1. Inoculate 200 ml of LB with 1–2% (V/V) of overnight grown seed-culture.
  2. Incubate at appropriate temperature with optimal shaking.
  3. Let the culture grow up to mid-log phase (OD600 ∼ 0.35–0.4).
Harvesting and Re-Suspension of Bacterial Cells
  1. Pellet the cells by centrifugation at 5,000 × g for 20 min.
  2. Gently decant the culture supernatant and re-suspend the bacterial pellet in 25–30 ml of phosphate buffer saline for washing.
  3. Pellet the cells by centrifugation at 5,000 × g for 20 min.
  4. Re-suspend the cells in 10 ml of chemotaxis buffer (OD600 ∼ 1.5)
Preparation of Semi-Solid Medium
  1. Add 0.3–0.4% (w/v) agar in 75 ml of Chemotaxis Buffer. Heat it to enough that the agar gets melted.
  2. Cool the agar to ∼50°C and then add the re-suspended bacterial cells from the above procedure to the cooled agar.
  3. Pour the agar with bacterial cells in 96 mm petri dish (making a semi-solid medium bed that is neither very thin nor very thick).
  4. The test substance is solubilised to a concentration of 1 M. 50 µL of the stock solution is applied on the respective spots. (Alternatively, for insoluble AA/reagents. Place 1–5 small size crystals/a pinch of powder of the test compound on the plate. (The amount of added compound should be in range of a 10–50 μg.)
  5. Incubate the plate at ambient temperature for 30 min.
Observation of Chemotaxis Rings (Swarm Rings)
  1. Monitor the petri dish after 30 min of incubation; Accumulation of bacterial cells around the compound in the form of concentric ring indicates a positive chemotactic response.
  2. In case of the weak chemoattractants, the concentric ring may be slightly feeble. In case of such chemicals the incubation time may be increased to 4–6 h.
  3. Take photos of each chemoattractant spot after 30 min. If no ring is visible let incubate for additional 2-4 h and document result.

Quantitative Analysis – µ-slide Chemotaxis Chamber (IBIDI GmbH, Martinsried, Germany)

To analyse swimming properties and speeds, a chip-based assay has been used. Adapted as described earlier (Elgamoudi et al., 2016). Basically, a chip consisting of two 60 µL reservoirs one filled with LMT-agarose infused with Cd2+, the other with bacteria and LMT-agarose are separated by a 10 µL stripe (transition zone) is imaged under a microscope. Different cadmium concentrations and other chemoattractants have been used to see desired chemotaxis-dependent migration from one reservoir to the other.



Fig 1.

Figure 2: Graphical representation of experimental set-up using µ-slide chemotaxis chamber (IBIDI GmbH, Martinsried, Germany). 10 µL of 1% autoclaved low melting (LMT) agarose gel solution is filled in the central transition zone chamber (observation chamber) and left at room temperature for 10 min. 60-65 µL of 1% low melting temperature (LMT) agarose gel infused with bacteria w and wo chemoattractant is applied to one liquid reservoir chamber. The second chamber only includes Cd2+ at different concentrations in 1% LMT agarose. (Elgamoudi et al., 2016).


Growing the Bacterial Inoculum for Chemotactic Test
  1. Inoculate 200 ml of LB with 1–2% (V/V) of overnight grown seed-culture (Chemotaxis active strain and negative control UU3330).
  2. Incubate at appropriate temperature with optimal shaking.
  3. Let the culture grow up to mid-log phase (OD600 ∼ 0.35–0.4).
    4. Prepare Chemotaxis Slides
  4. Fill transition zone (middle) with 10 µL of 1% LMT Agarose gel and leave at room temperature for 10 min.
  5. 60 µL of 1% LMT agarose + Chemoattractant is filled in the left reservoir of the chemotaxis slide.
    6. Harvesting and Re-Suspension of Bacterial Cells
  6. Harvest cells at OD600 = 0.35 – 0.4. Pellet the cells by centrifugation at 5,000 × g for 20 min.
  7. Gently decant the culture supernatant and re-suspend the bacterial pellet in 25–30 ml of phosphate buffer saline for washing.
  8. Pellet the cells by centrifugation at 5,000 × g for 20 min.
  9. Re-suspend the cells in 10 ml of chemotaxis buffer (CB2) adjust to OD600 ∼ 1.
    10. Apply Bacterial Cells in LMT Agar to right chamber
  10. Mix 500 µL of cells with 1% LMT agarose gel, adjust to OD600 = 0.5.
  11. Optionally add 0.1% Tween-20 to avoid cell adherence.
  12. Add 60 µL of bacterial solution to right reservoir chamber.
  13. Incubate for 60 min at microbic conditions.
    14. Data Acquisition via single cell tracking/colony tracking
  14. Place chemotaxis chip under microscope with 40x – 60x objective lens, focus centre of transition channel.
  15. Record 3x 3 min videos of bacterial migration with 15 frames per second.
  16. Repeat with negative control and only buffer filled chambers.

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