Coincubation Assay for Studying Competitive Interactions Between Bacterial Strains

Samenvatting

Source: Speare, L., et al. Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates. J. Vis. Exp. (2019)

This video demonstrates a co-incubation assay used to study how bacterial strains compete in a shared environment. It is widely applied in microbiology to identify mechanisms, such as contact-dependent or contact-independent killing, that drive these interactions.

Protocol

1. Coincubating strains (Figure 1B, C)
   1. Mix the reference strain and competitor strain in a 1:1 ratio (v/v) by adding 100 μL of each strain (normalized to OD 1.0) to a labeled 1.5 mL centrifuge tube. As a control, mix the reference strain with a differentially tagged version of itself (reference strain pVSV102 + reference strain pVSV208). This control is required for later statistical analysis to determine whether the presence of a competitor strain impacts the growth of the reference strain. Vortex the mixed-strain culture for 1-2 s.
      NOTE: This step may require optimization as the starting ratio can significantly impact results and may need to be adjusted.
   2. Repeat step 1 for each biological replicate. After completing this step, there should be a total of eight mixed-strain tubes: four biological replicates with differentially-tagged reference strains (control), and four biological replicates with differentially-tagged reference and competitor strains (experimental).
   3. Spot 10 μL of each control and experimental mixture onto Petri plates containing LBS agar; these culture spots will be used for fluorescence microscopy after the incubation.
   4. Allow the spots to dry completely on the bench until all liquid has been absorbed into the agar, and incubate the Petri plates at 24 °C for 24 h. A minimum of 15 h is required for V. fischeri strains tagged with pVSV102 and pVSV208 to grow to a high enough cell density to visualize green fluorescent protein (GFP) and red fluorescent protein (RFP), respectively, at the population level using a fluorescence dissecting microscope. Here, we use a 24 h incubation for imaging mixed spots.
      NOTE: It is important to use plates that are not too moist or too dry. If the plates are too moist, the coincubation spots will not be absorbed into the agar plate; avoid using plates poured on the same day. If the plates are too dry, small waves or cracks may form on the agar surface, making the coincubation spots irregular in shape.

Resultaten

Figure 1: Flowchart illustrating the coincubation assay. (A) Bacterial strains harborin...

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
1.5 mL Microcentrifuge Tubes	Fisher	05-408-129
10 μL multichannel pipette
100 μL multichannel pipette
300 μL multichannel pipette
10 μL single channel pipette
20 μL single channel pipette
200 μL single channel pipette
1000 μL single channel pipette
24-well plates	Fisher	07-200-84	sterile with lid
petri plates	Fisher	FB0875713	sterile with lid
TipOne 0.1-10 μL starter system	USA Scientific	1111-3500	10 racks
TipOne 200 μL starter system	USA Scientific	1111-500	10 racks
TipOne 1000 μL starter system	USA Scientific	1111-2520	10 racks
LBS media
1M Tris Buffer (pH ~7.5)			50 mL 1 M stock buffer (62 mL HCl, 938 mL DI water, 121 g Trizma Base)
Agar	Technical Fisher	DF0812-17-9	15 g (Add only for plates)
DI water			950 mL
Sodium Chloride	Fisher	S640-3	20 g
Tryptone	Fisher	BP97265	10 g
Yeast Extract	Fisher	BP9727-2	5 g