An In Vitro Assay to Study Microbial Interactions in Co-Culture

Abstract

Source: Temkin, M. I. et al. High Throughput Co-culture Assays for the Investigation of Microbial Interactions. J. Vis. Exp. (2019)

This video demonstrates an in vitro assay to study microbial interactions between Corynebacterium propinquum and Staphylococcus in a co-culture.

Protocol

1. Target Organism Inoculation

NOTE: After overnight incubation, ensure that the cultures are turbid. Some bacterial cultures may flocculate at the bottom of the culture tube. Vortex the culture tubes to disperse clumps and assess the culture turbidity.

1. Prepare the target plate by filling each well of an empty 12 well plate with 1.8 mL of Brain-heart-infusion (BHI) broth and 200 µL of the target overnight culture.
   NOTE: A target organism refers to the organism whose inhibition status is determined using the co-culture interaction assay. For this experiment, the target organisms are Staphylococcus spp. isolated from human nasal lavages samples.
2. Unwrap and place a sterile inoculation stamp into the target plate. Gently swirl the cultures around in the wells, taking care to ensure that the cultures do not cross contaminate neighboring wells.
3. Lift the inoculation stamp and ensure that there is a droplet of diluted target culture on each stamp tip.
4. Prepare a monoculture control plate by placing the inoculation stamp on an uninoculated bioassay plate and gently rock the stamp so that a culture drop inoculates each well. Once the inoculation stamp is removed, a droplet of culture should be visible in the wells of the bioassay plate.
   1. If any wells are not inoculated with the inoculation stamp due to uneven levels of media, spot 3 µL of diluted overnight culture onto the right side of the wells using a pipette.
5. Inoculate the bioassay plates as done above (see step 1.4.1), but align the stamp so that the tips align with the right side of the 12 well plate. Ensure that the stamp does not contact the existing bacterial colony when inoculating the wells.
6. Carefully remove the stamp and place it back into the target plate.
7. Repeat the inoculation for each bioassay plate until all the plates are inoculated.
8. Incubate bioassay plates upside down at the appropriate temperature for 7 days.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
12-well cell culture plate, sterile with lid	Greiner bio-one	665 180
Agar, bacteriological	VWR	J637
Brain Heart Infusion Broth	Dot Scientific	DSB11000-5000