High Throughput Co-culture Assays for the Investigation of Microbial Interactions

Our protocol provides a high throughput method for screening a large number of pairwise microbial interactions in tandem to identify interactions of interest which can aid microbiome research and antimicrobial discovery. This method is readily applicable to studying microbial interactions in a variety of systems, as long as the microorganisms are amiable to culture under laboratory conditions. The main advantage of this technique is that it allows users to screen a large number of microbial interactions in a relatively short period of time.

New users of this technique should take time, especially when inoculating the bioassay plates, as it is crucial to be careful to minimize overgrowth or contamination. Visual demonstration of this technique is critical, because inoculating the bioassay plates requires precision to ensure that the organisms do not overgrow the wells, and that the co-cultures are not contaminated. Begin by preparing overnight cultures.

Use a serological pipette to add three milliliters of BHI broth into 14-milliliter culture tubes. Then use a sterile one microliter inoculating loop to inoculate a bacterial colony into the broth. Swirl the loop to ensure that the clump disperses into the broth.

Briefly vortex the culture tubes, and incubate them overnight at 37 degrees Celsius on a shaker at 250 RPM. Once the bacterial cultures reach an OD 600 above one, vortex them to break up the clumps of cells. Prepare PHI media with 1.5%agar, and sterilize it by autoclaving.

Then cool the media to 55 degrees Celsius in a temperature-controlled water bath, and dispense three milliliters of molten BHI media into each of the 12 wells on a plate, ensuring that the wells are as even as possible. Then allow the agar to set overnight. Inoculate the test organism on a bioassay plate by inserting a sterile 10-microliter inoculating loop into the overnight culture, and streaking the culture droplet over the left third of a plate well.

Repeat until all 12 wells on the plate have been inoculated. Incubate the plates upside down at the appropriate temperature for seven days. If incubating at temperatures greater than or equal to 37 degrees Celsius, or in drier climates, store the plates in a humid container to prevent them from drying out.

After the six-day incubation of the bioassay plate, prepare overnight cultures of the target organism as previously described. Prepare the target plate by filling each well of an empty 12-well plate with 1.8 milliliters of BHI, and 200 microliters of the target overnight culture. Place a sterile inoculation stamp into the target plate.

Gently swirl the cultures around the wells, ensuring that they do not cross-contaminate neighboring wells. Lift the inoculation stamp, and ensure that there is a droplet of diluted target culture on each stamp tip. Place the inoculation stamp on an uninoculated bioassay plate, and gently rock the stamp so that the culture droplet inoculates each well.

Once the stamp is removed, a droplet of culture should be visible in the wells. If any wells are not inoculated with the inoculation stamp, spot three microliters of diluted overnight culture onto the wells using a pipette. Then inoculate the bioassay plates as previously described, but align the stamp tips with the right side of the 12-well plate, ensuring that the stamp does not contact the existing bacterial colony.

Carefully remove the stamp, and place it back into the target plate. If the agar has solidified in the wells unevenly, gently rock the stamp back and forth, but be careful not to shift the stamp into the test organism growth. Repeat the inoculation for each bioassay plate until all the plates are inoculated, then incubate the plates upside down at the appropriate temperature for seven days.

After co-culturing the test and target organism for one week, score the interactions based on visual assessment. Score the wells with the target organism growth that exhibits indistinguishable growth from the monoculture as zero. Score the wells with diminished growth as one, and no growth as two.

The co-culture assays described here were used to assess the inhibitory activity of the actinobacteria test organisms toward the staphylococcus species target organisms isolated from the human nasal cavity. A total of 812 pairwise combinations were tested. Notably, Corynebacterium propinquum strongly inhibited coagulase negative staphylococci, particularly in comparison to other Corynebacteria that either weakly inhibited coagulase negative staphylococci or had no inhibitory effects.

Through the use of comparative genomics, a biosynthetic gene cluster for siderophore production was identified in the Corynebacterium propinquum genome. Co-culture interaction assays using standard and iron-supplemented BHI medium determined that the inhibition phenotype between Corynebacterium propinquum and coagulase negative staphylococci was in fact iron-dependent. It is important to be careful when stamping the plate.

Double check each well after stamping, and make sure the inoculation stamp is aligned correctly to prevent contamination. After identifying interactions of interest, subsequent studies using microbial genetics and genomics, natural product isolation and characterization, or imaging mass spectrometry can be used to characterize mechanisms underlying these interactions. This protocol has recently been used to uncover siderophore-mediated competition among the human nasal microbiota, and aided in the discovery of a new antifungal molecule called cyphomycin.