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

Summary

The co-culture interaction assays presented in this protocol are inexpensive, high throughput, and simple. These assays can be used to observe microbial interactions in co-culture, identify interaction patterns, and characterize the inhibitory potential of a microbial strain of interest against human and environmental pathogens.

Abstract

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many approaches used for the study of microbial interactions require specialized equipment and are expensive and time intensive. This paper presents a protocol for co-culture interaction assays that are inexpensive, scalable to large sample numbers, and easily adaptable to numerous experimental designs. Microorganisms are cultured together, with each well representing one pairwise combination of microorganisms. A test organism is cultured on one side of each well and first incubated in monoculture. Subsequently, target organisms are simultaneously inoculated onto the opposite side of each well using a 3D-printed inoculation stamp. After co-culture, the completed assays are scored for visual phenotypes, such as growth or inhibition. These assays can be used to confirm phenotypes or identify patterns among isolates of interest. Using this simple and effective method, users can analyze combinations of microorganisms rapidly and efficiently. This co-culture approach is applicable to antibiotic discovery as well as culture-based microbiome research and has already been successfully applied to both applications.

Introduction

In nature, microorganisms rarely exist in isolation; consequently, they are constantly interacting with other organisms. Therefore, studying how microorganisms interact with each other is essential to understanding a multitude of microbial behaviors¹. Microbial interactions can be mutualistic, commensal, or antagonistic. These in teractions can affect not only the microorganisms themselves but also the environments and hosts that the microorganisms colonize^1,2.

Many scientists study microbial interactions to identify new antimicrobial molecules. One of the fi....

Protocol

Informed consent was obtained from the donor's parents, and the Human Subjects Committee at the University of Wisconsin-Madison approved the study (Institutional Review Board [IRB] approval number H-2013-1044).

1. Sample Culture

NOTE: This procedure is used here for the study of interactions among bacteria isolates from the human nasal cavity. In principle, the following methods are applicable to any culture condition. Brain-heart-infusion broth (BHI) is used for general propagation of nasal bacteria. All plates are solidified using 1.5% agar. For this study, samples are taken from saline solut....

Results

Co-culture interaction assays can be used to understand microbial interactions, identify patterns of interest, and uncover microbial isolates with intriguing activities. In these assays, a test organism is monocultured on one side of a 12 well agar plate and incubated for 7 days. Subsequently, a target organism is spotted next to the test organism and the two microbes were co-cultured for 7 days before scoring for the growth phenotype of the target organism. The assays are scored based on.......

Discussion

Antibiotics and other secondary metabolites that mediate microbial interactions are useful for a multitude of applications, including drug discovery. Herein, a protocol for co-culture assays to assess large numbers of microbial interactions is presented. These co-culture interaction assays are a simple, affordable, scalable, and high throughput means to investigate many pairwise combinations of microorganisms in tandem. Target organisms are spotted next to test organisms in a well of a 12 well plate using an inoculation .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 μL disposable polystyrene inoculating loops, blue	VWR	12000-806
10 μL disposable polystyrene inoculating loops, yellow	VWR	12000-810
12-well cell culture plate, sterile with lid	Greiner bio-one	665 180
14 mL polystyrene round bottom tube, 17 x 100 mm 2style, nonpyrogenic, sterile	Falcon	352057
2.0 self standing screw cap tubes with caps, sterile	USA scientific	1420-9710
25 mL serological pipet	Cell Treat	229225B
Agar, bacteriological	VWR	J637
Brain Heart Infusion Broth	Dot Scientific	DSB11000-5000
Polycarbonate filament, white, 3 mm diameter	Keene Village Plastics	12.1-3MM-WH-581.2-1KG-R
School Glue	Elmer's	EPIE304
Taz 6 3D printer	Lulzbot