1、 Actinomycetes

Actinomycetes are a group of prokaryotic organisms widely distributed in nature, and are an extremely important microbial resource closely related to human production and life. Their prominent characteristic is the ability to produce a large number and variety of antibiotics. Actinobacteria can form branching hyphae and conidia, growing in filamentous form, mainly through the formation of asexual spores for reproduction, or through the division of bacterial fragments for reproduction. When actinomycetes grow to a certain stage, a portion of the aerial hyphae form spore filaments, which mature and differentiate into many spores, known as conidia.

Figure 1: Pattern diagram of actinomycetes

Figure 2: The aerial hyphae and spore filaments of actinomycetes

Due to their small size, light weight, and easy dispersal, spores are prone to cross contamination between different monoclonal colonies on the same solid plate, posing a huge challenge to the cultivation and isolation of actinomycetes.

The high-throughput micro upgraded droplet culture omics system of Tianmu Biotechnology, MISS cell, can encapsulate cells (spores) in droplets to form independent culture units, avoiding hyphae or spores from scattering into other droplet clones, thus overcoming the problem of cross contamination between strains and improving the efficiency and purity of obtaining actinomycete clones. This experiment is based on the isolation and cultivation of actinomycetes using mycelium, providing a method for isolating and cultivating filamentous bacteria that do not produce spores.

2、 Isolation and cultivation of actinomycetes using MISS cells

1. Sample pretreatment to determine the concentration on the machine

Collect actinomycete mycelium (30 ℃, cultured for 4 days), add glass strain to break the mycelium and filter, count by hemocytometer, and dilute the sample concentration to 500-1000 CFU/mL with BM medium. The workflow is as follows:

Figure 3 Sample Processing Workflow

2. MISS cell isolation and cultivation

A total of 2500 droplets were generated in this experiment, and after 96 hours of cultivation, detection and sorting were carried out. After 60 minutes of sorting, about 500 bacterial containing droplets were obtained. The workflow of the MISS cell instrument is shown in the following figure.

Figure 4 MISS Cell Workflow

3. Experimental results

1) Comparison of growth rates:Observing the growth of liquid droplets and agar plates at different time points, growth appeared in the droplets after 1 day of liquid droplet cultivation, and mycelial growth appeared after 6 days of agar plate cultivation. The growth rate of actinomycetes in liquid droplet cultivation was significantly better than that in agar plate cultivation, and the optimal cultivation and sorting time for actinomycetes was 4 days.

Figure 5: Growth of Actinobacteria on Solid Plates

Figure 6 Growth of actinomycetes in droplets

2) Sorting results of actinomycete droplets

Figure 7 Sorting of actinomycetes cultured for 4 days in OD mode

Figure 8 Sorting of actinomycetes cultured for 4 days in fluorescence mode

3、 Tianmu Bioscience MISS Cell Instrument provides high-throughput and efficient automation solutions for the isolation, cultivation, and screening of actinomycetes

In this study, the MISS cell instrument of Tianmu Biotechnology was used to generate 2500 droplets, and about 500 single colonies were sorted, achieving automated cultivation and sorting of actinomycetes and effectively avoiding cross contamination between strains. Meanwhile, a method for isolating and cultivating filamentous bacteria that do not produce spores is also provided. A total of 8ml of culture medium was consumed in this experiment, and the entire process was automated to generate, culture, and screen droplets into a 96 well plate, greatly improving the efficiency of monoclonal acquisition while effectively avoiding cross contamination issues, providing an efficient tool platform for monoclonal screening of actinomycetes.

The Tianmu Bioscience MISS cell instrument provides an efficient automated experimental platform for monoclonal culture and high-throughput screening of bacteria containing spores, including actinomycetes, providing strong support for strain cultivation and screening!