A trap for in situ cultivation of filamentous actinobacteria

Abstract

The approach of growing microorganisms in situ, or in a simulated natural environment is appealing, and different versions of it have been described by several groups. The major difficulties with these approaches are that they are not selective for actinomycetes - a group of gram-positive bacteria well known as a rich source of antibiotics. In order to efficiently access actinomycetes, a trap for specifically capturing and cultivating these microorganisms in situ has been developed, based on the ability of these bacteria to form hyphae and penetrate solid environments. The trap is formed by two semi-permeable membranes (0.2-0.6 microm pore-size bottom membrane and 0.03 microm pore-size top membrane) glued to a plastic washer with sterile agar or gellan gum inside. The trap is placed on top of soil, and filamentous microorganisms selectively penetrate into the device and form colonies. Decreasing the size of the pores of the lower membrane to 0.2 microm restricted penetration of fungi. The trap produced more filamentous actinobacteria, and a higher variety of them, as compared to a conventional Petri dish cultivation from the same soil sample. Importantly, the trap cultivation resulted in the isolation of unusual and rare actinomycetes.

Fig. 1. Image and diagram of the trap

1, plastic or metal washer; 2, top membrane filter (0.03-μm pore-size); 3, bottom membrane filter (0.2- to 0.6-μm pore-size); 4, agar or gellan gum.

Fig. 2. Microorganisms growing in the trap

The traps were opened after 2 weeks of cultivation. a. Bacterial colonies and microfungal hyphae, agar-base trap with 0.4-μm pore-size bottom membrane. Bar, 1 mm. b. Actinomycetes-like microcolonies, gellan gum trap with 0.2-μm pore-size bottom membrane. Bar, 0.1 mm.

Fig. 3. Microcolonies of Actinobacteria from the trap

The trap was incubated for an additional week at room temperature after it was opened. Actinobacterial aerial (a, b, c) and substrate (d) mycelia. Gellan gum trap with 0.2-μm pore-size bottom membrane. Bar, 0.05 mm