Host Basics_ Microbacterium foliorum

An Introduction to the Host Bacteria, Microbacterium foliorum

As a network of researchers, SEA-PHAGES explores the genetic diversity and relationships of populations of bacteriophages that infect Actinobacteria, incluidng the bacterium Microbacterium foliorum (M. foliorum). There are many different strains of M. foliorum (isolated from different environmental samples). The M. foliorum strain widely used in SEA-PHAGES is called M. foliorum SEA B-24224 that was isolated from grass in Germany, and made available to you from the Agricultural Research Service of USDA. If you intend to isolate phages using this bacterium, then this will be your ‘host’ for phage isolation.

M. foliorum is one of more than 90 species that make up the genus Microbacterium, classified within the phylum Actinobacteria. Microbacteria are gram-positive rod-shaped bacteria that are slightly smaller than typical rod-shaped bacteria, hence the name microbacteria. Microbacteria

have been isolated from soil, water, and plants. Microbacteria have also been isolated from cheese rinds, where it may contribute to the flavor profile.

M. foliorum will grow and divide on many nutrient-rich media. In your research, you will grow M. foliorum on PYCa media at 28 – 30 ˚C. Under these conditions, M. foliorum grows rapidly, dividing approximately once every 90 minutes. This means that it typically takes about 2 – 3 days for an individual cell to form a colony on an agar plate. However, when preparing a bacterial lawn, you can

start by adding many bacterial cells (e. g., 10⁷ or more) so that a lush lawn grows within 24 hours. M. foliorum will also grow at room temperature (~ 22 ˚C), but it divides more slowly and therefore requires extra time to form either a colony or a lawn, typically an additional 24 hours. M. foliorum does not grow at 37 ˚C. For reproducibility of experiments, it is important that cultures used for a

given set of experiments be prepared at the same temperature.

M. foliorum colonies are tan to yellow in color, smooth and glossy (Figure 4.0-4). When incubated for prolonged periods, or when grown at room temperature, the color of the colonies are a brighter yellow. A liquid culture of M. foliorum inoculated from a single colony will take 24 hours to form a saturated liquid culture, exhibiting a tan to yellow color, which intensifies with prolonged incubation

or when grown at room temperature. The strain you will work with, M. foliorum, is not inhibited by the antifungal cyclohexamide. Therefore, cycloheximide can be added to the growth media to prevent other microorganisms from growing in your cultures.

On average, 30 % of attempts to isolate phage from soil by enrichment with M. foliorum yield phage.

Figure 4.0-4. Microbacterium foliorum growing on an agar plate.

If you are using this bacterium as your host for phage isolation, refer back to the list below for the specific growth and culture requirements for your experiments.

Growth media: PYCa

Growth temperature: optimal at 28 ˚C - 30 ˚C; grows well at room temperature (~ 22 ˚C); this host does not grow at 37˚C.

Antimicrobials: cycloheximide (10 μg/ml)

Phage Buffer: 10 mM Tris (pH 7.5), 10 mM MgSO₄, 68 mM NaCl, 1 mM CaCl₂, (10% glycerol, optional)

Restriction Enzymes: HaeIII, MseI, NspI, SacII, and SalI.

Note: Isoschizomers, which are different restriction enzymes that recognize the same DNA sequence, may be used in place of any of the enzymes listed above.