Cultivating Actinobacteria for Microscopic Analysis
In order to analyse actinobacteria using light microscopy, methods must take into account the multicellular nature of the test organism. Unlike unicellular organisms, actinobacteria are highly structured and grow in unique morphologies. To visualise this growth, it is possible to take a cross section from colonies by growing them along a coverslip on agar, which is then removed and mounted.
As the aerial hyphae and spores of many actinobacteria are hydrophobic, they will easily stick to a glass coverslip. After incubation, these coverslips will contain distinct zones of vegetative hyphae, aerial hyphae, and if given enough time to sporulate - spores.
- Pour an agar plate using a recipe that your strain of interest grows well on.
- Insert a sterile coverslip into the plate at a 45° angle to the surface of the agar.
- Pipette ~5μl of a 1:100 spore stock or liquid culture along the agar/coverslip interface.
- Allow to dry, then invert the plate and incubate at the optimal growth temperature until mature growth is observed.
- Carefully remove the coverslip from the agar, being mindful not to disturb the specimen.
- Lay the coverslip on a flat surface growth side up, and add 8-10μl of a 40% glycerol solution or PBS. Do not add water as this may lyse the cells.
- Lay a glass slide on the coverslip and mount the specimen.
- Seal the edges with clear nail varnish and allow to dry thoroughly.
- The slide is now ready to be imaged on the light microscope.
For long term storage at -20°C, specimens can also be fixed prior to mounting. See Staining of Streptomyces spp. Cell Wall and Nucleic Acids.