Staining of Streptomyces spp. Cell Wall and Nucleic Acids
The following staining technique is a useful method for visualising the actively growing regions of the cell wall, along with the nucleic acids within the cell. The method was developed by Schwedock et al.
This method is well established for Streptomyces coelicolor, S. lividans, S. lydicus, S. goldiniensis, S. clavuligerus and S. collinus. Furthermore, this protocol can also be used for other Actinomycetes such as Pseudonocardia.
Cultivation of Strains
Strains should be grown on sterile coverslips (Type 1.5 square coverslips) that have been inserted into agar at a 45° angle. It is advised that the strain of interest should be grown on a media on which it readily sporulates. Coverslips can be sterilised in ethanol or in an autoclave at 121°C.
After the insertion of the coverslip, 5µl of a 1:100 spore stock dilution is pipetted along the length of the agar-coverslip interface and incubated at the temperature at which the strain grows best (usually 30°C). It is advised to harvest coverslips at two different time points - one before sporulation and one after.
- Fixative - Store at 4°C
- PBS - Store at Room Temperature
- 2mg/ml lysozyme in GTE buffer - Store at Room Temperature, shake well before use
- 2% BSA in PBS - Store at 4°C
- FITC-WGA staining solution (2µg/ml FITC-WGA, 10µg/ml propidium iodide, 2% BSA in PBS) – stored at 4°C
- Propidium iodide staining solution (10µg/ml PI in PBS) – stored at 4°C
- 40% Glycerol in distilled water - Stored at 4°C
- Clear Nail Varnish
- NaCl 8g
- KCL 0.2g
- Na2HPO4 1.44g
- KH2PO4 0.24g
- ddH2O 800ml
- pH to 7.4 with HCL and make up to 1000ml ddH2
- 50mM Glucose
- 20mM Tris (pH 8.0)
- 20mM EDTA
- Made up in sterile distilled water
|Stains||Stock||Working Concentration||Volume of Stock in 10ml PBS|
|Stock Concentration||Working Concentration||Volume for 4ml||Volume for 20ml|
(Dilute with PBS)
- Add fixative to each coverslip and incubate at room temperature for 15 minutes.
- Wash coverslips twice with PBS and allow to air dry.
- Rehydrate each sample in PBS for five minutes.
- Cover each coverslip with a solution of 2mg/ml lysosome in GTE for one minute.(1)
- Wash with PBS.
- Incubate with 2% BSA in PBS for five minutes at room temperature.
(1)Strain dependent step, see notes.
In order to prevent photobleaching of stains, complete all following steps in the darkroom.
- Cover each sample in staining solution (2µg/ml fluorescein-WGA, 10µg/ml propidium iodide, 2% BSA in PBS) and incubate for one to three hours at room temperature.
- Wash each sample eight times with 10µg/ml propidium iodide in PBS.
- Remove excess solution and add 8µl of slow fade solution or 40% glycerol in ddH2O.
- Mount the coverslip on a clean glass slide and seal with clear nail varnish.