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.

Method

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.

Reagents

Required Reagents

• 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

PBS

• 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

GTE

• 50mM Glucose
• 20mM Tris (pH 8.0)
• 20mM EDTA
• Made up in sterile distilled water

Stains

Stains	Stock	Working Concentration	Volume of Stock in 10ml PBS
Fluorescein-WGA	1mg/ml	2µg/ml	20µl
Propidium Iodide	12.5mg/ml	10µg/ml	8µl

Fixative

	Stock Concentration	Working Concentration	Volume for 4ml	Volume for 20ml
Glutaraldehyde	25%	0.0045%	0.7µl	3.5µl
Formaldehyde	40%	2.8%	280µl	1.4ml

(Dilute with PBS)

Staining

1. Add fixative to each coverslip and incubate at room temperature for 15 minutes.
2. Wash coverslips twice with PBS and allow to air dry.
3. Rehydrate each sample in PBS for five minutes.
4. Cover each coverslip with a solution of 2mg/ml lysosome in GTE for one minute.(1)
5. Wash with PBS.
6. 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.

1. 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.
2. Wash each sample eight times with 10µg/ml propidium iodide in PBS.
3. Remove excess solution and add 8µl of slow fade solution or 40% glycerol in ddH₂O.
4. Mount the coverslip on a clean glass slide and seal with clear nail varnish.