Staining of Streptomyces spp. Cell Wall and Nucleic Acids: Difference between revisions
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==Cultivation of Strains== | ==Cultivation of Strains== | ||
Strains should be grown on sterile 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. | 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. | 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. | ||
==Staining== | ==Staining== | ||
# 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.* | |||
# Wash with PBS. | |||
# Incubate with 2% BSA in PBS for five minutes at room temperature. | |||
*Strain dependent step, see notes. | |||
==Visualisation== | ==Visualisation== | ||
Revision as of 15:30, 26 July 2019
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.
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.
Staining
- 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.*
- Wash with PBS.
- Incubate with 2% BSA in PBS for five minutes at room temperature.
- Strain dependent step, see notes.
