ActinoBase - User contributions [en-gb]

Cultivating Actinobacteria for Microscopic Analysis

2020-01-31T14:51:57Z

Jordanmurray:

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.

==Method==

* 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| Staining of ''Streptomyces'' spp. Cell Wall and Nucleic Acids]].

Cultivating Actinobacteria for Microscopic Analysis

2020-01-31T14:49:55Z

Jordanmurray:

Cultivating Actinobacteria for Microscopic Analysis

2020-01-31T14:49:17Z

Jordanmurray: /* Method */

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.

==Method==

* 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, specimens can also be fixed prior to mounting. See [[Staining of ''Streptomyces'' spp. Cell Wall and Nucleic Acids]].

Cultivating Actinobacteria for Microscopic Analysis

2020-01-31T14:47:03Z

Jordanmurray: Created page with "In order to analyse actinobacteria using light microscopy, methods must take into account the multicellular nature of the test organism. Unlike unicellular organisms, actinoba..."

Protocols

2020-01-31T14:31:07Z

Jordanmurray: /* Microscopy */

[[File:Actinobase-icons-trans_Protocols.png|150px|Protocols|link=Protocols]]

=Protocols organised by category=

==[[Biochemistry]]==

*[[GUS assay]] (β-Glucuronidase assay)

==Microbiology==

*[[Characterisation of Actinomycetes | General Characterisaton of Actinomycetes]]

*[[Exploration | ''Streptomyces'' Exploration]]

*[[General Growth and Maintenance of Streptomyces sp.]]

*[[Isolating Actinomycetes from the environment]]

For a comprehensive guide to actinomycete characterisation see the [https://www.dsmz.de/collection/catalogue/microorganisms/special-groups-of-organisms/compendium-of-actinobacteria Compendium of Actinobacteria] from [https://www.helmholtz-hzi.de/en/research/research-topics/anti-infectives/microbial-strain-collection/our-research/#anchorsection Prof. Joachim Wink] at the German Collection of Microorganisms & Cell Cultures ([https://www.dsmz.de/ DSMZ]). This resouce provides protocols for characterising a wide range of features for new isolates, but may also be applicable more broadly for example for phenotyping experiments. The tests include colony morphology, melanin production, salt tolerance, carbon utlilisation and much more. To download the guide please see the link below.

[https://actinobase.org/downloads/DSMZ.pdf DSMZ Compendium of Actinobacteria]

==[[Microscopy]]==

*[[Staining of ''Streptomyces'' spp. Cell Wall and Nucleic Acids| Staining of ''Streptomyces'' spp. Cell Wall and Nucleic Acids]]

*[[Cultivating Actinobacteria for Microscopic Analysis]]

==[[Mutagenesis]]==

*[[CRISPR/Cas9-mediated genome editing]]

*[[Chemical mutagenesis]]

*[[Depletion strains]]

*[[Lambda-red mediated recombination (PCR-targeting system a.k.a. "Redirect")]]

*[[Lambda-red mediated recombination using ssDNA]]

*[[Meganuclease I-SceI based system for gene deletions]]

*[["Quikchange" site-directed mutagenesis]]

*[[Random mutagenesis using a mutator strain]]

*[[Random mutagenesis using error-prone PCR]]

*[[Suicide vectors]]

*[[Transposons]]

*[[UV mutagenesis]]

==[[Natural Products]]==

*[[Cloning, Transformation and Expression of Gene Clusters]]

*[[Metabolite extractions]]

*[[Production of microbial volatile organic compounds]]

==[[Next Generation Sequencing]]==

*[[ChIP Sequencing]]
*[[ChIP-exo sequencing]]
https://www.nature.com/articles/s41467-018-05265-7/
*[[RNA Sequencing]]
*[[Differential RNA sequencing]]

==[[Nucleic Acid Methods]]==

*[[Codon Optimising Sequences for Heterologous Expression in Streptomyces]]

*[[High GC PCR]]

*[[RNA extraction from Actinobacteria]]

*[[RNA interference]]

*[[Salting Out Genomic DNA Extraction Method]]

*[[Southern Blotting]]

==[[Protein Methods]]==

*[[Isolating extracellular protein from Streptomyces]]

=Alphabetical list of protocols=

[[Characterisation of Actinomycetes]]

[[Cloning, Transformation and Expression of Gene Clusters]]

[[CRISPR/Cas9-mediated genome editing]]

[[Chemical mutagenesis]]

[[ChIP Seq]]

[[Codon Optimising Sequences for Heterologous Expression in Streptomyces]]

[[Depletion strains]]

[[Exploration | ''Streptomyces'' Exploration]]

[[General Growth and Maintenance of Streptomyces sp.]]

[[GUS assay]] (β-Glucuronidase assay)

[[High GC PCR]]

[[Isolating Actinomycetes from the environment]]

[[Isolating extracellular protein from Streptomyces]]

[[Lambda-red mediated recombination (PCR-targeting system a.k.a. "Redirect")]]

[[Lambda-red mediated recombination using ssDNA]]

[[Meganuclease I-SceI based system for gene deletions]]

[[Metabolite extractions]]

[["Quikchange" site-directed mutagenesis]]

[[Random mutagenesis using a mutator strain]]

[[Random mutagenesis using error-prone PCR]]

[[RNA extraction from Actinobacteria]]

[[Salting Out Genomic DNA Extraction Method]]

[[Southern Blotting]]

[[Staining of ''Streptomyces'' spp. Cell Wall and Nucleic Acids| Staining of ''Streptomyces'' spp. Cell Wall and Nucleic Acids]]

[[Suicide vectors]]

[[Transposons]]

[[UV mutagenesis]]

Protocols

2020-01-31T14:30:54Z

Jordanmurray: /* Microscopy */

[[File:Actinobase-icons-trans_Protocols.png|150px|Protocols|link=Protocols]]

=Protocols organised by category=

==[[Biochemistry]]==

*[[GUS assay]] (β-Glucuronidase assay)

==Microbiology==

*[[Characterisation of Actinomycetes | General Characterisaton of Actinomycetes]]

*[[Exploration | ''Streptomyces'' Exploration]]

*[[General Growth and Maintenance of Streptomyces sp.]]

*[[Isolating Actinomycetes from the environment]]

For a comprehensive guide to actinomycete characterisation see the [https://www.dsmz.de/collection/catalogue/microorganisms/special-groups-of-organisms/compendium-of-actinobacteria Compendium of Actinobacteria] from [https://www.helmholtz-hzi.de/en/research/research-topics/anti-infectives/microbial-strain-collection/our-research/#anchorsection Prof. Joachim Wink] at the German Collection of Microorganisms & Cell Cultures ([https://www.dsmz.de/ DSMZ]). This resouce provides protocols for characterising a wide range of features for new isolates, but may also be applicable more broadly for example for phenotyping experiments. The tests include colony morphology, melanin production, salt tolerance, carbon utlilisation and much more. To download the guide please see the link below.

[https://actinobase.org/downloads/DSMZ.pdf DSMZ Compendium of Actinobacteria]

==[[Microscopy]]==

*[[Staining of ''Streptomyces'' spp. Cell Wall and Nucleic Acids| Staining of ''Streptomyces'' spp. Cell Wall and Nucleic Acids]]

*[[Cultivating Actinobacteria for Microscopic Analysis]]*

==[[Mutagenesis]]==

*[[CRISPR/Cas9-mediated genome editing]]

*[[Chemical mutagenesis]]

*[[Depletion strains]]

*[[Lambda-red mediated recombination (PCR-targeting system a.k.a. "Redirect")]]

*[[Lambda-red mediated recombination using ssDNA]]

*[[Meganuclease I-SceI based system for gene deletions]]

*[["Quikchange" site-directed mutagenesis]]

*[[Random mutagenesis using a mutator strain]]

*[[Random mutagenesis using error-prone PCR]]

*[[Suicide vectors]]

*[[Transposons]]

*[[UV mutagenesis]]

==[[Natural Products]]==

*[[Cloning, Transformation and Expression of Gene Clusters]]

*[[Metabolite extractions]]

*[[Production of microbial volatile organic compounds]]

==[[Next Generation Sequencing]]==

*[[ChIP Sequencing]]
*[[ChIP-exo sequencing]]
https://www.nature.com/articles/s41467-018-05265-7/
*[[RNA Sequencing]]
*[[Differential RNA sequencing]]

==[[Nucleic Acid Methods]]==

*[[Codon Optimising Sequences for Heterologous Expression in Streptomyces]]

*[[High GC PCR]]

*[[RNA extraction from Actinobacteria]]

*[[RNA interference]]

*[[Salting Out Genomic DNA Extraction Method]]

*[[Southern Blotting]]

==[[Protein Methods]]==

*[[Isolating extracellular protein from Streptomyces]]

=Alphabetical list of protocols=

[[Characterisation of Actinomycetes]]

[[Cloning, Transformation and Expression of Gene Clusters]]

[[CRISPR/Cas9-mediated genome editing]]

[[Chemical mutagenesis]]

[[ChIP Seq]]

[[Codon Optimising Sequences for Heterologous Expression in Streptomyces]]

[[Depletion strains]]

[[Exploration | ''Streptomyces'' Exploration]]

[[General Growth and Maintenance of Streptomyces sp.]]

[[GUS assay]] (β-Glucuronidase assay)

[[High GC PCR]]

[[Isolating Actinomycetes from the environment]]

[[Isolating extracellular protein from Streptomyces]]

[[Lambda-red mediated recombination (PCR-targeting system a.k.a. "Redirect")]]

[[Lambda-red mediated recombination using ssDNA]]

[[Meganuclease I-SceI based system for gene deletions]]

[[Metabolite extractions]]

[["Quikchange" site-directed mutagenesis]]

[[Random mutagenesis using a mutator strain]]

[[Random mutagenesis using error-prone PCR]]

[[RNA extraction from Actinobacteria]]

[[Salting Out Genomic DNA Extraction Method]]

[[Southern Blotting]]

[[Staining of ''Streptomyces'' spp. Cell Wall and Nucleic Acids| Staining of ''Streptomyces'' spp. Cell Wall and Nucleic Acids]]

[[Suicide vectors]]

[[Transposons]]

[[UV mutagenesis]]

PMS82

2019-10-25T13:32:25Z

Jordanmurray: /* Use */

{{DISPLAYTITLE: pMS82}}

==Use==

This integration vector allows for conjugal transfer of DNA into the φBT1 phage attachment site of Streptomyces genomes from E. coli. This vector is derived from the pSET152 backbone. It contains the oriT from Streptomyces as well as the hygromycin resistance cassette.

This vector can be manipulated to contain genes of interest for the expression of homologous proteins, photoproteins, natural products etc.

==Features==
*Hygromycin resistance cassette
*φBT1 integration site & integrase

==History==

==Map==
[[File:pMS82.png]]

==Sequence links==

==References==
===Needs formatting!!===
[https://jb.asm.org/content/185/17/5320.long here]

Staining of Streptomyces spp. Cell Wall and Nucleic Acids

2019-08-30T14:27:32Z

Jordanmurray:

{{DISPLAYTITLE: 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'''

{| class="wikitable"
! 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'''

{| class="wikitable"
!
! 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==

# 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.

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.

Staining of Streptomyces spp. Cell Wall and Nucleic Acids

2019-08-30T14:27:12Z

Jordanmurray:

{{DISPLAYTITLE: 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'''

{| class="wikitable"
! 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'''

{| class="wikitable"
!
! 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==

# 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.

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.

Staining of Streptomyces spp. Cell Wall and Nucleic Acids

2019-08-30T14:20:28Z

Jordanmurray: /* Method */

{{DISPLAYTITLE: 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.''

=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'''

{| class="wikitable"
! 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'''

{| class="wikitable"
!
! 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==

# 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.

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.

Staining of Streptomyces spp. Cell Wall and Nucleic Acids

2019-08-30T14:18:04Z

Jordanmurray: /* Reagents */

{{DISPLAYTITLE: 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.''

=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.

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.

==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'''

{| class="wikitable"
! 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'''

{| class="wikitable"
!
! 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 of Streptomyces spp. Cell Wall and Nucleic Acids

2019-08-30T14:05:50Z

Jordanmurray: /* Reagents */

{{DISPLAYTITLE: 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.''

=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.

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.

==Reagents==

'''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

{| class="wikitable"
!
! Stock
! Working Concentration
! Volume of Stock in 10ml PBS
|-
| Fluorescein-WGA
| 1mg/ml
| 2µg/ml
| 20µl
|-
| Propidium Iodide
| 12.5mg/ml
| 10µg/ml
| 8µl
|}

Staining of Streptomyces spp. Cell Wall and Nucleic Acids

2019-08-30T13:55:55Z

Jordanmurray: /* Reagents */

Staining of Streptomyces spp. Cell Wall and Nucleic Acids

2019-08-30T13:55:11Z

Jordanmurray: /* Reagents */

Staining of Streptomyces spp. Cell Wall and Nucleic Acids

2019-08-30T13:54:32Z

Jordanmurray: /* Reagents */

Staining of Streptomyces spp. Cell Wall and Nucleic Acids

2019-08-30T13:54:00Z

Jordanmurray: /* Reagents */

Media recipes

2019-07-26T15:53:48Z

Jordanmurray:

{{DISPLAYTITLE:Media Recipes}}

[[File:Actinobase-icons-trans_Media-recipes.png|150px|Media Recipes|link=Media_recipes]]

[[A1]]

[[CM]] (Complete medium)

[[DNA]] (Difco Nutrient Agar) | [[DNB]] (Difco Nutrient Broth) | [[SNA]] (soft nutrient agar)

[[Emerson medium]]

[[Gause's No. 1]]

[[GYM]] (Glucose + Yeast Extract + Malt Extract)

[[HT]] (Hickey-Tresner)

[[HV]]

[[IMA]] (Smash)

[[ISP2]]

[[ISP3]]

[[ISP4]]

[[ISP5]]

[[ISP6]]

[[ISP7]]

[[LB]] (Lysogeny Broth) | [[L]] (L agar)

[[MM]] (Minimal medium)

[[MYM]] (Maltose + Yeast Extract + Malt Extract)

[[NMMP]] (minimal liquid medium)

[[OT]] (oligotrophic agar)

[[PGA]] (Potato Glucose)

[[R2]] | [[R2YE]] | [[R5]]

[[SC]] (Starch Casein agar)

[[SFM]] (Soy Flour Mannitol)

[[SMM]] (supplemented liquid minimal medium) | [[SMMS]] (supplemented minimal medium, solid)

[[SPY]] (Salt Peptone Yeast)

[[TSB]] (Tryptone Soy Broth)

[[YEME]] (Yeast Extract + Malt Extract)

[[YP]] (Yeast Peptone) | [[YPD]] (Yeast Peptone Dextrose)

[[2X YT]]

[[MC agar]] (Milk Casein Agar)

Media recipes

2019-07-26T15:51:52Z

Jordanmurray:

{{DISPLAYTITLE: Media Recipes}}

[[File:Actinobase-icons-trans_Media-recipes.png|150px|Media Recipes|link=Media_recipes]]

[[A1]]

[[CM]] (Complete medium)

[[DNA]] (Difco Nutrient Agar) | [[DNB]] (Difco Nutrient Broth) | [[SNA]] (soft nutrient agar)

[[Emerson medium]]

[[Gause's No. 1]]

[[GYM]] (Glucose + Yeast Extract + Malt Extract)

[[HT]] (Hickey-Tresner)

[[HV]]

[[IMA]] (Smash)

[[ISP2]]

[[ISP3]]

[[ISP4]]

[[ISP5]]

[[ISP6]]

[[ISP7]]

[[LB]] (Lysogeny Broth) | [[L]] (L agar)

[[MM]] (Minimal medium)

[[MYM]] (Maltose + Yeast Extract + Malt Extract)

[[NMMP]] (minimal liquid medium)

[[OT]] (oligotrophic agar)

[[PGA]] (Potato Glucose)

[[R2]] | [[R2YE]] | [[R5]]

[[SC]] (Starch Casein agar)

[[SFM]] (Soy Flour Mannitol)

[[SMM]] (supplemented liquid minimal medium) | [[SMMS]] (supplemented minimal medium, solid)

[[SPY]] (Salt Peptone Yeast)

[[TSB]] (Tryptone Soy Broth)

[[YEME]] (Yeast Extract + Malt Extract)

[[YP]] (Yeast Peptone) | [[YPD]] (Yeast Peptone Dextrose)

[[2X YT]]

[[MC agar]] (Milk Casein Agar)

Media

2019-07-26T15:51:01Z

Jordanmurray: Changed redirect target from Media Recipes to Media recipes

#REDIRECT [[Media recipes]]

Media

2019-07-26T15:50:35Z

Jordanmurray: Redirected page to Media Recipes

#REDIRECT [[Media Recipes]]

Staining of Streptomyces spp. Cell Wall and Nucleic Acids

2019-07-26T15:49:26Z

Jordanmurray:

Staining of Streptomyces spp. Cell Wall and Nucleic Acids

2019-07-26T15:30:31Z

Jordanmurray:

Staining of ''Streptomyces'' spp. Cell Wall and Nucleic Acids

2019-07-26T15:09:40Z

Jordanmurray: Redirected page to Staining of Streptomyces spp. Cell Wall and Nucleic Acids

#REDIRECT [[Staining of Streptomyces spp. Cell Wall and Nucleic Acids]]

Staining of Streptomyces spp. Cell Wall and Nucleic Acids

2019-07-26T14:58:47Z

Jordanmurray:

Staining of Streptomyces spp. Cell Wall and Nucleic Acids

2019-07-26T14:37:08Z

Jordanmurray:

Microscopy

2019-07-26T14:19:42Z

Jordanmurray: /* Streptomyces */

=Microscopy of Actinobacteria=

Listed here are some of the Standard Operating Procedures (SOPs) for microscopy of Actinobacteria.

=Streptomyces=

* Staining
** [[Staining of ''Streptomyces'' spp. Cell Wall and Nucleic Acids| Staining of ''Streptomyces'' spp. Cell Wall and Nucleic Acids]]

Staining of Streptomyces spp. Cell Wall and Nucleic Acids

2019-07-26T14:18:09Z

Jordanmurray: Created page with "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..."

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.''

Microscopy

2019-07-26T14:05:51Z

Jordanmurray: /* Microscopy of Actinobacteria */

=Microscopy of Actinobacteria=

Listed here are some of the Standard Operating Procedures (SOPs) for microscopy of Actinobacteria.

=Streptomyces=

* Staining
** [[Staining of Streptomyces spp. Cell Wall and Nucleic Acids| Staining of ''Streptomyces'' spp. Cell Wall and Nucleic Acids]]

Microscopy

2019-07-26T14:00:19Z

Jordanmurray: /* Streptomyces */

=Microscopy of Actinobacteria=

Listed here are some of the Standard Operating Procedures (SOPs) for microscopy of Actinobacteria.

=Streptomyces=

* Staining
** [[Staining of Streptomyces spp. Cell Wall and Nucleic Acids| Staining of ''Streptomyces'' spp. Cell Wall and Nucleic Acids]]

Microscopy

2019-07-26T13:55:32Z

Jordanmurray: /* Streptomyces */

=Microscopy of Actinobacteria=

Listed here are some of the Standard Operating Procedures (SOPs) for microscopy of Actinobacteria.

==Streptomyces==

* Staining
** [[Staining of ''Streptomyces'' spp. Cell Wall and Nucleic Acids]]
*[[Salmonella enterica subsp. arizonae|''S. enterica'' subsp. ''arizonae'']]

Microscopy

2019-07-26T13:43:56Z

Jordanmurray: /* Streptomyces */

Microscopy

2019-07-26T13:43:05Z

Jordanmurray: /* Microscopy of Actinobacteria */

=Microscopy of Actinobacteria=

Listed here are some of the Standard Operating Procedures (SOPs) for microscopy of Actinobacteria.

==Streptomyces==

* Staining
** [[Staining of Streptomyces spp. Cell Wall and Nucleic Acids]]

Microscopy

2019-07-26T13:31:07Z

Jordanmurray: Created page with "=Microscopy of Actinobacteria= ==Streptomyces=="

=Microscopy of Actinobacteria=

==Streptomyces==

Protocols

2019-07-26T13:28:37Z

Jordanmurray:

[[File:Actinobase-icons-trans_Protocols.png|150px|Protocols|link=Protocols]]

[[Cloning and Expression of Gene Clusters]]

[[CRISPR/Cas9-mediated genome editing]]

[[General Growth and Maintenance of Streptomyces sp.]]

[[High GC PCR]]

[[Mutagenesis]]

[[Nucleic Acid Methods]]

[[Protein Methods]]

[[Microscopy]]