ActinoBase - User contributions [en-gb]

Metabolite extractions

2019-09-27T09:31:48Z

Laia: /* Bacterial cultures in HP-20 resin and metabolite extraction */

==HP-20 resin Ethyl Acetate (EtOAc) extractions from liquid cultures==

===HP-20 resin and Media Preparation===

HP-20 resin activation: Cover 100 g of dried HP-20 resin with approximately 300 mL of EtOAc in a clean glass bottle with polypropylene lid and shake for 10 minutes. Filter the HP-20 resin using a Buchnar funnel and filter paper and discard the EtOAc into an appropriate flammable solvent waste container. Add HP-20 resin back into the used bottle and cover again with 300 mL ethyl acetate.

Adding HP-20 resin to media: Dispense 50 mL of media into 250 mL conical flasks. Use a Buchner funnel and filter paper to weigh out 5% w/v of HP-20 resin (2.5 g of HP-20 resin per 50 mL of media). Once completely dry (no resin clumping), add 2.5 g oh HP.20 resin per 50 mL of media and autoclave.

Notes: the HP-20 resin should be completely dry. Never contact plastic with EtOAc or HP-20 resin that has been stored in EtOAc. Add HP-20 resin to fermentation media prior to autoclaving.

===Bacterial cultures in HP-20 resin and metabolite extraction===

Add 2.5 mL of a bacterial pre-culture into the conical flask containing 50 mL of media and 5% of HP-20. Incubate the culture for the required time in a shaker. After incubation, transfer the culture from the conical flask into a 50 mL Falcon tube. Centrifuge for 10 mins at 4°C and 5,000 rpm. Remove the tube from the centrifuge, discard the supernatant and freeze at - 80°C the cell pellet/HP-20 resin overnight. After frozen, place the tube in a freeze-dryer until completely dry (no clumps / humidity should be observed). Transfer the dry cell pellet/HP-20 resin to a clean Erlenmeyer flask (50 mL). Add 20 mL of EtOAc and shake overnight (100 rpm). Filter the EtOAc into a pre-weighted 20 mL glass scintillation vial with filter paper. Again, add 20 mL of EtOAc and do the same process as before. Evaporate EtOAc each time under nitrogen and record the extract weight.

Notes: for scale-up experiments, scale-up the bacterial cultures and add the required amount of HP-20 resin and EtOAc.

==Ethyl Acetate (EtOAc) extractions from solid media cultures==
For bacterial cultures from solid media, transfer the wished cultures to a Falcon tubes (50 mL). Freeze-dry samples as previously explained. Transfer sample into a clean Erlenmeyer flask (50 mL) and extract metabolites twice with 20 mL EtOAc. Remove EtOAc each time into a pre-weighted 20 mL glass scintillation vial. Evaporate under nitrogen and record the extract weight.

Notes: for scale-up experiments, scale-up the bacterial cultures and add the required amount of EtOAc.

Metabolite extractions

2019-09-27T09:31:34Z

Laia: /* Ethyl Acetate (EtOAc) extractions from solid media cultures */

==HP-20 resin Ethyl Acetate (EtOAc) extractions from liquid cultures==

===HP-20 resin and Media Preparation===

HP-20 resin activation: Cover 100 g of dried HP-20 resin with approximately 300 mL of EtOAc in a clean glass bottle with polypropylene lid and shake for 10 minutes. Filter the HP-20 resin using a Buchnar funnel and filter paper and discard the EtOAc into an appropriate flammable solvent waste container. Add HP-20 resin back into the used bottle and cover again with 300 mL ethyl acetate.

Adding HP-20 resin to media: Dispense 50 mL of media into 250 mL conical flasks. Use a Buchner funnel and filter paper to weigh out 5% w/v of HP-20 resin (2.5 g of HP-20 resin per 50 mL of media). Once completely dry (no resin clumping), add 2.5 g oh HP.20 resin per 50 mL of media and autoclave.

Notes: the HP-20 resin should be completely dry. Never contact plastic with EtOAc or HP-20 resin that has been stored in EtOAc. Add HP-20 resin to fermentation media prior to autoclaving.

===Bacterial cultures in HP-20 resin and metabolite extraction===

Add 2.5 mL of a bacterial pre-culture into the conical flask containing 50 mL of media and 5% of HP-20. Incubate the culture for the required time in a shaker. After incubation, transfer the culture from the conical flask into a 50 mL Falcon tube. Centrifuge for 10 mins at 4°C and 5,000 rpm. Remove the tube from the centrifuge, discard the supernatant and freeze at - 80°C the cell pellet/HP-20 resin overnight. After frozen, place the tube in a freeze-dryer until completely dry (no clumps / humidity should be observed). Transfer the dry cell pellet/HP-20 resin to a clean Erlenmeyer flask (50 mL). Add 20 mL of EtOAc and shake overnight (100 rpm). Filter the EtOAc into a pre-weighted 20 mL glass scintillation vial with filter paper. Again, add 20 mL of EtOAc and do the same process as before. Evaporate EtOAc each time under nitrogen and record the extract weight.

==Ethyl Acetate (EtOAc) extractions from solid media cultures==
For bacterial cultures from solid media, transfer the wished cultures to a Falcon tubes (50 mL). Freeze-dry samples as previously explained. Transfer sample into a clean Erlenmeyer flask (50 mL) and extract metabolites twice with 20 mL EtOAc. Remove EtOAc each time into a pre-weighted 20 mL glass scintillation vial. Evaporate under nitrogen and record the extract weight.

Notes: for scale-up experiments, scale-up the bacterial cultures and add the required amount of EtOAc.

Metabolite extractions

2019-09-27T09:30:35Z

Laia: /* HP-20 resin Ethyl Acetate (EtOAc) extractions from liquid cultures */

==HP-20 resin Ethyl Acetate (EtOAc) extractions from liquid cultures==

===HP-20 resin and Media Preparation===

HP-20 resin activation: Cover 100 g of dried HP-20 resin with approximately 300 mL of EtOAc in a clean glass bottle with polypropylene lid and shake for 10 minutes. Filter the HP-20 resin using a Buchnar funnel and filter paper and discard the EtOAc into an appropriate flammable solvent waste container. Add HP-20 resin back into the used bottle and cover again with 300 mL ethyl acetate.

Adding HP-20 resin to media: Dispense 50 mL of media into 250 mL conical flasks. Use a Buchner funnel and filter paper to weigh out 5% w/v of HP-20 resin (2.5 g of HP-20 resin per 50 mL of media). Once completely dry (no resin clumping), add 2.5 g oh HP.20 resin per 50 mL of media and autoclave.

Notes: the HP-20 resin should be completely dry. Never contact plastic with EtOAc or HP-20 resin that has been stored in EtOAc. Add HP-20 resin to fermentation media prior to autoclaving.

===Bacterial cultures in HP-20 resin and metabolite extraction===

Add 2.5 mL of a bacterial pre-culture into the conical flask containing 50 mL of media and 5% of HP-20. Incubate the culture for the required time in a shaker. After incubation, transfer the culture from the conical flask into a 50 mL Falcon tube. Centrifuge for 10 mins at 4°C and 5,000 rpm. Remove the tube from the centrifuge, discard the supernatant and freeze at - 80°C the cell pellet/HP-20 resin overnight. After frozen, place the tube in a freeze-dryer until completely dry (no clumps / humidity should be observed). Transfer the dry cell pellet/HP-20 resin to a clean Erlenmeyer flask (50 mL). Add 20 mL of EtOAc and shake overnight (100 rpm). Filter the EtOAc into a pre-weighted 20 mL glass scintillation vial with filter paper. Again, add 20 mL of EtOAc and do the same process as before. Evaporate EtOAc each time under nitrogen and record the extract weight.

==Ethyl Acetate (EtOAc) extractions from solid media cultures==
For bacterial cultures from solid media, transfer the wished cultures to a Falcon tubes (50 mL). Freeze-dry samples as previously explained. Transfer sample into a clean Erlenmeyer flask (50 mL) and extract metabolites twice with 20 mL EtOAc. Remove EtOAc each time into a pre-weighted 20 mL glass scintillation vial. Evaporate under nitrogen and record the extract weight.

Notes: for scale-up experiments, scale-up the bacterial cultures and add the required amount of HP-20 resin and EtOAc.

Protocols

2019-08-30T15:31:48Z

Laia:

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

[[Characterisation of Actinomycetes]]

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

[[CRISPR/Cas9-mediated genome editing]]

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

[[High GC PCR]]

[[Isolating Actinomycetes from the environment]]

[[Microscopy]]

[[Mutagenesis]]

[[Nucleic Acid Methods]]

[[Protein Methods]]

[[Protoplasts Formation, Regeneration and Transformation]]

[[Metabolite extractions]]

Metabolite extractions

2019-08-30T15:28:07Z

Laia:

Actinobacteria isolation from marine environments

2019-08-30T15:06:01Z

Laia: /* Media selection */

=Pre-treatment methods of marine samples=
Pre-treatment methods for the isolation of marine Actinobacteria have been applied to eliminate the growth of terrestrial actinobacteria, gram-negative bacteria and fungi (1). These methods include drying the samples using laminar air flow hood, mixing the samples with seawater, applying UV irradiation, applying high frequency radiation and the use of certain antibiotics to kill gram negative bacteria.

==Drying marine samples with laminar air flow hood==
Several drying pre-treatment methods can be used to enhance the isolation of Actinobacteria from marine environments (2):

# Drying sediment overnight in a laminar flow hood until clumping occurs. Press a sterile foam plug against the sediment sample and onto the surface of an agar plate making a serial dilution (2).

(2#) Drying sediment overnight in a laminar flow hood until clumping occurs. Dilute dried sediment with 5 ml of sterile seawater and mix well. Add 50 µl of the solution onto an agar plate and spread with an spreader (2).

==Mixing the samples with seawater==
Media can be enriched with 2% of seawater or samples can be added directly grown in sterile (autoclaved) seawater (3).

==Applying UV irradiation==
Directly apply UV‐irradiation to the sediment suspension (5 ml) for 30 s. Use a wavelength of 254 nm and leave a distance of 20 cm (4).

==Applying high frequency radiation==
Apply superhigh frequency radiation directly to sediment samples by placing the sediment solution in a Eppendorf tube and microwave it for 45 seconds at 2460 MHz and 80 W (4).

==Dilute / Heat==
Add dry sediment to 3 mL of seawater (dilutions 1:3 or 1:6), heat it to 55°C for 6 min, and inoculate 50-70 µL of the resulting suspension onto an agar plate (2).

=Media selection=
Soil agar (4):

Filtered soil extract (mix 200 g of soil in 1 L of water and boil for 30 min) – 1 L

Agar – 20 g

Soil agar with 3% sea salt added

Modified organic agar 2 Gause (4):

Peptone – 5.0 g

Trypton – 3.0 g

Glucose – 10 g

NaCl – 5.0 g

Tap water – 1 L

Modified organic agar 2 Gause with 3% sea salt added (4)

Mineral agar 1 Gause (4):

Starch‐soluble – 20.0 g

K2HPO4– 0.5 g

MgSO4– 0.5 g

KNO3– 1.0 g

NaCl – 0.5 g

FeSO4−0.01 g

Agar – 20.0 g

Tap water – 1 L

Soy‐bean meal agar (4):

Soy‐bean meal – 3.0 g

KNO3– 0.2 g

K2HPO4– 0.5 g

MgSO4– 0.4 g

Agar 20.0 g

Tap water – 1 L

Soy‐bean meal agar with 3% sea salt added (4)

Yeast‐corn‐starch agar (4):

Starch‐soluble – 10.0 g

Yeast extract – 10 g

Corn meal extract – 10.0 g

NaCl – 2.0 g

Agar – 20.0 g

Tap water – 1 L

Pea‐meal agar (4):

Pea meal – 10.0 g

Glucose – 10.0 g

NaCl – 5.0 g

CaCO3– 1.0 g

Agar – 20 g

Tap water – 1.0 L

Adjust to pH to 7.0 - 7.5 and supplement with nalidixic acid (10 μg ml−1) and nystatin (50 μg ml−1) to inhibit the growth of Gram-negative bacteria and fungi.

M3 (5):
Agar – 18 g
Monopotassium phosphate – 0.466 g

Disodium phosphate – 0.732 g

Potassium Nitrate – 0.10 g

Magnesium Sulfate heptahydrate - 0.10 g

Calcium Carbonate – 0.02 g

Iron sulfate heptahydrate – 200 µg

Zinc sulfate heptahydrate – 180 µg

Sodium propionate – 0.20g

Nystatin – 50 µg/ml

Nalidixic acid –10 µg/ml

==References==
1- Subramani, R. and Sipkema, D. (2019) ‘Marine rare actinomycetes: A promising source of structurally diverse and unique novel natural products’, Marine Drugs. doi: 10.3390/md17050249.

2- Jensen, P. R. et al. (2005) ‘Culturable marine actinomycete diversity from tropical Pacific Ocean sediments’, Environmental Microbiology. doi: 10.1111/j.1462-2920.2005.00785.x.

3- Mincer, T. J., Fenical, W. and Jensen, P. R. (2005) ‘Culture-dependent and culture-independent diversity within the obligate marine actinomycete genus Salinispora’, Applied and Environmental Microbiology. doi: 10.1128/AEM.71.11.7019-7028.2005.

4- Bredholt, H. et al. (2008) ‘Actinomycetes from sediments in the Trondheim fjord, Norway: Diversity and biological activity’, Marine Drugs. doi: 10.3390/md6010012.

5- Maldonado et al. (2005) 'Diversity of culturable Actinobacteria in geographically widespread marine sediments', Antonie Van Leeuwenhoek. doi: 10.1007/s10482-004-6525-0

Actinobacteria isolation from marine environments

2019-08-30T14:59:18Z

Laia: /* Pre-treatment methods of marine samples */

=Pre-treatment methods of marine samples=
Pre-treatment methods for the isolation of marine Actinobacteria have been applied to eliminate the growth of terrestrial actinobacteria, gram-negative bacteria and fungi (1). These methods include drying the samples using laminar air flow hood, mixing the samples with seawater, applying UV irradiation, applying high frequency radiation and the use of certain antibiotics to kill gram negative bacteria.

==Drying marine samples with laminar air flow hood==
Several drying pre-treatment methods can be used to enhance the isolation of Actinobacteria from marine environments (2):

# Drying sediment overnight in a laminar flow hood until clumping occurs. Press a sterile foam plug against the sediment sample and onto the surface of an agar plate making a serial dilution (2).

(2#) Drying sediment overnight in a laminar flow hood until clumping occurs. Dilute dried sediment with 5 ml of sterile seawater and mix well. Add 50 µl of the solution onto an agar plate and spread with an spreader (2).

==Mixing the samples with seawater==
Media can be enriched with 2% of seawater or samples can be added directly grown in sterile (autoclaved) seawater (3).

==Applying UV irradiation==
Directly apply UV‐irradiation to the sediment suspension (5 ml) for 30 s. Use a wavelength of 254 nm and leave a distance of 20 cm (4).

==Applying high frequency radiation==
Apply superhigh frequency radiation directly to sediment samples by placing the sediment solution in a Eppendorf tube and microwave it for 45 seconds at 2460 MHz and 80 W (4).

==Dilute / Heat==
Add dry sediment to 3 mL of seawater (dilutions 1:3 or 1:6), heat it to 55°C for 6 min, and inoculate 50-70 µL of the resulting suspension onto an agar plate (2).

=Media selection=
Soil agar (4):

Filtered soil extract (mix 200 g of soil in 1 L of water and boil for 30 min) – 1 L

Agar – 20 g

Soil agar with 3% sea salt added

Modified organic agar 2 Gause (4):

Peptone – 5.0 g

Trypton – 3.0 g

Glucose – 10 g

NaCl – 5.0 g

Tap water – 1 L

Modified organic agar 2 Gause with 3% sea salt added (4)

Mineral agar 1 Gause (4):

Starch‐soluble – 20.0 g

K2HPO4– 0.5 g

MgSO4– 0.5 g

KNO3– 1.0 g

NaCl – 0.5 g

FeSO4−0.01 g

Agar – 20.0 g

Tap water – 1 L

Soy‐bean meal agar (4):

Soy‐bean meal – 3.0 g

KNO3– 0.2 g

K2HPO4– 0.5 g

MgSO4– 0.4 g

Agar 20.0 g

Tap water – 1 L

Soy‐bean meal agar with 3% sea salt added (4)

Yeast‐corn‐starch agar (4):

Starch‐soluble – 10.0 g

Yeast extract – 10 g

Corn meal extract – 10.0 g

NaCl – 2.0 g

Agar – 20.0 g

Tap water – 1 L

Pea‐meal agar (4):

Pea meal – 10.0 g

Glucose – 10.0 g

NaCl – 5.0 g

CaCO3– 1.0 g

Agar – 20 g

Tap water – 1.0 L

Adjust to pH to 7.0 - 7.5 and supplement with nalidixic acid (10 μg ml−1) and nystatin (50 μg ml−1) to inhibit the growth of Gram-negative bacteria and fungi.

==References==
1- Subramani, R. and Sipkema, D. (2019) ‘Marine rare actinomycetes: A promising source of structurally diverse and unique novel natural products’, Marine Drugs. doi: 10.3390/md17050249.

2- Jensen, P. R. et al. (2005) ‘Culturable marine actinomycete diversity from tropical Pacific Ocean sediments’, Environmental Microbiology. doi: 10.1111/j.1462-2920.2005.00785.x.

3- Mincer, T. J., Fenical, W. and Jensen, P. R. (2005) ‘Culture-dependent and culture-independent diversity within the obligate marine actinomycete genus Salinispora’, Applied and Environmental Microbiology. doi: 10.1128/AEM.71.11.7019-7028.2005.

4- Bredholt, H. et al. (2008) ‘Actinomycetes from sediments in the Trondheim fjord, Norway: Diversity and biological activity’, Marine Drugs. doi: 10.3390/md6010012.

Actinobacteria isolation from marine environments

2019-08-30T13:50:58Z

Laia: /* Media selection */

=Pre-treatment methods of marine samples=
Pre-treatment methods for the isolation of marine Actinobacteria have been applied to eliminate the growth of terrestrial actinobacteria, gram-negative bacteria and fungi (1). These methods include drying the samples using laminar air flow hood, mixing the samples with seawater, applying UV irradiation, applying high frequency radiation and the use of certain antibiotics to kill gram negative bacteria.

==Drying marine samples with laminar air flow hood==
Several drying pre-treatment methods can be used to enhance the isolation of Actinobacteria from marine environments (2):

# Drying sediment overnight in a laminar flow hood until clumping occurs. Press a sterile foam plug against the sediment sample and onto the surface of an agar plate making a serial dilution (2).

(2#) Drying sediment overnight in a laminar flow hood until clumping occurs. Dilute dried sediment with 5 ml of sterile seawater and mix well. Add 50 µl of the solution onto an agar plate and spread with an spreader (2).

==Mixing the samples with seawater==
Media can be enriched with 2% of seawater or samples can be added directly grown in sterile (autoclaved) seawater (3).

==Applying UV irradiation==
Directly apply UV‐irradiation to the sediment suspension (5 ml) for 30 s. Use a wavelength of 254 nm and leave a distance of 20 cm (4).

==Applying high frequency radiation==
Apply superhigh frequency radiation directly to sediment samples by placing the sediment solution in a Eppendorf tube and microwave it for 45 seconds at 2460 MHz and 80 W (4).

=Media selection=
Soil agar (4):

Filtered soil extract (mix 200 g of soil in 1 L of water and boil for 30 min) – 1 L

Agar – 20 g

Soil agar with 3% sea salt added

Modified organic agar 2 Gause (4):

Peptone – 5.0 g

Trypton – 3.0 g

Glucose – 10 g

NaCl – 5.0 g

Tap water – 1 L

Modified organic agar 2 Gause with 3% sea salt added (4)

Mineral agar 1 Gause (4):

Starch‐soluble – 20.0 g

K2HPO4– 0.5 g

MgSO4– 0.5 g

KNO3– 1.0 g

NaCl – 0.5 g

FeSO4−0.01 g

Agar – 20.0 g

Tap water – 1 L

Soy‐bean meal agar (4):

Soy‐bean meal – 3.0 g

KNO3– 0.2 g

K2HPO4– 0.5 g

MgSO4– 0.4 g

Agar 20.0 g

Tap water – 1 L

Soy‐bean meal agar with 3% sea salt added (4)

Yeast‐corn‐starch agar (4):

Starch‐soluble – 10.0 g

Yeast extract – 10 g

Corn meal extract – 10.0 g

NaCl – 2.0 g

Agar – 20.0 g

Tap water – 1 L

Pea‐meal agar (4):

Pea meal – 10.0 g

Glucose – 10.0 g

NaCl – 5.0 g

CaCO3– 1.0 g

Agar – 20 g

Tap water – 1.0 L

Adjust to pH to 7.0 - 7.5 and supplement with nalidixic acid (10 μg ml−1) and nystatin (50 μg ml−1) to inhibit the growth of Gram-negative bacteria and fungi.

==References==
1- Subramani, R. and Sipkema, D. (2019) ‘Marine rare actinomycetes: A promising source of structurally diverse and unique novel natural products’, Marine Drugs. doi: 10.3390/md17050249.

2- Jensen, P. R. et al. (2005) ‘Culturable marine actinomycete diversity from tropical Pacific Ocean sediments’, Environmental Microbiology. doi: 10.1111/j.1462-2920.2005.00785.x.

3- Mincer, T. J., Fenical, W. and Jensen, P. R. (2005) ‘Culture-dependent and culture-independent diversity within the obligate marine actinomycete genus Salinispora’, Applied and Environmental Microbiology. doi: 10.1128/AEM.71.11.7019-7028.2005.

4- Bredholt, H. et al. (2008) ‘Actinomycetes from sediments in the Trondheim fjord, Norway: Diversity and biological activity’, Marine Drugs. doi: 10.3390/md6010012.

Actinobacteria isolation from marine environments

2019-08-30T13:46:16Z

Laia: /* Media selection */

=Pre-treatment methods of marine samples=
Pre-treatment methods for the isolation of marine Actinobacteria have been applied to eliminate the growth of terrestrial actinobacteria, gram-negative bacteria and fungi (1). These methods include drying the samples using laminar air flow hood, mixing the samples with seawater, applying UV irradiation, applying high frequency radiation and the use of certain antibiotics to kill gram negative bacteria.

==Drying marine samples with laminar air flow hood==
Several drying pre-treatment methods can be used to enhance the isolation of Actinobacteria from marine environments (2):

# Drying sediment overnight in a laminar flow hood until clumping occurs. Press a sterile foam plug against the sediment sample and onto the surface of an agar plate making a serial dilution (2).

(2#) Drying sediment overnight in a laminar flow hood until clumping occurs. Dilute dried sediment with 5 ml of sterile seawater and mix well. Add 50 µl of the solution onto an agar plate and spread with an spreader (2).

==Mixing the samples with seawater==
Media can be enriched with 2% of seawater or samples can be added directly grown in sterile (autoclaved) seawater (3).

==Applying UV irradiation==
Directly apply UV‐irradiation to the sediment suspension (5 ml) for 30 s. Use a wavelength of 254 nm and leave a distance of 20 cm (4).

==Applying high frequency radiation==
Apply superhigh frequency radiation directly to sediment samples by placing the sediment solution in a Eppendorf tube and microwave it for 45 seconds at 2460 MHz and 80 W (4).

=Media selection=
Soil agar:
Filtered soil extract (mix 200 g of soil in 1 L of water and boil for 30 min) – 1 L
Agar – 20 g
Adjust to pH – 7.0

Soil agar with 3% sea salt added

Modified organic agar 2 Gause:
Peptone – 5.0 g
Trypton – 3.0 g
Glucose – 10 g
NaCl – 5.0 g
Tap water – 1 L

Modified organic agar 2 Gause with 3% sea salt added

Mineral agar 1 Gause:
Starch‐soluble – 20.0 g
K2HPO4– 0.5 g
MgSO4– 0.5 g
KNO3– 1.0 g
NaCl – 0.5 g
FeSO4−0.01 g
Agar – 20.0 g
Tap water – 1 L

Soy‐bean meal agar:
Soy‐bean meal – 3.0 g
KNO3– 0.2 g
K2HPO4– 0.5 g
MgSO4– 0.4 g
Agar 20.0 g
Tap water – 1 L

Soy‐bean meal agar with 3% sea salt added

Yeast‐corn‐starch agar
Starch‐soluble – 10.0 g
Yeast extract – 10 g
Corn meal extract – 10.0 g
NaCl – 2.0 g, agar – 20.0 g
Tap water – 1 L

Pea‐meal agar:
Pea meal – 10.0 g
Glucose – 10.0 g
NaCl – 5.0 g
CaCO3– 1.0 g
Agar – 20 g
Tap water – 1.0 L

Adjust to pH to 7.0 - 7.5 and supplement with nalidixic acid (10 μg ml−1) and nystatin (50 μg ml−1) to inhibit the growth of Gram-negative bacteria and fungi.

==References==
1- Subramani, R. and Sipkema, D. (2019) ‘Marine rare actinomycetes: A promising source of structurally diverse and unique novel natural products’, Marine Drugs. doi: 10.3390/md17050249.

2- Jensen, P. R. et al. (2005) ‘Culturable marine actinomycete diversity from tropical Pacific Ocean sediments’, Environmental Microbiology. doi: 10.1111/j.1462-2920.2005.00785.x.

3- Mincer, T. J., Fenical, W. and Jensen, P. R. (2005) ‘Culture-dependent and culture-independent diversity within the obligate marine actinomycete genus Salinispora’, Applied and Environmental Microbiology. doi: 10.1128/AEM.71.11.7019-7028.2005.

4- Bredholt, H. et al. (2008) ‘Actinomycetes from sediments in the Trondheim fjord, Norway: Diversity and biological activity’, Marine Drugs. doi: 10.3390/md6010012.

Isolating Actinomycetes from the environment

2019-08-30T13:24:10Z

Laia:

[https://actinobase.org/index.php?title=Isolating_Streptomyces_from_plant_roots Isolating ''Streptomyces'' from plant roots]

[https://actinobase.org/index.php?title=Actinobacteria_isolation_from_marine_environments Isolating ''Actinomycetes'' from marine environments]

Isolating Actinomycetes from the environment

2019-08-30T13:22:50Z

Laia:

[https://actinobase.org/index.php?title=Isolating_Streptomyces_from_plant_roots Isolating ''Streptomyces'' from plant roots]
[https://actinobase.org/index.php?title=Actinobacteria_isolation_from_marine_environments Isolating ''Actinomycetes'' from marine environments]

Actinobacteria isolation from marine environments

2019-07-26T16:04:09Z

Laia:

Actinobacteria isolation from marine environments

2019-07-26T16:03:39Z

Laia:

Actinobacteria isolation from marine environments

2019-07-26T16:03:10Z

Laia: /* Drying marine samples with laminar air flow hood */

Actinobacteria isolation from marine environments

2019-07-26T16:02:04Z

Laia:

Actinobacteria isolation from marine environments

2019-07-26T15:59:40Z

Laia:

Category:Actinobacteria isolation from marine environments

2019-07-26T15:57:12Z

Laia: Laia moved page Category:Actinobacteria isolation from marine environments to Actinobacteria isolation from marine environments over redirect

#REDIRECT [[Actinobacteria isolation from marine environments]]

Actinobacteria isolation from marine environments

2019-07-26T15:57:12Z

Laia: Laia moved page Category:Actinobacteria isolation from marine environments to Actinobacteria isolation from marine environments over redirect

Actinobacteria isolation from marine environments

2019-07-26T15:55:58Z

Laia: Laia moved page Actinobacteria isolation from marine environments to Category:Actinobacteria isolation from marine environments

Actinobacteria isolation from marine environments

2019-07-26T15:47:24Z

Laia: /* Applying high frequency radiation */

Actinobacteria isolation from marine environments

2019-07-26T15:47:07Z

Laia: /* Pre-treatment methods of marine samples */