Isolating Streptomyces from plant roots - Revision history

Jake Newitt at 14:36, 26 October 2021

2021-10-26T14:36:30Z

← Older revision		Revision as of 14:36, 26 October 2021
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	<h2><strong>Uses</h2></strong>		<h2><strong>Uses</h2></strong>
	*Nucleic acid isolation for microbial community analysis		*[[Nucleic acid isolation from wheat roots]] for microbial community analysis
	*General isolation of novel root associated microorganisms		*General isolation of novel root associated microorganisms
	*[[~~Plant root colonisation~~ assay (~~preliminary~~)]]		*[[Root colonization assay – colony forming unit (CFU) count method]]

	<h2><strong>References</h2></strong>		<h2><strong>References</h2></strong>

Jake Newitt at 14:32, 26 October 2021

2021-10-26T14:32:59Z

← Older revision		Revision as of 14:32, 26 October 2021
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	{{DISPLAYTITLE: Isolating ''Streptomyces'' from plant roots}}		{{DISPLAYTITLE: Isolating ''Streptomyces'' from plant roots}}

	=Sampling of plant roots for isolation of ''Streptomyces'' and other microorganisms from the plant rhizosphere and endosphere=		<h2><strong>Sampling of plant roots for isolation of ''Streptomyces'' and other microorganisms from the plant rhizosphere and endosphere</h2></strong>

	This method can be used to sample plant roots for total or selective bacterial isolation, as well as nucleic extraction for various experiments.		This method can be used to sample plant roots for total or selective bacterial isolation, as well as nucleic extraction for various experiments.
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	Depending on soil type and number of plants being sampled, this protocol can take up to a full day.		Depending on soil type and number of plants being sampled, this protocol can take up to a full day.

	==Preparation and reagents needed==		<h2><strong>Preparation and reagents needed</h2></strong>
	*Phosphate buffer saline (PBS)		*Phosphate buffer saline (PBS)
	**6.33g NaH<sub>2</sub>PO<sub>4</sub>.H<sub>2</sub>O		**6.33g NaH<sub>2</sub>PO<sub>4</sub>.H<sub>2</sub>O
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	*Disposable gloves		*Disposable gloves

			<h2><strong>Root sampling protocol</h2></strong>
	==Root sampling protocol==
	#Set out foil and wipe down with 70% Ethanol		#Set out foil and wipe down with 70% Ethanol
	#Take plants from pots: shake off large soil particles in a gloved hand (make sure gloves have been disinfected with ethanol), cut off the stems aseptically.		#Take plants from pots: shake off large soil particles in a gloved hand (make sure gloves have been disinfected with ethanol), cut off the stems aseptically.
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	#For DNA extraction from endophytic community, remove roots from PBS and separate into 2 samples for each plant. Snap freeze in liquid nitrogen and crush with a sterile mortar and pestle. Store at -80° until sample is extracted. Use MPBio fast spin kit for soil to extract from crushed roots.		#For DNA extraction from endophytic community, remove roots from PBS and separate into 2 samples for each plant. Snap freeze in liquid nitrogen and crush with a sterile mortar and pestle. Store at -80° until sample is extracted. Use MPBio fast spin kit for soil to extract from crushed roots.

	==Wheat root sterilisation protocol==		<h2><strong>Wheat root sterilisation protocol</h2></strong>
	#For sterilisation of wheat (''Triticum aestivum''), transfer the PBS washed roots into a falcon containing 10-20ml 70% ethonol and incubate at room temperature for 30 seconds.		#For sterilisation of wheat (''Triticum aestivum''), transfer the PBS washed roots into a falcon containing 10-20ml 70% ethonol and incubate at room temperature for 30 seconds.
	#Transfer the roots to a fresh falcon tube containing 10-20ml 3% bleach solution and incubate at room temperature for 10 minutes.		#Transfer the roots to a fresh falcon tube containing 10-20ml 3% bleach solution and incubate at room temperature for 10 minutes.
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	#Continue with steps 10 or 11 of root sampling protocol above.		#Continue with steps 10 or 11 of root sampling protocol above.

	==Uses==		<h2><strong>Uses</h2></strong>
	*Nucleic acid isolation for microbial community analysis		*Nucleic acid isolation for microbial community analysis
	*General isolation of novel root associated microorganisms		*General isolation of novel root associated microorganisms
	*[[Plant root colonisation assay (preliminary)]]		*[[Plant root colonisation assay (preliminary)]]

	==References==		<h2><strong>References</h2></strong>

	Root sampling protocol adapted from:-		Root sampling protocol adapted from:-

J Newitt: /* References */

2019-08-29T15:16:23Z

References

← Older revision		Revision as of 15:16, 29 August 2019
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	*Larran, S., Perello, A., Simon, M.R. and Moreno, V., 2002. Isolation and analysis of endophytic microorganisms in wheat (Triticum aestivum L.) leaves. World Journal of Microbiology and Biotechnology, 18(7), pp.683-686.		*Larran, S., Perello, A., Simon, M.R. and Moreno, V., 2002. Isolation and analysis of endophytic microorganisms in wheat (Triticum aestivum L.) leaves. World Journal of Microbiology and Biotechnology, 18(7), pp.683-686.

	Protocol adapted for ActinoBase by Jake T. Newitt~~, PhD student at~~ the University of East Anglia.		Protocol adapted for ActinoBase by Jake T. Newitt from the University of East Anglia.

J Newitt: /* Uses */

2019-08-15T15:34:34Z

Uses

← Older revision		Revision as of 15:34, 15 August 2019
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	*Nucleic acid isolation for microbial community analysis		*Nucleic acid isolation for microbial community analysis
	*General isolation of novel root associated microorganisms		*General isolation of novel root associated microorganisms
	*[[Plant root colonisation assay (~~crude~~)]]		*[[Plant root colonisation assay (preliminary)]]

	==References==		==References==

J Newitt at 15:27, 15 August 2019

2019-08-15T15:27:44Z

← Older revision		Revision as of 15:27, 15 August 2019
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	#Transfer to a new falcon with PBS (25ml) and sonicate for 15-20 minutes in a sonicating water bath.		#Transfer to a new falcon with PBS (25ml) and sonicate for 15-20 minutes in a sonicating water bath.
	#If you are sampling from ''Arabidopsis'' roots, continue onto step 10 or 11. If you are sampling from thicker roots like ''Triticum aestivum'', see root sterilisation section below before continuing.		#If you are sampling from ''Arabidopsis'' roots, continue onto step 10 or 11. If you are sampling from thicker roots like ''Triticum aestivum'', see root sterilisation section below before continuing.
	#For culturing endophytic bacteria: crush the roots in 1-2ml (depending on root size) in sterile 1G with a sterile pestle and mortar, transfer to an MCT. Make serial dilutions (0,-1,-2) and plate out onto replicate agar plates. [[SFM]] ~~is generally a good media to use for the isolation of Actinomycetes~~.		#For culturing endophytic bacteria: crush the roots in 1-2ml (depending on root size) in sterile 1G with a sterile pestle and mortar, transfer to an MCT. Make serial dilutions (0,-1,-2) and plate out onto replicate agar plates. It is possible to partially select for filamentous actinomycetes by plating on [[SFM]] with nystatin, cyclohexamide and naladixic acid.
	#For DNA extraction from endophytic community, remove roots from PBS and separate into 2 samples for each plant. Snap freeze in liquid nitrogen and crush with a sterile mortar and pestle. Store at -80° until sample is extracted. Use MPBio fast spin kit for soil to extract from crushed roots.		#For DNA extraction from endophytic community, remove roots from PBS and separate into 2 samples for each plant. Snap freeze in liquid nitrogen and crush with a sterile mortar and pestle. Store at -80° until sample is extracted. Use MPBio fast spin kit for soil to extract from crushed roots.

Sam Prudence: /* Uses */

2019-08-15T15:20:15Z

Uses

← Older revision		Revision as of 15:20, 15 August 2019
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	==Uses==		==Uses==
	*[[Nucleic acid isolation for microbial community analysis]]		*Nucleic acid isolation for microbial community analysis
	*~~[[Isolation~~ of novel root associated microorganisms]]		*General isolation of novel root associated microorganisms
	*[[Plant root colonisation assay (crude)]]		*[[Plant root colonisation assay (crude)]]

Sam Prudence: /* Root sterilisation protocol */

2019-08-15T15:17:34Z

Root sterilisation protocol

← Older revision		Revision as of 15:17, 15 August 2019
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	#For DNA extraction from endophytic community, remove roots from PBS and separate into 2 samples for each plant. Snap freeze in liquid nitrogen and crush with a sterile mortar and pestle. Store at -80° until sample is extracted. Use MPBio fast spin kit for soil to extract from crushed roots.		#For DNA extraction from endophytic community, remove roots from PBS and separate into 2 samples for each plant. Snap freeze in liquid nitrogen and crush with a sterile mortar and pestle. Store at -80° until sample is extracted. Use MPBio fast spin kit for soil to extract from crushed roots.

	==~~Root~~ sterilisation protocol==		==Wheat root sterilisation protocol==
	#For sterilisation of wheat (''Triticum aestivum''), transfer the PBS washed roots into a falcon containing 10-20ml 70% ethonol and incubate at room temperature for 30 seconds.		#For sterilisation of wheat (''Triticum aestivum''), transfer the PBS washed roots into a falcon containing 10-20ml 70% ethonol and incubate at room temperature for 30 seconds.
	#Transfer the roots to a fresh falcon tube containing 10-20ml 3% bleach solution and incubate at room temperature for 10 minutes.		#Transfer the roots to a fresh falcon tube containing 10-20ml 3% bleach solution and incubate at room temperature for 10 minutes.

J Newitt: /* Root sampling protocol */

2019-08-14T17:42:34Z

Root sampling protocol

← Older revision		Revision as of 17:42, 14 August 2019
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	#Transfer to a new falcon with PBS (25ml) and sonicate for 15-20 minutes in a sonicating water bath.		#Transfer to a new falcon with PBS (25ml) and sonicate for 15-20 minutes in a sonicating water bath.
	#If you are sampling from ''Arabidopsis'' roots, continue onto step 10 or 11. If you are sampling from thicker roots like ''Triticum aestivum'', see root sterilisation section below before continuing.		#If you are sampling from ''Arabidopsis'' roots, continue onto step 10 or 11. If you are sampling from thicker roots like ''Triticum aestivum'', see root sterilisation section below before continuing.
	#For culturing endophytic bacteria: crush the roots in 1-2ml (depending on root size) in sterile 1G with a sterile pestle and mortar, transfer to an MCT. Make serial dilutions (0,-1,-2)~~. Plate~~ out onto replicate agar plates. [[SFM]] is generally a good media to use for the isolation of Actinomycetes.		#For culturing endophytic bacteria: crush the roots in 1-2ml (depending on root size) in sterile 1G with a sterile pestle and mortar, transfer to an MCT. Make serial dilutions (0,-1,-2) and plate out onto replicate agar plates. [[SFM]] is generally a good media to use for the isolation of Actinomycetes.
	#For DNA extraction from endophytic community, remove roots from PBS and separate into 2 samples for each plant. Snap freeze in liquid nitrogen and crush with a sterile mortar and pestle. Store at -80° until sample is extracted. Use MPBio fast spin kit for soil to extract from crushed roots.		#For DNA extraction from endophytic community, remove roots from PBS and separate into 2 samples for each plant. Snap freeze in liquid nitrogen and crush with a sterile mortar and pestle. Store at -80° until sample is extracted. Use MPBio fast spin kit for soil to extract from crushed roots.

J Newitt: /* Root sampling protocol */

2019-08-14T17:40:45Z

Root sampling protocol

← Older revision		Revision as of 17:40, 14 August 2019
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	#Transfer the roots to a fresh tube of PBS, repeat step 3 (wash or vortex).		#Transfer the roots to a fresh tube of PBS, repeat step 3 (wash or vortex).
	#If sampling rhizosphere, take the previous tube and centrifuge soil/PBS mixture at 3200g for 15 minutes to form a pellet. The resulting pellet can be resuspended in a smaller volume of PBS and diluted for spread plating, if the end goal is to isolate live bacteria. Alternatively, if the sampling is for nucleic acid isolation, snap freeze pellet in liquid nitrogen.		#If sampling rhizosphere, take the previous tube and centrifuge soil/PBS mixture at 3200g for 15 minutes to form a pellet. The resulting pellet can be resuspended in a smaller volume of PBS and diluted for spread plating, if the end goal is to isolate live bacteria. Alternatively, if the sampling is for nucleic acid isolation, snap freeze pellet in liquid nitrogen.
	#Repeat steps 3 and 4 until no further particles are removed by the washing.		#Repeat steps 3 and 4 with the root samples until no further particles are removed by the washing.
	#If there are still soil particles attached to the root, transfer to a sterile petri dish and remove as much soil debris as possible with ethanol flame sterilised tweezers.		#If there are still soil particles attached to the root, transfer to a sterile petri dish and remove as much soil debris as possible with ethanol flame sterilised tweezers.
	#Transfer to new falcon with PBS (25ml) and sonicate for 15-20 minutes in a sonicating water bath.		#Transfer to a new falcon with PBS (25ml) and sonicate for 15-20 minutes in a sonicating water bath.
	#If you are sampling from ''Arabidopsis'' roots, continue onto step 10 or 11. If you are sampling from thicker roots like ''Triticum aestivum'', see root sterilisation section below before continuing.		#If you are sampling from ''Arabidopsis'' roots, continue onto step 10 or 11. If you are sampling from thicker roots like ''Triticum aestivum'', see root sterilisation section below before continuing.
	#For culturing endophytic bacteria: crush the roots in ~~1ml~~ sterile 1G in sterile pestle and ~~mortars~~, ~~place in eppendorfs~~. Make serial dilutions (0,-1,-2). Plate out onto replicate agar plates.		#For culturing endophytic bacteria: crush the roots in 1-2ml (depending on root size) in sterile 1G with a sterile pestle and mortar, transfer to an MCT. Make serial dilutions (0,-1,-2). Plate out onto replicate agar plates. [[SFM]] is generally a good media to use for the isolation of Actinomycetes.
	#For DNA extraction from endophytic community ~~instead:~~ remove roots from PBS~~. Separate~~ into 2 samples for each plant and ~~snap freeze, place~~ at -80 ~~degrees~~. Use MPBio fast spin kit for soil to extract from crushed roots.		#For DNA extraction from endophytic community, remove roots from PBS and separate into 2 samples for each plant. Snap freeze in liquid nitrogen and crush with a sterile mortar and pestle. Store at -80° until sample is extracted. Use MPBio fast spin kit for soil to extract from crushed roots.

	==Root sterilisation protocol==		==Root sterilisation protocol==

J Newitt: /* Sampling of plant roots to isolate Streptomyces from rhizosphere and endosphere compartments */

2019-08-14T17:34:03Z

Sampling of plant roots to isolate Streptomyces from rhizosphere and endosphere compartments

← Older revision		Revision as of 17:34, 14 August 2019
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	{{DISPLAYTITLE: Isolating ''Streptomyces'' from plant roots}}		{{DISPLAYTITLE: Isolating ''Streptomyces'' from plant roots}}

	=Sampling of plant roots ~~to isolate~~ ''Streptomyces'' from rhizosphere and endosphere ~~compartments~~=		=Sampling of plant roots for isolation of ''Streptomyces'' and other microorganisms from the plant rhizosphere and endosphere=

	This method can be used to sample plant roots for ~~the~~ total or selective bacterial isolation, as well as nucleic extraction for various experiments.		This method can be used to sample plant roots for total or selective bacterial isolation, as well as nucleic extraction for various experiments.

	Depending on soil type and number of plants being ~~samples~~, this protocol can take up to a full day.		Depending on soil type and number of plants being sampled, this protocol can take up to a full day.

	==Preparation and reagents needed==		==Preparation and reagents needed==