Isolation of phages from single plaques - Revision history

Paulhoskisson at 12:17, 5 May 2020

2020-05-05T12:17:09Z

← Older revision		Revision as of 12:17, 5 May 2020
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	This protocol has been confirmed to work for the following organisms:		This protocol has been confirmed to work for the following organisms:
	*<em>[[Streptomyces coelicolor]]</em> with ΦC31		*<em>[[Streptomyces coelicolor]]</em> with ΦC31, ΦBT1, ΦHau3, ΦR4
			*<em>[[Streptomyces lividans]]</em> with ΦC31, ΦBT1, ΦHau3, ΦR4
	*<em>[[Streptomyces venezuelae]]</em> with ΦSV1		*<em>[[Streptomyces venezuelae]]</em> with ΦSV1

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	#The phage lysates remain viable for long periods when stored at 4°C (I have recovered phage from filtrates ~10 years old!).		#The phage lysates remain viable for long periods when stored at 4°C (I have recovered phage from filtrates ~10 years old!).
	#It is not necessary to add chloroform to the phage lysates.		#It is not necessary to add chloroform to the phage lysates.
			#Plaques can be very variable in size (0.1-0.8 cm), shape and turbidity - this may also change from the initial isolation plate through to stable populations of the phage. Turbid plaques are characteristic of temperate phages, clear plaques of lytic phages. It is not uncommon to see clear plaques that are turbid at the edges.

MFeeney at 10:55, 5 May 2020

2020-05-05T10:55:16Z

← Older revision		Revision as of 10:55, 5 May 2020
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	Materials needed:		Materials needed:
	#Plates containing the bacteriophage grown on a permissive host strain (see either [[Streptomyces ~~bacteriophage~~ plaque assay]] or [[Isolation of phages from environmental samples]])		#Plates containing the bacteriophage grown on a permissive host strain (see either [[Streptomyces Bacteriophage plaque assay]] or [[Isolation of phages from environmental samples]])
	#Liquid [[DNB]] medium		#Liquid [[DNB]] medium
	#0.45µm syringe filter		#0.45µm syringe filter

MFeeney at 10:45, 5 May 2020

2020-05-05T10:45:54Z

← Older revision		Revision as of 10:45, 5 May 2020
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	Instructions:		Instructions:
	#The bacteriophage of choice should be grown on a permissive host ~~strange~~, following either the enrichment procedure or bacteriophage plaque assay protocol.		#The bacteriophage of choice should be grown on a permissive host strain, following either the enrichment procedure or bacteriophage plaque assay protocol.
	#Identify the plaque(s) you wish to isolate.		#Identify the plaque(s) you wish to isolate. Ideally, these should be well-separated from any other plaques.
	#Using a ‘yellow pipette tip’ pick the plaque from the SNA overlay and place it into 1 mL of DNB broth in a Bijoux bottle.		#Using a ‘yellow pipette tip’ pick the plaque from the SNA overlay and place it into 1 mL of DNB broth in a Bijoux bottle.
	#Shake at room temperature for 2 hrs.		#Shake at room temperature for 2 hrs.

MFeeney: Created page with "=Isolation of phages from single plaques= This protocol has been confirmed to work for the following organisms: **Streptomyces coelicolor* with..."

2020-05-05T10:44:32Z

Created page with "=Isolation of phages from single plaques= This protocol has been confirmed to work for the following organisms: *Streptomyces coelicolor with..."

New page

=Isolation of phages from single plaques=

This protocol has been confirmed to work for the following organisms:
*[[Streptomyces coelicolor]] with ΦC31
*[[Streptomyces venezuelae]] with ΦSV1

===Isolation of phages from single plaques===

It is often very useful to isolate phage from single plaques (this is the equivalent of working with single colonies of bacteria). At an appropriate multiplicity of infection (MOI), each plaque will represent a single infection event (with all phage progeny being, in theory at least, genetically identical to the "parent" phage.)

Thus, when isolating novel phage from the environment, it is essential to go through at least one round of isolation from a single plaque.

===Protocol===

Materials needed:
#Plates containing the bacteriophage grown on a permissive host strain (see either [[Streptomyces bacteriophage plaque assay]] or [[Isolation of phages from environmental samples]])
#Liquid [[DNB]] medium
#0.45µm syringe filter
#Sterile yellow pipette tips, Bijoux bottles, etc.

Instructions:
#The bacteriophage of choice should be grown on a permissive host strange, following either the enrichment procedure or bacteriophage plaque assay protocol.
#Identify the plaque(s) you wish to isolate.
#Using a ‘yellow pipette tip’ pick the plaque from the SNA overlay and place it into 1 mL of DNB broth in a Bijoux bottle.
#Shake at room temperature for 2 hrs.
#Filter sterilise using a 0.45 µm syringe filter, and store at 4°C.
#This phage lysate can be used to generate high-titre preparations and for host range studies.

===Notes===
#The phage lysates remain viable for long periods when stored at 4°C (I have recovered phage from filtrates ~10 years old!).
#It is not necessary to add chloroform to the phage lysates.

Protocol adapted for ActinoBase by Paul Hoskisson from the University of Strathclyde.

Isolation of phages from single plaques - Revision history

Paulhoskisson at 12:17, 5 May 2020

MFeeney at 10:55, 5 May 2020

MFeeney at 10:45, 5 May 2020

MFeeney: Created page with "=Isolation of phages from single plaques= This protocol has been confirmed to work for the following organisms: *Streptomyces coelicolor with..."

MFeeney: Created page with "=Isolation of phages from single plaques= This protocol has been confirmed to work for the following organisms: **Streptomyces coelicolor* with..."