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Escherichia coli bacteriophage MS2

15597-B1

Product category
Viruses
Product type
Bacteriophage
Preceptrol culture
Strain designation
MS2
Type strain
No
Applications
Water testing
Product format
Freeze-dried
Storage conditions
2°C to 8°C
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ATCC determines the biosafety level of a material based on our risk assessment as guided by the current edition of Biosafety in Microbiological and Biomedical Laboratories (BMBL), U.S. Department of Health and Human Services. It is your responsibility to understand the hazards associated with the material per your organization’s policies and procedures as well as any other applicable regulations as enforced by your local or national agencies.

ATCC highly recommends that appropriate personal protective equipment is always used when handling vials. For cultures that require storage in liquid nitrogen, it is important to note that some vials may leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vial exploding or blowing off its cap with dangerous force creating flying debris. Unless necessary, ATCC recommends that these cultures be stored in the vapor phase of liquid nitrogen rather than submersed in liquid nitrogen.

Required Products

These products are vital for the proper use of this item and have been confirmed as effective in supporting functionality. If you use alternative products, the quality and effectiveness of the item may be affected.

Detailed product information

General

Specific applications
Water testing
Control Culture Ref USEPA Manuual of Method for Virology, Chapeter 16. Washington, DC:Environmental Protection Agency;EPA EPA 600/4-84/013 (N16), 2001
coliphages in water Ref 9224 C: Male-specific coliphage assay using Escherichia coli Famp. Washington, DC:American Public Health Association;Standard Methods for the Examination of Water and Wastewater, 2005 APHA9224 - Detection of Coliphages;.
control strain RefEnvironmental Protection Agency Male-specific (F+) and Somatic Coliphage in Water by Two-Step Enrichment Procedure. Washington, DC:Environmental Protection Agency;EPA EPA Method 1601, 2001 RefEnvironmental Protection Agency Male-specific (F+) and Somatic Coliphage in Water by Single Agar Layer (SAL) Procedure. Washington, DC:Environmental Protection Agency;EPA EPA Method 1602, 2001
detection of bacteriophages Ref Water quality --- Detection and enumeration of bacteriophages --- Part 1: Enumeration of F-specific RNA bacteriophages [BS EN ISO 10705-1:2001]. London, UK:British Standards Institution;British Standard BS 6068-4.11:1996.
testing Ref Inactivation of MS2 virus in Drinking Water. Washington, DC:Environmental Protection Agency;EPA EPA NSF 02/03/EPADWCTR. Ref Protocal for Equipment Verification Testing for Physical Removal of Microbiological and Particulate Contaminants. Washington, DC:Environmental Protection Agency;EPA EPA 05/9205/EPADWCTR.
Preceptrol
Yes

Characteristics

Comments
male-specific (F+) coliphage

Handling information

Host
Escherichia coli C-3000 (ATCC 15597)
Medium
Temperature
37°C
Atmosphere
Aerobic
Handling procedure
  1. Follow general procedures given below for phage propagation. 
  2. Escherichia coli strain C-3000 (ATCC 15597) is the recommended host. 
GENERAL PROCEDURES FOR THE PROPAGATION OF BACTERIOPHAGE. 

To recover phage from freeze-dried or frozen vial:

  1. Before opening the phage vial, prepare an actively growing culture of the recommended host strain. The host should be 16-18 hours old. 
  2. Pick one colony from the isolation plate and homogenize in 5 mL of the appropriate broth. Incubate at 37°C while shaking (160-180 rpm) until the growth reaches OD600 of 0.1 to 0.4. 
  3. Add approximately 1.0 mL of the recommended broth to a freeze-dried bacteriophage vial or 0.5 mL to a liquid cryovial. Infect each 5 mL culture with 100 µL of the bacteriophage. Shake at 160-180 rpm in 37°C overnight. After 16 to 18 hours, centrifuge phage culture at 4000 g for 10 minutes. Filter the lysate with a 0.2 µm or 0.45 µm PES sterile filter.  The filtrate can be stored at 4°C. 
  4. Prior to performing a spot titer, warm one or two plates at 37°C.
  5. Melt the soft agar (0.5% agar added to the recommended medium) and maintain at 43°C to 45°C until ready to use. It is best to allow the melted agar to remain at this temperature for about an hour to ensure that it has cooled to 43°C to 45°C. Warmer temperatures may kill the host.
  6. Add 50 to 100 µL of the host culture from step 1 to each 9 mL (approximately) of soft agar from step 5 above. Immediately overlay the surface of each plate with 2.5 mL. Allow the overlay to harden for 10 to 20 minutes.
  7. The phage lysate can be serial diluted in a 96 well plate in quadruplicate (if desired). Aliquot 90 µL of broth medium into each well. Add 10 µL of phage filtrate from step 3 to each well and mix. Transfer 10 µL from each well of the first dilution to each well of the second dilution and mix. Continue to the desired number of dilutions.
  8. Spot 2 µL of each dilution on the plate from step 6. Up to 8 dilutions can fit on a 90 mm petri dish. After overnight incubation, lysis should be visible.  At the higher dilutions, individual plaques should be countable. To calculate pfu/mL, use the following formula: pfu/mL = average plaque count / [(dilution factor) (2x10-3 mL)]
  9. Spotting the phage on plates makes visualizing the lysis easier. If phage is added directly to soft-agar before pouring plates, hazy or tiny plaques may be difficult to see. Resistant host bacteria may also mask plaque formation.
To propagate phage: 
  1. Determine the total volume needed and place this amount of broth in a flask.  Add a small amount of overnight host culture to the flask and incubate at 37°C while shaking until the growth reaches OD600 of 0.1 to 0.4.   
    2. Infect with the calculated volume of phage lysate using the following formula. Volume of phage to add (ml) = (8x108 x total culture volume in ml x OD600 x MOI) / phage titer (PFU/ml). Shake at 160-180 rpm at 37°C overnight.
    3. Centrifuge phage culture at 4000 g for 10 minutes. Filter the lysate with a 0.2 µm or 0.45 µm PES sterile filter.  The filtrate can be stored at 4°C. 
    4. Lysates should remain viable under refrigeration for long periods. They may also be frozen with or without cryoprotectant.  If available, liquid nitrogen storage is the best method for long term storage. Most phage can also be freeze-dried. ATCC uses double strength skim milk mixed half and half with the filtrate.
    Note: To achieve the highest PFU and total volume, the broth method detailed above has demonstrated the best results if the process is followed exactly. However, if any of the equipment are not available or if the technique is not possible for other reasons, the Adam’s Overlay method described below will provide adequate results for smaller volumes at satisfactory titer count. (Adams agar-overlay method as described in M. H. Adams' Bacteriophages, Interscience Publishers, Inc., New York, 1959)

 

To recover phage using the Adam’s Overlay method:

  1. Before opening the phage vial, prepare an actively growing broth culture of the recommended host strain. The host should be in early log phase. Times vary according to species but usually no more than 16 to 18 hours incubation.
  2. Pre-warm plates of the recommended medium in a 37°C incubator.
  3. Melt the soft agar (0.5% agar added to the recommended medium) and maintain at 43°C to 45°C until ready to use. It is best to allow the melted agar to remain at this temperature for about an hour to ensure that it has cooled to 43°C to 45°C. Warmer temperatures may kill the host.
  4. Add one to two drops of the host culture from step 1 to each 9 mL (approximately) of soft agar from step 3 above. Immediately, overlay the surface of each plate with 2.5 mL. Allow the overlay to harden for 10 to 20 minutes.
  5. Add approximately 1.0 mL of the recommended broth to a freeze-dried phage vial or 0.5 mL to a liquid cryovial. Transfer the entire contents of the vial into a test tube of the recommended broth medium. Perform a 1:10 serial dilution for as many passages as desired.
  6. One drop of each dilution is spotted on the surface of a prepared overlay plate. Allow to dry. Up to four dilutions can be placed on each plate, however, it may be difficult to keep the spots from running into each other. This is up to the discretion of the biologist.
  7. Incubate at the recommended temperature for 16 to 18 hours. For slower growing hosts, 24 hours may be needed.

To propagate phage:

  1. Phage may be propagated by preparing overlay plates as in steps 3 & 4 above and covering the surface with approximately 0.5 mL of the concentrated phage. Alternatively, you may add the phage directly to the melted agar with host before pouring over the plates. 
  2. For a larger amount, large-sized T-flasks can be prepared with the recommended agar and approximately 12.0 mL of melted soft agar with host poured over the surface. Allow the overlay to harden. Add approximately 1.0 mL of the recommended broth to a freeze-dried phage vial or 0.5 mL to a liquid cryovial. Transfer the entire contents of the vial to the T-flask and allowed to run over the hardened surface. 
  3. Incubate overnight. When lysis is observed, scrape the soft agar off the surface of the agar plates or T-flask. Centrifuge at about 1000 rpm for 25 minutes to sediment the cellular debris and agar. Draw off the supernatant and keep.
  4. The supernatant is passed through a .22 μm Millipore filter and the filtrate is kept and stored at 4°C to 8°C. A second filtration may be necessary to completely remove the host. 
 

 

Handling notes

Additional information on this culture is available on the ATCC® web site at www.atcc.org.

 

History

Deposited as
MS2
Depositors
JH Strauss
Chain of custody
ATCC <-- JH Strauss <-- A Clark

Legal disclaimers

Intended use
This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use.
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The product is provided 'AS IS' and the viability of ATCC® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid.  Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.

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Permits & Restrictions

Import Permit for the State of Hawaii

If shipping to the U.S. state of Hawaii, you must provide either an import permit or documentation stating that an import permit is not required. We cannot ship this item until we receive this documentation. Contact the Hawaii Department of Agriculture (HDOA), Plant Industry Division, Plant Quarantine Branch to determine if an import permit is required.

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References

Curated Citations

Strauss H Jr., Sinsheimer RL. Purification and properties of bacteriophage MS2 and of its ribonucleic acid. J. Mol. Biol. 7: 43-54, 1963. PubMed: 13978804

Environmental Protection Agency Male-specific (F+) and Somatic Coliphage in Water by Two-Step Enrichment Procedure. Washington, DC:Environmental Protection Agency;EPA EPA Method 1601, 2001

Environmental Protection Agency Male-specific (F+) and Somatic Coliphage in Water by Single Agar Layer (SAL) Procedure. Washington, DC:Environmental Protection Agency;EPA EPA Method 1602, 2001

Inactivation of MS2 virus in Drinking Water. Washington, DC:Environmental Protection Agency;EPA EPA NSF 02/03/EPADWCTR.

Protocal for Equipment Verification Testing for Physical Removal of Microbiological and Particulate Contaminants. Washington, DC:Environmental Protection Agency;EPA EPA 05/9205/EPADWCTR.

View All Curated Citations for this Product

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