Pseudomonas sp. bacteriophage Ps-G3

1. Carefully follow procedures given below for phage propagation. 
2. Pseudomonas sp. G3 (ATCC 49780) is the recommended host. 

PROCEDURE FOR THE PROPAGATION OF BACTERIOPHAGE 49780-B1.

To recover phage from freeze-dried or frozen vial:

1. Prepare an actively growing culture of the recommended host strain before opening the phage specimen. The host should be 18-24 hours old. 
2. Pick one colony from the #1822 plate and homogenize in 5 mL of #1822 broth. Incubate at 30°C while shaking (170-200 rpm) until growth reaches OD₆₀₀ of 0.07 to 0.1. 
3. Add approximately 0.5 mL of the recommended broth to a freeze-dried bacteriophage vial. For a frozen vial, thaw completely. Infect each 5 mL culture with 100 µL of the bacteriophage. Shake at 170-180 rpm in 30°C overnight. 
4. Sub culture the host and incubate at 30°C overnight.
5. After 16 to 18 hours, centrifuge phage culture at 4000 g for 10 minutes. Filter the lysate two times with a 0.2 µm or 0.45 µm PES sterile filter.  The filtrate can be stored at 4°C. 
6. Melt the 0.5% #1822 agar completely. The melted soft agar can be stored at 55°C for up to a week before use.
7. To perform a spot titer, warm one or two plates at 37°C. The soft agar should be brought to 43°C to 45°C until ready to pour. It may be advisable to use a water bath. Overlay the surface with 2.5 mL of melted 0.5% #1822 agar containing 100 µL of the overnight host culture.  Allow overlay to harden. This usually won’t take longer than 15 to 30 minutes.
8. The phage lysate can be serial diluted in a 96 well plate in quadruplicate (if desired). Aliquot 90 µL of #1822 broth medium into each well. Add 10 µL of phage lysate to each well and mix. Pass 10 µL to each of the next set of wells and mix. Continue to the desired number of passages.
9. Spot 2 µL of each dilution on the plate from step g. Up to 8 dilutions can fit on a 90 mm petri dish. Allow spots to dry then incubate inverted plates at 30°C. This temperature is important to prevent the host from over growing. After overnight incubation, lysis should be visible.  At the higher dilutions, individual plaques should be countable. 
10. To calculate: pfu/mL = average plaque count / [(dilution factor) (2x10^-3mL)]
    NOTE: 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 30°C while shaking until the growth reaches OD₆₀₀ of 0.07 to 0.1.   
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 170-180 rpm at 30°C overnight.
3. Centrifuge phage culture at 4000 g for 10 minutes. Filter the lysate two times 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.

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.

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. Any proposed commercial use is prohibited without a license from ATCC.

While ATCC uses reasonable efforts to include accurate and up-to-date information on this product sheet, ATCC makes no warranties or representations as to its accuracy. Citations from scientific literature and patents are provided for informational purposes only. ATCC does not warrant that such information has been confirmed to be accurate or complete and the customer bears the sole responsibility of confirming the accuracy and completeness of any such information.

This product is sent on the condition that the customer is responsible for and assumes all risk and responsibility in connection with the receipt, handling, storage, disposal, and use of the ATCC product including without limitation taking all appropriate safety and handling precautions to minimize health or environmental risk. As a condition of receiving the material, the customer agrees that any activity undertaken with the ATCC product and any progeny or modifications will be conducted in compliance with all applicable laws, regulations, and guidelines. This product is provided 'AS IS' with no representations or warranties whatsoever except as expressly set forth herein and in no event shall ATCC, its parents, subsidiaries, directors, officers, agents, employees, assigns, successors, and affiliates be liable for indirect, special, incidental, or consequential damages of any kind in connection with or arising out of the customer's use of the product. While reasonable effort is made to ensure authenticity and reliability of materials on deposit, ATCC is not liable for damages arising from the misidentification or misrepresentation of such materials.

Please see the material transfer agreement (MTA) for further details regarding the use of this product. The MTA is available at www.atcc.org.