Standard Operating Procedures for T1-Phage Testing Assay

 

I. Introduction:

This assay uses a lawn of phage-susceptible E. coli (DH10B) embedded in a layer of agarose. This top agarose lays on a bed of standard LB agar. If phage is present in a clone streaked or stamped onto the top agarose lawn, the lawn there will be lysed on incubation overnight.

 

II.   Materials and Equipments:

• DH10B (original glycerol stock) or DH10B cultured stock for phage test.
• DH10B-T1 phage infected cells (positive control).
• Bacto-Tryptone, DIFCO, Becton Dickinson
• Bacto-Yeast Extract, DIFCO, Becton Dickinson
• Bacto-Agar, DIFCO, Becton   Dickinson
• NaCl, Angus Buffers & Biochemicals
• Agarose, GibcoBRL, Life Technologies
• Grade 190 Ethanol, Rossville
• 50 ml polypropylene tubes, Falcon, Corning
• Q-tray, Genetix
• 96-pins stamping tool, V & P Scientific, Inc.
• Autoclave, refrigerator, incubator, waterbath, and laminar flow hood.

 

III. Procedures:

Method to prepare the bottom LB agar:

1. Add 15 g tryptone peptone, 7.5 g yeast extract and 7.5 g NaCl into 2 liter of flask containing 1.5 liter of deionized distilled water.

   1 liter = 4 Q-trays	10g tryp, 5g yeast, 10 g NaCl, 15g Agar	200-250ml each tray	200 ml LB Broth + 1.6 g Agarose
   2 liter = 8 Q-trays	10g tryp, 5g yeast, 10 g NaCl, 15g Agar x2	200-250ml each tray	200 ml LB Broth + 1.6 g Agarose x2
   3 liter = 12 Q-trays	10g tryp, 5g yeast, 10 g NaCl, 15g Agar x3	200-250ml each tray	200 ml LB Broth + 1.6 g Agarose x3
   4 liter = 16 Q-trays	10g tryp, 5g yeast, 10 g NaCl, 15g Agar x4	200-250ml each tray	200 ml LB Broth + 1.6 g Agarose x4
   5 liter = 20 Q-trays	10g tryp, 5g yeast, 10 g NaCl, 15g Agar x5	200-250ml each tray	200 ml LB Broth + 1.6 g Agarose x5
   6 liter = 24 Q-trays	10g tryp, 5g yeast, 10 g NaCl, 15g Agar x6	200-250ml each tray	200 ml LB Broth + 1.6 g Agarose x6
   7 liter = 28 Q-trays	10g tryp, 5g yeast, 10 g NaCl, 15g Agar x7	200-250ml each tray	200 ml LB Broth + 1.6 g Agarose x7

2. Mix with a magnetic stirring bar until the powder is completely dissolved.
3. Add 680 m l 5N NaOH to bring pH to ~7.2.
4. Add 22.5 g bacto-agar and stir the solution for 5 minutes.
5. Cover the bottle with aluminum foil . If a bottle with screw cap is used instead of flask, be sure to loose the cap prior to starting autoclave. Autoclaving liquid in the bottles requires loosening the cap to facilitate leaking air pressure out from the bottle during the cycle. Failure to do this may cause a breakage of the bottle during the cycle due to the intense high pressure.
6. Autoclave the medium still containing the magnetic stirring bar at 121° C for 60 minutes.
7. Once the autoclave cycle is finished, take the bottle out from the autoclave carefully. Be sure that the temperature is below 98° C.
8. Stir the medium on a stirrer and cool the medium to 55° C . It is possible to place the bottle in a bucket containing water to help cooling. Do not use ice-cold water, because the glassware may crack or break because of the immediate temperature change.
9. Pour ~225 ml media into a Q-tray using a 500 ml sterile cylinder. It is important to minimize time and height of lifting the cover to avoid contamination from the air while pouring.
10. Flame the surfaces of the media in Q-tray briefly with a Bunsen burner while lifting the cover slightly. Flaming the media facilitates eliminating bubbles from the surface.
11. Leave the plates at room temperature for ~45 min to solidify.
12. Wrap the plates in a plastic bag and store them upside-down at 4° C.

 

Method to prepare the top agarose as bacterial lawn:

1. Grow freshly the phage-susceptible E. coli clone culture using LB media the day before making bacterial lawns. This step can be skipped if we have stored culture at -80� C (see materials).
2. Pull out required number of prepared Q-trays with bottom LB agar and place them upside-down in 37� C incubator for an hour.
3. Find a waterbath and set temperature at 45� C prior to melting agarose.
4. For making 4 Q-trays of bacterial lawns, pour 200 ml LB media into 500 ml flask and add 1.6g of agarose. Autoclave at 121� C for 20 min.
5. Divide the 200 ml 0.8% agarose/LB into 4 aliquots of 45-50 ml in 50 ml Falcon tubes.
6. Place all Falcon tubes in a waterbath set at 45� C for at least 30 min to attain total equilibrated temperature.
7. Add ~1.2 ml lawn culture into each Falcon tube and mix gently & quickly .
8. Take (under laminar flow hood ) one Q-tray out of the incubator at a time and pour on the top agarose with lawn culture in an evenly fashion. Be sure to spread the top agarose homogeneously .
9. Leave the plates at room temperature for at least 10 min. Keep the plates upside-down at room temperature until use.
10. Method to test for phage on lawn culture by streaking:
11. Clean inside of a hood with 10% bleach, deionized water and 70% ethanol and sterilize the inside with UV light for at least 20 minutes.
12. Streak (Under the hood) each clone onto the lawn using a sterile tip or toothpick. Discard the used tip into bleach immediately .
13. Seal the lawn Q-tray with Sealwrap or saranwrap.
14. Incubate overnight at 37� C. and read the results tomorrow. (Lawn Q-trays should NEVER be opened after incubation if lysis is observed, thus preventing the spread of the phage throughout the laboratory ).
15. Clean inside of the hood with 10% bleach, deionized water and 70% ethanol thoroughly and sterilize the inside with UV light for at least 30 minutes.

 

Method to test for phage on lawn culture by stamping:

1. Clean inside of a hood with 10% bleach, deionized water and 70% ethanol and sterilize the inside with UV light for at least 20 minutes.
2. Sterilize the 96 or 384-pins stamping tool by using our standard sterilization sequences: 1X in 10% bleach, 1X on medium level deionized water, and 1X on high level 95% ethanol. The use of ascending level of bleach, water, and ethanol is to eliminate the possibility of cross contamination by phage between each stamping. Bleach kills the phage and the clones. Water cleans up the bleach. And ethanol removes the water .
3. Dip stamping tool into testing block or plate very carefully .
4. Pick up the stamping tool and lay it on top of the lawn Q-tray. Take a corner of the Q-tray as the A1 position for the block .
5. Put the stamping tool immediately after each usage into the 10% bleach.
6. Repeat the above steps on all testing blocks, using one lawn Q-tray for every four blocks.
7. Seal the lawn Q-tray with Sealwrap or saranwrap.
8. Incubate overnight at 37� C and read the results tomorrow. Lawn Q-trays should not be opened after incubation if lysis is observed, thus preventing the spread of the phage throughout the laboratory .
9. Clean inside of the hood with 10% bleach, deionized water and 70% ethanol thoroughly and sterilize the inside with UV light for at least 30 minutes.

 

Result Interpretation:

• For a phage infected clone, the underlying lawn will be lysed where the clone was streaked or stamped.
• For a phage infected and possibly resistant clone, the underlying lawn will be lysed with clones growing on top of the lysis area. These clones can be rescued by using our standard rescue procedures.
• For a phage infected and possibly non-resistant clone, the underlying lawn will be lysed without any clone growing on top of the lysis area.

 

References:

Phage Testing Assay Protocol by HGMP Resource Center, UK , funded by MRC.