Plaque Assays with Soft Agar

Preparing Soft Agar

The soft agar should be prepared first, likely at least an hour before you pour

1. Make sure the water bath has enough water and is warming to 42C
2. Put a heating block in the hood; set to 42C and place sterile 13mm test tubes in it to warm up; use the number of plates expected, plus 1 test tube.
3. You will use about 4 mL of soft agar per plate, calculate total volume of agar needed accordingly
   • Always prep at least one extra plate
4. Loosen the cap of the soft agar bottle and microwave the bottle of agar until it is fully liquid:
   • Check for bubbling about every 15 seconds
   • You will know when it is fully melted because:
     • There will be no more “cloudiness” or bits
     • Shaking or swirling will cause bubbles to rise directly to the top (not being blocked by solid material)
5. After microwaving, place bottle into the preheated water bath
6. After preparing agar, prepare count out enough 13 ml tubes for your plates, and place in the heating block inside the virus hood

Preparing Petri Dishes

1. Use plain LB plates (no antibiotics)
2. Dish should have: Date, Your name, Strain, Dilution, and Duplicate ID
3. Try to label with small handwriting around the edges to make future plaque counting easier
4. The dilution labels on the petri dishes should only correspond to the serial dilutions that you wish to plate (for example: 10^6 -> 10^8, so 3 plates), do not plate every serial dilution
5. If you are working in multiples (like triplicates), calculate total amount of plates needed, ID them with letters
6. After labeling plates, place them in an incubator to warm

Pouring Soft Agar

The top layer will consist of an equally sized layer made of the same broth and soft agar (.8%) mixed with 30 uL of bacterial culture and 100 uL of phage dilution

• To prepare for pouring the phage layer:

1. 1. Prepare all phage pipettes
   2. Prepare filtered tips for phage
   3. Check that your dilutions are represent and organized
   4. Check that you collected all your warmed plates from the incubator
   5. Aliquot 4 mL of soft agar into your warming glass tubes
   6. Place big soft agar bottle on styrofoam to prevent rapid heat diffusion
   7. Make sure your vortexer is plugged in and the outlet is switched on

• Quickly but carefully (1 by 1), for full-plate phage dilutions:

1. 1. Pipette 100 uL of phage into the same glass tube
   2. Vortex your phage tube to confirm homogenous mixture
   3. Pipette 30 uL of bacterial culture into a glass tube
   4. Vortex for about 5 seconds ( look for the tornado)
   5. Open pre-labeled plate and pour mixture onto the LB layer
   6. Swirl gently to allow soft agar to spread evenly, cover, then set aside
   7. Allow about 15 seconds for a poured plate to set before moving
   8. Repeat for desired number of plates
   9. Incubate overnight at 37C with ambient CO2

• Quickly but carefully (1 by 1), for spot phage dilutions:

1. 1. Pipette 60 uL of overnight bacterial culture into 13mm test tube
   2. Vortex for about 5 seconds (look for the tornado)
   3. Open pre-labeled plate and pour mixture onto the LB layer
   4. Swirl gently to allow soft agar to spread evenly, cover, then set aside
   5. Allow about 15 seconds for a poured plate to set before moving
   6. Plate 5 μl of each phage sample and dilution according to previously made circles
   7. After adding phage we need to allow liquid to dry since it is on top of the solidified soft agar. While it would dry faster if the plate was uncovered, we do not want the phage to contaminate the hood in any way, so let liquid dry with the lid on the plate.
   8. Allow to dry for about 1 hour to ensure no drying issues
   9. Place plates in 37C incubator and check the next day.

Notes and Troubleshooting

• If the plaques (lawn clearings) are not visible:
  • Potentially adjust amount of phage added
  • Ask for help and consider substituting soft agar for an even lower concentration
• Titer- The concentration of infective phages within a stock
  • Titer is calculated in PFU/mL
  • PFU: Plaque Forming Units/ mL
• Remember that for a viral plaque assays, we are looking for “clear spots”, not colonies
• Overnight incubation of E.coli cells in a flask is recommended for optimal results
• This protocol makes use of LB plates, make sure you calculate how many you need before starting
• Conduct all cell work under sterile conditions
• Conduct all phage work under the designated hood with designated instruments