TITLE: Plaque Assay
AUTHORS: Alice Lee

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Bacteriophages (phage) are viruses that infect bacteria and can be found anywhere that bacteria is found. Performing plaque assays is a technique to purify a population of viruses and can also be used to determine the viral concentration of a given sample. This protocol will outline the steps to carry out plaque assays.</div></div>

[STEPS]
?. [Inoculating a liquid bacterial culture overnight]
The day before performing the plaque assay experiment, grow the bacteria associated with the phage of interest.
?. [Inoculating a liquid bacterial culture overnight]
Take out an already streaked bacteria plate from the   room.
4 °C
?. [Inoculating a liquid bacterial culture overnight]
Take a snap cap tube and place it on a rack. Open the cap and aseptically transfer  of BHI media into the tube.
2 ml
?. [Inoculating a liquid bacterial culture overnight]
Take a disposable inoculating loop and scrape a colony from your bacteria streak plate and transfer into the snap cap tube with  of BHI media. Recap the snap cap tube and throw the used inoculating loop in the red biohazard bin.
2 ml
?. [Inoculating a liquid bacterial culture overnight]
Transfer the snap cap tube to the  incubator room and place it on the shaker. Then turn the shaker on to . When leaving the room, remember to turn off the light switch on the outside of the room.
37 °C
Centrifuge: 200 33
?. [Inoculating a liquid bacterial culture overnight]
Grow the bacteria on  shaker overnight.
37 °C
?. [Inoculating liquid bacterial culture day of experiment]
The day of the experiment, take the overnight grown bacteria and transfer to fresh media to grow so that we will be working with log-phase bacteria.
?. [Inoculating liquid bacterial culture day of experiment]
Take a new snap cap tube and aseptically addof fresh BHI media into the tube.
2 ml
?. [Inoculating liquid bacterial culture day of experiment]
Take the overnight grown bacteria from the  incubator. Aseptically transfer  of the overnight culture into the new tube with  of BHI media.
37 °C
100 µl
2 ml
?. [Inoculating liquid bacterial culture day of experiment]
Transfer this snap cap tube to the  incubator room and place it on the shaker. Then turn on the shaker to .
37 °C
Centrifuge: 200 33
?. [Inoculating liquid bacterial culture day of experiment]
Incubate the sample on the shaker for ~3 hours or until the sample has become cloudy indicating bacterial growth.
?. [Preparing BHI plates]
Take out the required number of BHI plates from the  incubator room. Wipe down the hood with 70% ethanol. Place the plates inside the hood with the lid halfway open. This will dry out the liquid droplets from the plate.
4 °C
?. [Phage Serial Dilution]
In order to determine the concentration of the phage stock, you will need to perform a serial dilution of the stock.
?. [Phage Serial Dilution]
Take the phage stock tube from the  freezer and let it thaw at room temperature. The location of the phage stocks can be determined by looking at the MissionControl document on Google Drive.
-80 °C
?. [Phage Serial Dilution]
To perform a 1:10 dilution of the stock sample, add  of SM buffer into a 1.5mL tube then add  of the stock phage into the tube. Mix well.
90 µl
10 µl
?. [Phage Serial Dilution]
To perform a 1:100 dilution of the stock sample, add  of SM buffer into a 1.5mL tube then add  of the 1:10 phage dilution from Step 4.2 into the tube. Mix well.
90 µl
10 µl
?. [Phage Serial Dilution]
Continue performing the serial dilutions for how many samples you will be observing.
?. [Phage Serial Dilution]
Create a tube for the negative control by adding  of SM buffer and  of Ultra Pure Water (UPW) into a 1.5mL tube.
90 µl
10 µl
?. [Plaque Assay]
Combine the log phase bacteria and serially diluted phages then plate them.
?. [Plaque Assay]
Take a bottle of BHI + 0.35% LMP agarose and microwave until boil. The microwave is located in BSL1 so make sure to remove all BSL2 PPE before entering BSL1. Start off with  then gradually add more time to prevent boil over of the media onto the microwave. Use paper towels or an autoclave glove to handle the glassware and transfer it back to BSL2.
?. [Plaque Assay]
Ensure that the BHI + 0.35% LMP agarose cools down before using it or it may heat shock the bacteria and kill it.
?. [Plaque Assay]
Take a glass culture tube and label it appropriately with the sample.
?. [Plaque Assay]
Add  of log-phase bacteria from Step 2 into the glass tube. Then add  of the phage dilution into the same glass tube. Let sit for 15-20 minutes.
100 µl
10 µl
?. [Plaque Assay]
Use a serological pipette and measure  of cooled down BHI + 0.35% LMP agarose. Aseptically transfer to the glass tube and mix by pipetting up and down but not quickly to prevent any bubble formation.
3.5 ml
?. [Plaque Assay]
Transfer the contents of the glass tube onto a dried BHI plate and swirl the plate around to properly disperse the contents.
?. [Plaque Assay]
There is a green taped X mark on the sink counter which is leveled. Place the plate on top of the X mark and let it solidify.
?. [Plaque Assay]
Continue for the rest of the samples. Don't forget to always perform a negative control plate.
?. [Plaque Assay]
After the plates have solidified, store in the  incubator upside down. Incubate overnight.
37 °C
?. [Determine the phage concentration]
The next day, observe the plates to determine the concentration of the viral sample from the stock.
?. [Determine the phage concentration]
Look at the plates and find one where there are enough phage forming units (PFU) that can be counted. Count the number of plaques on the plate.
?. [Determine the phage concentration]
Use this formula: PFU/uL = Number of Plaques Counted/ (Dilution x Volume of Diluted Virus Added)Example: There are 28 plaques on a 10^-2 phage dilution plate. You had plated 10uL of this along with 100uL of bacteria. 28/10^-2 x 10uL = 280 = 2.8 x 10^2 PFU/uL