Plate Reader Assay and Growth Curve

Day 1: Streaking Bacteria From Freezer Stocks

• Refer to Eric to determine which strains will need to be streaked out. Some of the strains are NOT in the strain database; therefore, it is important to coordinate the experiment with Eric so he can help in the streaking process. Strains located in the strain database, refer to the protocol below.

1. Practicing Bio safety 2 protocols, bring an ice box to the -80 degree Celsius freezer (located in S223 instrument room). Obtain desired strains and put in the ice box.

2. Under a sterile hood, use an inoculating loop to pick the desired strain. Streak out onto pre-labeled blood plate (date, strain number, and strain code).

3. Dispose of contaminated inoculating look in 5% virkon S solution.

4. Place streaked blood plates face down in the 37 degree Celcius incubator and allow them to incubate overnight.

Day 1B: Maintaining Stocks of S. suis

1. Removed old S. suis stocks from the 37°C incubator and place under a sterile hood.

2. Using an inoculating loop, pick a single colony from the old stock plate containing grown bacteria.

3. Spread inoculating loop over the surface of a new "TSA Only" plate labeled with the date, strain number, and strain code.

4. Dispose of contaminated inoculating look in 5% virkon S solution.

Day 2: Overnight Culture

• All work done in the hood while practicing sterile technique.

1. Obtain autoclaved empty culture tubes and Todd Hewitt Broth (THB).

2. Pipette 3ml of THB into culture tubes. One tube will be used as a control/blank and all other tubes will be used to culture individual strains overnight.

3. Remove culture plate from the 37-degree Celsius incubator. Place under a sterile hood.

4. Label each culture tube with the corresponding strain number on the blood plate.

5. Using an inoculating loop, pick a single colony from the blood plate. Place loop in corresponding culture tube and move loop up and down in the THB media to mix. Place loop in the Virkon S for disposal.

6. Repeat step 5 for all other samples.

7. Place culture tubes/ plates back into the 37-degree Celsius incubator and allow to sit overnight.

Day 3: Plate Reader Assay'

1. Remove all strains from the 37 degree Celsius incubator and place under sterile hood.

2. In a deep 96-well plate, pipette 900 uL of 1 x PBS into each well.

3. For each of the strains under investigation, we will want to create a 1:10 dilution series. To do this:

    a. Vortex the original overnight culture. 
    b. Pipette 100 uL of cells from the overnight into the first well of the dilution series (This creates a 10^-1 dilution).
    c. To create subsequent dilutions remove 100 uL from the previous dilution and place in the next well in the series. Go up to a 10^-4 dilution.  

4. Once dilution series is complete, set up a normal 96-well plate.

5. Fill a reagent reservoir with THB broth.

6. Using a multichannel pipette, pipette 190 uL of THB and put into all 96 wells on the plate.

7. For the current investigation, use the repeating pipette to take in 40uL of sample from the 10^-4 dilution.

8. Dispense sample four times in the designated wells for each experimental sample under investigation (B1-B4 : Strain #1; C1-C4 : Strain #2; ect.)

    a. To take in sample with the repeating pipette, set sample uptake to 40 uL and press the elctronic plunger button
    b. Press plunger button once to remove air bubble from the pipette tip. 
    c. Press plunger tip to dispense sample. Repeat for the necessary number of samples (4 presses = 10 uL dispenses 4 times). 
    d. Once empty, press plunger button in order to dispose of tips in the 2% Virkion S. solution. 

9. Repeat steps 7-8 until all samples are added to the original 96-well plate.

Day 3 Continued: Operating The Plate Reader

• Plate reader located in the Bio super lab room (E 104)

1. Turn on the plate reader.

2. Log into the computer with the Tecan username

2. Open the icontrol 110 software.

3. Select File > Open. Open the script "GrowthRate_595nm_37deg"

4. Load the plate into the plate reader.

5. Start the analysis. Check the excel spreadsheet shortly after running the sample to ensure that the machine is running correctly.

6. The next day, return to the plate reader. Save the data, remove the 96-well plate from the machine, turn off the console, and transport the samples back to lab following bio safety two protocols.

Day 4: RStudio Code

1. Data must be converted to the following format:

time Blank.Rep1 Blank.Rep2 Ect….

time.pt1 Insert.Abs1 Insert.Abs2 Insert.Ect.

• Make sure to label the time column as “time” and label each column with the corresponding strain.

2. Copy and paste data into a new excel spreadsheet (if you don’t do this, RStudio may interpret empty cells as incomplete data entry. Save file as tab delineated (.txt) or comma delineated (.csv)

3. Now we can open RStudio and analyze the data.

To plot graphs of the 96-well plate:

• Make sure that the growth curver program is turned on. To do this navigate to Packages >> User Library >> growthcurver. Make sure the box next to growthcurver is checked.

> setwd("/Users/maure/desktop")

> file_name <- "/Users/maure/desktop/AC_PlateReader_Exp1_RStudio.txt"

> d <- read.table(file_name, header = TRUE, sep = "\t", stringsAsFactors = FALSE) # Convert the "time" column from seconds to hours

> d$time <- d$time / 60 / 60

> summary_plate <-SummarizeGrowthByPlate(plate = d)

> plot(summary_plate)

> summary_plate

> firstexp <-SummarizeGrowthByPlate(plate = d, plot_fit=TRUE)

> firstexp <-SummarizeGrowthByPlate(plate = d, plot_fit=TRUE, plot_file = "firstexp_plots.pdf")

To export data table:

> output_file_name <- "/Users/maure/Desktop/36.5_Exp1_24hrs_platereader_outputtable.txt"

> write.table(firstexp, file = output_file_name, quote = FALSE, sep = "\t", row.names = FALSE)