A High-throughput Shigella-specific Bactericidal Assay

This protocol follows the guidelines of the WRAIR Human Subject’s Protection Board. Samples used in this study are human serum samples collected as part of WRAIR protocol number 1328, in compliance with all institutional and federal regulations governing the protection of human subjects. Samples were de-identified, and the use of these anonymous samples was classified as non-human subject research by the UAB IRB (protocol number N150115001).

1. Prepare Assay Reagents

1. Prepare 1% Gelatin by adding 1 g of gelatin to 100 mL of water. Autoclave and store at room temperature.
2. Prepare 100 mg/mL TTC (2,3,5-Triphenyltetrazolium chloride) stock solution (1,000x) by adding 5 g of TTC to 40 mL water. When the TTC is fully dissolved, adjust the volume to 50 mL with water and sterile filter using 0.2 µm filter. Store the solution at 4 °C and protect from light.
   NOTE: TTC colorizes the bacterial colonies and makes them much easier to count. TTC solution has a slight yellow color. If TTC solution develops a red color, discard and prepare fresh.
3. Prepare 10% sodium azide (NaN₃) stock solution (100x) by adding 5 g NaN₃ to 40 mL of water. After complete dissolution, add water to 50 mL. Store at room temperature.
   CAUTION: Sodium azide is a poison and may be toxic if ingested or absorbed through the skin or eyes. It may react with lead and copper plumbing to form highly explosive metal azides. On disposal of reagents containing sodium azide, flush with a large volume of water to prevent azide build-up or discard in a biohazard bag.
4. Prepare LBA plate (LB agar plate) by adding 35 g of LB agar to 1 L of water and autoclave. Add 25 mL to each square petri dish (120 x 120 mm²). Incubate plates at RT for 10-20 min to allow agar to solidify. Place plates back into plastic bags and store at 4 °C for up to 1 month.
5. Prepare Overlay Agar by adding 7.5 g of agar to 1,000 mL of water and autoclave. Incubate in 56 °C water bath until needed. Right before use, add 1 mL of 100 mg/mL TTC and 10 mL of 10% NaN₃ and mix well.
   NOTE: Each LBA plate needs 25 mL of Overlay Agar. Overlay Agar can be prepared up to one month in advance and melted in a microwave or on a hot plate as needed for the assay. Ensure that agar temperature is ~55 °C before application to LBA plates.
6. Prepare Assay Buffer by adding 5 mL of 10x Hanks’s Balanced Salt Solution (HBSS) with Ca²⁺/Mg²⁺ and 5 mL of 1% gelatin to 40 mL of water. Store at room temperature.

2. Prepare Complement and Target Bacteria

1. Prepare baby rabbit complement (BRC)
   NOTE: Detailed criteria for complement lot selection can be found here: https://www.vaccine.uab.edu/uploads/mdocs/UAB-MOPA.pdf
   1. Obtain frozen BRC and thaw with running cold water. Physically mix the BRC every ~10-20 min until completely thawed. Do not subject BRC to repeated freeze thaw cycles.
   2. While BRC is thawing, label 1.5 mL, 5mL, or 15 mL centrifuge tubes. Place labeled tubes on ice to pre-cool. Aliquot proper volume BRC to the pre-cooled centrifuge tubes (after filling, immediately return each tube to the ice). Store aliquots at ≤ -70 °C in the freezer.
      NOTE: Approximately 1 mL of complement is needed for each assay plate. Complement aliquots are for single use and should be aliquoted in volumes appropriate to assay layouts.
   3. To prepare Heat-inactivated BRC, thaw one aliquot of active BRC. Prepare a 56 °C water bath. After BRC is completely thawed, transfer it to the water bath and incubate for 30 min.
   4. After incubation, remove heat-inactivated BRC from the water bath and allow to cool at RT for 10-15 min. Mix vigorously, and aliquot ~150 µL to 1.5 mL microcentrifuge tubes. Store aliquots at ≤ -10 °C.
2. Prepare target bacteria stock
   NOTE: The procedure below is used to prepare 48 aliquots of target bacteria stock; if more aliquots are needed the protocol can be scaled up.
   1. Remove the bacteria master stock vial from the freezer and scrape the frozen bacterial surface to remove a small amount of ice from the vial onto a blood agar plate. Immediately return the master stock vial to the freezer.
   2. Streak this small aliquot of bacterial stock onto the blood agar plate and cover with the plate lid. Incubate the plate upside down overnight in 37 °C/5% CO₂ incubator.
   3. Transfer ~10 isolated smooth colonies to a 50 mL tube containing 30 mL of LB broth. Incubate for 3-5 h at 37 °C with gentle shaking until the culture broth has an OD₆₀₀ of ~0.6-0.7.
   4. Harvest the top 12.5 mL of the culture and transfer to a fresh 50 mL tube. Centrifuge the culture at 15,000 x g for 2 min using a table top micro-centrifuge. Discard the supernatant and re-suspend the pellet in 25 mL of 15% sterile glycerol-LB.
   5. Mix well and dispense 0.5 mL aliquots into sterile 1.5 mL micro centrifuge tubes (~48 tubes). Store aliquots at ≤ -70 °C in freezer.
   6. Confirm the bacterial identity using the agglutination test before use.
3. Determination of optimal dilution factor for target bacteria stock
   NOTE: Each batch of target bacteria stock must be titrated in assay conditions to determine the dilution necessary to yield ~120 CFU/spot on LB plates.
   1. Get a microtiter plate (Dilution Plate) and add 135 µL of Assay Buffer to well 1A. Add 120 µL of Assay butter to wells 1B-1H.
   2. Remove a vial of frozen target bacteria from the freezer and thaw at room temperature. Add 15ul of thawed bacterial stock to well 1A to make a 10-fold dilution of the bacterial stock.
   3. Transfer 30 µL of bacterial solution from well 1A to well 1B to perform a 5-fold serial dilution. Continue 5-fold serial dilutions to well 1H for a total of 8 dilutions (1:10; 1:50; 1:250; 1:1 250; 1:6 250; 1: 31 250; 1: 156 250; 1:781 250).
   4. Get another microtiter plate (Assay Plate) and add 20 µL of Assay Buffer to all well in columns 1 and 2 in the Assay Plate.
   5. Transfer 10 µL of diluted bacteria from each well in column 1 of the Dilution plate into the corresponding wells in columns 1 and 2 of the Assay Plate. 10 µL of bacteria is transferred from well 1A of the dilution plate into well 1A and 2A in the Assay Plate, etc.
   6. Continue with the assay as described in Serum Bactericidal Assay (SBA) below for Control A and Control B, steps 3.6-3.12.
   7. After the plates have been incubated on ice, use a multichannel pipette with 8 pipette tips to spot 10 µL from the wells in column 1 onto an LBA plate. Also, spot the wells from column 2 onto the LBA plate.
   8. Continue with the assay as described below in steps 3.14-4.6.
   9. Determine the bacteria dilution that yields ~120 CFU/spot in Control B, this dilution will be used in the assay.

3. Serum Bactericidal Assay (SBA)

NOTE: The procedure described below is for one assay plate, but the number of Assay Plates can be increased.

1. Heat-inactivate test samples by incubating samples in a 56 °C water bath for 30 min.
   NOTE: Test samples must be heat-inactivated prior to the test to abrogate any endogenous complement activity. This can be done ahead of the assay and inactivated samples can be re-frozen or stored at 4 °C until tested.
2. Get an Assay Plate and add 20 µL of Assay Buffer to columns 1 through 12 of rows A through G. Add 20 µL of Assay Buffer to columns 1 and 2 of row H, see Table 1.
3. Load 30 µL of each test sample, in duplicate, to row H of the Assay Plate. For example, dispense 30 µL of sample 1 into wells 3H and 4H, and dispense 30 µL of sample 2 into wells 5H and 6H, etc.
4. Perform 3-fold serial dilutions of test samples using a multichannel pipette.
   1. Remove 10 µ of the sample from wells 3H-12H and transfer to corresponding wells in row G and mix the sample well by pipetting up and down 8-10 times.
   2. Then remove 10 µ from wells 3G-12G and transfer to corresponding wells in row F and mix well.
   3. Continue these serial dilutions through row A. After mixing the wells in row A, remove and discard 10 µl from wells 3A-12A so that all wells contain 20 µl.
      NOTE: Because 20 µL of serum is used in a total assay volume of 80 µL, there is a 4-fold additional dilution in the assay. This dilution must be taken into account during the calculation of an SBA titer by multiplying the dilution of serum by 4. For example, if a starting dilution of 1:2 is used, the actual dilution being tested is 1:8.
5. Remove one vial of frozen Target Bacteria Stock and thaw at room temperature. Dilute the bacteria in 20 mL of Assay Buffer according to the pre-determined optimal dilution factor (this dilution factor was determined in section 2.3). Add 10 µL of diluted bacteria to each well of the assay plate using a multichannel pipette.
6. Remove one vial of frozen BRC and one vial of frozen Heat-inactivated BRC, thaw at room temperature with running cold water or place on the grate of a biological safety cabinet with blowing air to thaw quickly.
7. Prepare a 20% solution of heat-inactivated BRC. Mix 100 µL of heat-inactivated BRC with 400 µL of Assay Buffer. Add 50 µL of this 20% heat-inactivated BRC solution to all wells in column 1 (Control A wells).
   NOTE: Heat-inactivated BRC is used as a control to monitor non-specific killing (NSK) in the assay.
8. Prepare a 20% solution of native BRC. Mix 1 mL of native BRC with 4 mL of Assay Buffer. Add 50 µL of this mixture to all wells in columns 2 through 12 (Control B and test sample wells).
   NOTE: The final concentration of BRC in the reaction mixture is 12.5%.
9. Briefly mix Assay Plate by shaking gently for 10-15 s on a plate shaker or mix by pipetting up and down 8 times using a multichannel pipette.
10. Put the Assay Plate in 37 °C microbiological incubator for 2 h (without shaking).
11. Dry 2 LBA plates by removing lids and placing plates face up in biological safety cabinet for 40-60 min.
12. When the 2 h incubation is complete, move the Assay Plate to wet ice and incubate for 10-20 min to stop the reaction.
13. Using a 12-channel pipette, mix the wells in row H, and spot plate 10 µL of the reaction mixture onto the bottom of an LBA plate. Immediately tilt the plate and allow the spots to run for ~1.5-2 cm. Repeat this procedure for row G, F, and E, spotting them above the previous row on the LBA plate. Row E, F, G, and H are spotted onto one LBA plate, and row A, B, C, and D are spotted onto a second LBA plate in the same manner, see Figure 1.
14. Incubate the LBA plates at room temperature until the solution is adsorbed into the LBA plates (10-15 min). Put the lids on the LBA plates and place the LBA plates in the microbiological incubator upside-down to incubate overnight (~16-18 h). Incubate S. flexneri 2a and 3a at 29 °C and incubate S. sonnei is at 26 °C.
    NOTE: These temperatures yield smaller “micro-colonies” with sizes suitable for accurate counting by a colony counter⁹.
15. After overnight incubation, add 25 mL of Overlay Agar (at ~55 °C) containing 100 µg/mL TTC and 0.1% NaN₃ to each LBA plate.
16. Incubate the LBA plates at 37 °C for 2 h to allow the surviving bacteria to develop red color, see Figure 1 below.
17. Photograph plates using a digital camera and transfer images to a computer where NIST’s Integrated Colony Enumerating Software (NICE) has been installed, see Figure 2.
    NOTE: NICE colony-counting software is available at no charge. See Materials List for details and installation instructions.

4. Count Bacterial Colonies

1. Open NICE Software and input Operator Name, Experiment information, and any assay Notes into the empty fields. Once this data is entered click the Done button.
2. Import photographed plates by clicking the Open button and selecting the correct files from the computer.
3. Adjust the assay parameters by setting the number of Rows to 4 and the number of Columns to 12. Adjust the Background setting to -3 sigma and the Resolution to Low. See Figure 2.
4. Move the Regions of Interest (ROIs) by clicking and dragging so that each ROI is directly over one sample spot. Ensure that all bacterial colonies are inside the ROI with no overlap between samples. Once one plate is complete, double click the next plate in the Stored data Images list and adjust the ROIs. See Figure 2.
5. When all plates have been adjusted, click the green Count button, see Figure 2 and Figure 3. When counting is finished click the Export button to export the data in .xls/.xlsx format. Name and save the .xls/.xlsx file for data analysis.
6. Arrange exported data into a table format so that counts are organized in a table representing the 96-well assay plate, see Table 2.

5. Calculate SBA Titer (KI) and Non-specific Killing (NSK)

NOTE: SBA titer, or killing index (KI) is defined as the reciprocal of the serum dilution that kills 50% of the target bacteria.

1. Calculate the 50% killing index (KI) cutoff value by averaging the CFU of active complement control wells (Control B) and dividing by 2. Calculate the average CFU for each dilution of each sample that was run in duplicate, see Table 3.
2. Because a serum dilution will rarely yield exactly this 50% KI value, it can be interpolated from two sequential serum dilutions, one that kills less than 50% and one that kills more than 50%, see Figure 4. The formula for calculating the interpolated SBA KI is shown below:
   
   NOTE: The bactericidal titer, or KI, can also be calculated automatically using Opsotiter software developed by UAB. To Request Opsotiter, contact Dr. Moon Nahm or Mr. Rob Burton. See https://www.vaccine.uab.edu for contact information.
3. Calculate the NSK value by taking 1 minus the average of Control B divided by the average of Control A.