To begin, incubate the samples in a warm water bath at 56 degrees Celsius for 30 minutes to heat-inactivate the test samples. Obtain an assay plate and 20 microliters of assay buffer to columns 1 through 12 of rows A through G. Add 20 microliters of assay buffer to columns 1 and 2 of row H. Load 30 microliters of each test sample in duplicate to row H of the assay plate.
To begin performing three-fold serial dilutions of the test samples, use a multichannel pipette to remove 10 microliters from wells 3H through 12H. Transfer the samples to the corresponding wells in row G, and pipette up and down 8 to 10 times to mix the sample well. Then, remove 10 microliters from these wells, transfer to the corresponding wells in row F, and pipette up and down 8 to 10 times to mix. Continue this serial dilution process through row A. After mixing the wells in row A, remove and discard 10 microliters from wells 3A through 12A so that the final volume in all wells is 20 microliters.
Retrieve one vial of frozen target bacterial stock, and thaw it at room temperature. Then, dilute the bacteria in 20 ml of assay buffer according to the predetermined optimal dilution factor. Using a multichannel pipette, add 10 microliters of diluted bacteria to each well of the assay plate.
Retrieve one vial of frozen baby rabbit complement and one vial of frozen heat-inactivated BRC. Use cold running water to thaw the vials to room temperature. Then, mix 100 microliters of heat and heat-inactivated BRC with 400 microliters of assay buffer. Add 50 microliters of this 20% heat-inactivated BRC solution to all wells in column 1. Mix 1 milliliter of native BRC with 4 milliliters of assay buffer. Add 50 microliters of this 20% mixture to all the wells in columns 2 through 12. Place the plate on a plate shaker for 10 to 15 seconds to gently mix the assay plate.
Incubate the assay plate in a microbiological incubator for two hours. Meanwhile, remove the lids from two LB agar plates, and place the plates face up in a biological safety cabinet for 40 to 60 minutes to dry. When the incubation is complete, transfer the assay plate to wet ice, and incubate for 10 to 20 minutes to stop the reaction. Using a 12-channel pipette, mix the wells in row H and spot-plate 10 microliters of the reaction mixture onto the bottom of an LBA plate. Immediately tilt the plate, and allow the spots to run for about 1.5 to 2 centimeters.
Repeat this procedure for rows G, F, and E, spotting them above the previous row. Incubate the LBA plates at room temperature until the solution is absorbed into the LBA plates. Then, put the lids on the plates, and transfer them upside down into a microbiological incubator to incubate overnight.
The next day, add 25ml of overlay agar at 55 degrees Celsius containing 100 micrograms per milliliter TTC and 0.1% sodium azide to each LBA plate. Incubate at 37 degrees Celsius for two hours to allow the surviving bacteria to develop a red color. Then, use a digital camera to photograph the plates. Transfer the images to a computer, and use NIST's integrated colony enumerating software to analyze the images as outlined in the text protocol.
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