The overall goal of this experiment is to test the comparative efficacy of handwashing options on hands that have been experimentally inoculated with an organism of interest. This method can provide information on key questions to help prevent of infectious diseases. By allowing researchers to investigate the efficacy of handwashing for particular organisms of interest.
The main advantage of this technique is that it is flexible and can be adapted to allow for investigation of many different organisms. We first had the idea for this method when we realized chlorine solutions were being used for handwashing in Ebola contexts and there was no evidence for the efficacy of this at the time. After recruiting eligible subjects and preparing handwashing solutions according to the text protocol, prepare bacteria by streaking a non-pathogenic strand of E.Coli onto LB agar plates and incubate the cultures at 37 degrees celsius for 24 hours to obtain single colonies.
One day before the start of the experiment, use a sterile lube to grab a single colony from the plate and inoculate 10 milliliters of LB broth. Incubate the cultures at 37 degree celsius, with shaking, overnight. On the morning of the experiment, add one milliliter of the overnight culture to 20 milliliters of fresh LB broth.
Incubate the cultures for approximately two and half hours to achieve a salt density greater than 10 to the eighth CFU per milliliter. Then, use a spectrophotometer to estimate the concentration of the culture. Immediately prior to testing, prepare an inoculum composed of 68%bacteria or viral suspension and 32%sodium chloride or soil load.
Swirl or gently vortex to mix. After preparing the volunteers according to the text protocol, test the pH of each volunteer's skin by placing a flat tip skin pH probe in the webbed space between the pointer and the middle fingers. Ensure that the electrode is flat against the skin and record the pH reading.
Next, have the volunteer cup both hands together. Then, carefully pipette 750 microliters of the inoculate into each palm to spike the hands. When spiking volunteers'hands, it is important none of the inoculum is spilled.
Remind volunteers this is important and give them the opportunity to practice with water before beginning the procedure. Have the volunteers gently rub their hands together until all surfaces of the hand are coded with the inoculate, while subjecting the hands to as little friction as possible. Instruct the volunteers to hold their hands still and away from their body for an additional 30 seconds to allow the inoculate to dry.
Then wash the hands using one of the methods described in the text protocol. For hand washing with soap, use 10 milliliters of Ultrapure water to wet the hands. Have the volunteers lather their hands with soap and then rub their hands together for an additional 20 seconds.
Rinse the volunteer's hands by pouring 500 milliliters of Ultrapure water at room temperature through a funnel at a flow rate of 1.5 liters per minute into a large sample collection bag and process the sample within two hours. For all chlorine solutions, pour 200 milliliters of chlorine solution over the volunteer's hands through a funnel at a flow rate of 1.5 liters per minute and have the volunteers rub their hands thoroughly. As another hand washing method, have the volunteers use hand sanitizer to wash their hands.
It's important all surfaces of the hand are gently rubbed, instruct participants to focus on each part, palms, fingernails, in between fingers as they rub and then ensure the entire hand has been rinsed. For control A, after inoculation, do not perform a handwashing step. For control B, use only Ultrapure water at room temperature to perform handwashing through a funnel with a known flow rate.
After handwashing, immediately place each volunteer's hand selected for testing, into a sample bag containing 75 milliliters of Leeuwin up to the wrist. Hold the top of the bag tightly around the wrist. Have the volunteers gently rub their hand in the solution for 30 seconds, taking care to reach in between the fingers and underneath the fingernails.
Gently massage the hand from outside the bag for 30 seconds to ensure that the entire hand is thoroughly rinsed in the Leeuwin, all the way up to the wrist. Seal the bag and process it according to the appropriate essay. Before repeating the process with each handwashing method, have the volunteers use soap in warm water to thoroughly wash their hands in the sink.
Finally, use 70%ethanol to spray the volunteer's hands until they are coated on both sides, and allow them to dry. To analyze the samples, set up a flame and a filtration manifold with sterile filtration funnels and a vacuum connection. Sterilize forceps by dipping them in ethanol and flaming them.
Use the forceps to place a 0.45 micron filter onto the filtration manifold with the grid facing up. Then use a small amount of sterile PBS to wet the filter. Place the funnel on the base and pipette or pour the sample directly onto the filter.
Engage the vacuum until the entire sample has passed through the membrane. Then, use sterile PBS to rinse the sides of the funnel and engage the vacuum again. Next, remove the funnel, flame sterilize the forceps and lift the filter from the base.
Then place the filter gently on the medium in a petri dish with the grid facing up, ensuring that the filter lies flush against the surface. Invert the plates and incubate at 37 degrees celsius for 24 hours. Following the incubation, count the E.Coli colonies and record the data.
Carry out data analysis according to the text protocol. The protocol described in this video was completed with 18 volunteers tested with E.Coli and Phi6, for E.Coli without soil load, handwashing with HTH, NaDCC and stabilized NaOCl, all resulted in significantly greater log reductions than handwashing with water only. With soil load, HTH resulted in a significantly greater log reduction of E.Coli than water only handwashing with soap and ABHS.
There was no significant difference between methods for Phi6 without soil load. However, for Phi6 with soil load, water alone resulted in a greater log reduction than ABHS or stabilized NaOCl, and handwashing with soap in a greater log reduction than ABHS, stabilized NaOCl and generated NaOCl. HTH also had a greater log reduction than ABHS and stabilized NaOCl and NaDCC resulted in a greater log reduction than stabilized NaOCl and ABHS.
As demonstrated in these graphs, chlorine resulted in a significantly greater log reduction of E.Coli persisting in the rinse water than handwashing with soap. Finally, the same pattern was found in Phi6 without soil load with all chlorine solutions resulting in a significantly greater reduction of Phi6 in rinse water than handwashing with soap. There were no significant decreases in rinse water with Phi6 and soil load.
While attempting this procedure, it's important to remember efficacy results for each condition are calculated by comparing the results for the two controls. This means it is very important to perform the steps consistently for each handwashing option.
