Hypochlorous Acid: An Ideal Wound Care Agent With Powerful Microbicidal, Antibiofilm, and Wound Healing Potency

Serhan Sakarya, MD; Necati Gunay, MS; Meltem Karakulak, MS; Barcin Ozturk, MD; Bulent Ertugrul, MD


Wounds. 2014;26(12):342-350. 

In This Article

Materials and Method


Hypochlorous acid is generated from sodium hypochlorite and hydrogen peroxide reverse reaction. The concentration used in this study was 218 ppm, pH 7.1, ORP 871 MV and its stability was 24 months (NPS Biosidal, Istanbul, Turkey).

Cell Lines and Microorganism Strains

Skin fibroblast cell line (BJ ATCC CRL-2522, American Type Culture Collection, Manassas, VA) was grown in Eagle's Minimum Essential Medium with 10% fetal bovine serum (Biological Industries, Kibbutz Beit-Haemek, Israel). Human skin keratinocyte cell line (CCD 1101 quarter, American Type Culture Collection, Manassas, VA) was grown in keratinocyte serum-free medium (Life Technologies, Carlsbad, CA). Slime producing Staphylococcus aureus (ATCC 35556) and Pseudomonas aeruginosa (ATCC 15692-PAO-1) were obtained from the Leibniz-Institut DSMZ GmbH (Braunschweig, Germany) and Candida albicans (ATCC 90028) from the American Type Culture Collection. Clinical isolates were obtained from the clinical microbiology laboratory of Adnan Menderes University Hospital Aydın, Turkey.

Minimum Bactericidal Concentration and Time Kill Assay

Inocula were prepared following the described guidelines of Clinical and Laboratory Standards Institute.[20] The stabilized HOCl solution inactivated with organic materials and HOCl solution with serial dilutions of 1/2, 1/4, 1/8, 1/16, 1/32, and 1/64 (109, 55, 22.5, 11, 5.5, and 2.75 ppm, respectively) were prepared in sterile phosphate buffered saline (PBS) solution. Sterile PBS alone was used as a control. Hypochlorous acid dilutions and control were prepared in 10 ml tubes and inoculums were added with a final cell density of 105 cfu/ml and incubated at 37°C for 60 minutes for minimum bacterial concentration (MBC). A 10 μl sample was removed and plated on Mueller Hinton (MH) or Sabouraud dextrose agar (SDA) plates. After incubating for 24 hours at 37°C, the growths were observed. The concentration at which there was a complete absence of colony growth was determined to be the MBC.

In time-kill (TK) studies, MBC concentrations of HOCl were inoculated with 105 cfu/ml of each organism and incubated for 0, 5, 10, 15, 20, 30, 60, and 90 minutes at 37°C. For each time point, 10 μl of inoculum was transferred to agar and incubated at 37°C for 24 hours. The time at which there was a complete absence of colony growth was determined to be the TK.

To determine the accurate killing time of bacteria, green fluorescent protein (GFP)-transfected P. aeruginosa were exposed to 1/32 dilution (5.5 ppm) of HOCl and video recorded through fluorescence microscopy (Olympus, Tokyo, Japan).

Antibiofilm and Microbicidal Effects of Hypochlorous Acid Within Biofilm

The ability of microorganisms to form biofilm on abiotic surfaces was quantified as described in Christensen et al.[21] Briefly, microorganisms were grown overnight in Triptic soy broth (Sigma-Aldrich, St. Louis, MO) or Sabouraud dextrose broth (Sigma-Aldrich, St. Louis, MO) with 0.25% glucose at 37°C. The culture was diluted 1:40 in proper media, and 200 μL of this cell suspension was used to inoculate into wells of 2 groups of sterile 96-well polystyrene U-bottom microtiter plates. After 48 hours incubation at 37°C, wells were gently washed 3 times with 200 μL of sterile PBS and a 2-fold serially diluted HOCl and sterile PBS alone were added to each well and incubated for 24 hours at 37°C.

One group of wells was used for biofilm eradication and the other for the microbicidal effect within the biofilm. After incubation, a biofilm-eradication group of wells was washed 3 times with PBS, dried in an inverted position and stained with 200 μL of 1% crystal violet for 15 minutes. Wells were rinsed and dried, and crystal violet was solubilized in ethanol-acetone (80:20, vol/vol). The optical density (OD) of well contents was determined at 595 nm using a microplate reader (Thermo Fisher Scientific, Milford, MA). A group of wells used to evaluate the bactericidal effect within the biofilm group was washed 3 times and each well was scraped and sonicated for 5 minutes in 100μl sterile PBS. Then, 10 μl of the contents of each well was plated on Mueller-Hinton or Sabouraud dextrose agar plates. Growth was observed after incubating for 24 hours at 37°C.

Skin Fibroblast and Keratinocyte Cell Migration in a Wound Healing Assay

Cells were cultured to confluence in the wells of 24-well plates (Corning Inc, Corning, NY). After 48 hours, a sterile 10 μl pipette tip was used to create a single wound across the diameter of each monolayer, and the medium was replaced with povidone iodine and HOCl solutions with serial dilutions of 1/2, 1/4, 1/8, 1/16, and 1/32 with PBS and PBS alone as a control, as previously described.[22] The cells were then incubated at 37°C and 5% CO2. Images of each monolayer were captured at 0, 4, 8, and 24 hours.

For determining the short time exposure effect of povidone iodine and HOCl, cells were treated with the serial dilutions of povidone iodine and HOCl solution for 10 minutes and rinsed with PBS. The cells were incubated in proper media at 37°C and 5% CO2 and images were captured at the time interval indicated above. Cell migration into the wound was calculated using the publicly available Image J software. Cell migration into each wound after 4, 8, and 24 hours was compared to the wounded monolayer at 0 hours.