Investigation of Bactericidal and Fungicidal Effects of Stabilized Ozone Gel

Mediplus Pharma, Inc. (Formerly VMC Corporation) Gotaro Shiota
Yoshiaki Maruyama, Kikumi Suzuki
Showa University School of Dentistry, Department of Prosthodontics, Akihiko Shiba
Tokyo Medical and Dental University Graduate School of Medical and Dental Sciences, Shiba Kiyoko, Okamura Noboru,
Chida Toshio
Division of Laboratory Medicine, Chiba University Hospital, Asako Kimura

Development Background:

Disinfection with ozone gas or ozonated water is more useful than conventional disinfectants because it is effective on a wide spectrum of bacteria, yeast, mold, viruses, etc. and does not leave residue, preventing the emergence of resistant strains. However, ozone has a short half-life of less than one hour, which means that ozone must be generally produced where it is used, thus limiting its scope of application.
To overcome this shortcoming of ozone, we have developed a new ozone compound, stabilized ozone gel, using glycerin as a solvent, which can be stored long-term and has an expanded range of applications.


・Investigation of the bactericidal and fungicidal effects of stabilized ozone gel (OG) on various microbial species and inhibition of their growth over time.
・Examination of OG’s sterilization concentration and effects of storage.

Test strains for OG disinfection:
Standard strains
Escherichia coli ATCC25922
Pseudomonas aeruginosa ATCC27853
Staphylococcus aureus ATCC25923
Candida albicans IAM4966

Clinical isolates
Methicillin-resistant S. aureus (MRSA)
Staphylococcus epidemidis
Klebsiella pneumoniae
Salmonella enteritidis
Legionella pneumophila
Bacillus subtilis


We developed an effective disinfectant OG that can be stored.
The oxidation power of purified OG was extremely strong, and its bactericidal and fungicidal effects remained effective after dilution.
Similar to conventional ozonated water, it effectively eliminated a wide spectrum of microorganisms, and did so in a relatively short time.

Future outlook:

The applications of OG which have been limited thus far, will be expanded with this storable form. Furthermore, applications that take advantage of the properties of the solvent glycerin (hydrosolubility and moisture-retention) will also be explored for its potential as a treatment for pressure sores, as a mouthwash, or as a hand disinfectant.

Bactericidal and fungicidal effects of ozone gel

Methods: To determine the bactericidal and fungicidal effects of OG, Okamura’s semi-quantitative viable count method using a 96-well plate was used. First, about 108 CFU/ml of the microbial solution with 10 times the amount of OG are mixed, and after exposure time, sodium thiosulfate is mixed to stop the reaction, after which 10 µl of that admixture is placed in the 1st column prepared with the culture medium to make a 10-fold dilution series up to 12 columns sequentially. Then, the culture is incubated overnight at 37°C to observe how many columns have developed the bacterial and fungal growth, and calculate the count from the dilution ratio to take the semi-quantitative measurement.
With this measurement method, the limit of detection for the viable count is “102 CFU/ml or less,” which is not very precise. However, this method was selected aiming to present an introductory overview of the bactericidal and fungicidal effects of the novel compound OG.

・Effects of storage on the bactericidal effect of ozone gel

The results for S. aureus and E. coli are shown on the left and right graphs. The vertical axis shows the viable count, and the horizontal axis represents the exposure time. Compared to the controls (sterilized water and glycerin), newly purified OG and OG stored for 2 months after purification both demonstrated strong bactericidal effects that reduced the viable count below the limit of detection.

Bactericidal effects of ozone gel by concentration

・ Undiluted ozone gel

The bactericidal power of undiluted OG was investigated in 6 species shown in the Table. With the exception of B. subtilis, a significant bactericidal effect was observed immediately after contact with OG until the viable count was below the limit of detection.
The oxidizing power of undiluted OG was several hundred units, equivalent to several hundred ppm in ozone concentration. This exceeded the concentration of ozonated water, which is used as a disinfectant, so we diluted OG with glycerin to investigate its bactericidal effects at concentrations comparable to existing products.

・Bactericidal and fungicidal effects of 100 ppm OG

The bactericidal and fungicidal power of 100 ppm ozone gel was investigated in 6 species.
We observed the bactericidal and fungicidal effect over time up to an exposure time of 120 minutes. The results are shown on the slide. With the exception of B. subtilis, all other species reached the limit of detection within 1 min.

・Bactericidal and fungicidal effect of 10ppm ozone gel

The bactericidal and fungicidal power of 10 ppm ozone gel was investigated in 6 species.
We observed the bactericidal and fungicidal effect of OG over an exposure time upto 360 minutes. The results are shown on the slide. C. albicans, E. coli, P.aeruginosa, K. pneumoniae, and S. epidemidis reached the limit of detection at 1, 30, 30, 120, and 180 min, respectively.