University Medical Center Goettingen Departement Medical Microgiology Drinking water and general hygiene laboratory Dr. Ulrich Schmelz, M.D., Food Chemist and Engineer Head of the laboratory www.hygiene-goettingen.de UMG, Dr.med.U.Schmelz Humboldtallee 34A, D-37073 Göttingen Firma DIOP GmbH & Co. KG Dieselstraße 5-6 D-61191 Rosbach Germany Laboratory and testing center: Humboldtallee 34A D-37073 Goettingen Dr. Ulrich Schmelz, M.D. Tel.: 0049 / 551 / 39 4973 E-Mail: Ullischmelz@aol.com Labor: Telefon: 0049 / 551 / 39-4970 Fax: 0049 / 551 / 39-4957 Date: 21th January 2013 Laboratory Report Determination of microbiocide inactivation performance of the DIOSOL-Process within quantitative Suspension Test with practical fomites 1. Question: Within the quantitative Suspension Test microbiocide inactivation performance of the DIOSOL-Process should be tested with practical fomites. The DIOSOL-Process (DIOSOL-Generator Standard) is based on a selected cold-fumigation of DIOSOLdilution (Hydrogenperoxide dilution w=3% with colloidal silver 35ppm) in a room for the purpose of room- and surface disinfection. In this connection a defined volume of DIOSOL dilution is filled into an aerosol as a function of the volume of space; the aerosol is exposed to the surfaces of the room over a selected exposure time. Following testing germs will be investigated: Staphylococcus aureus Enterococcus faecalis Pseudomonas aeruginosa Escherichia coli Candida albicans Trichophyton rubrum Aspergillus niger Thus both gram-positive and gram-negative bacteria, yeast, dermatophytes and mould are detected. As practical fomites stainless steel plates with 10*10cm length of the edge (surface = 100cm²) are used. The stainless steel plates possess a roughness depth of 100µm.
For preparation of the inactivation test fomites are sterilized at first by steam according to DIN EN 285. Afterwards fomites are spread on one site with a suspension of the respective testing germ. Every preparation is applied twice. One of both preparations is directly loaded with the germ suspension ( in the following described as fomites ), the other preparation is covered with a suspension that additionally contains 3,0% of beefserumalbulin and 0,3% of sheep-erythrocyte (below described as fomites + organic load ). After application of the respective testing germ fomites are dried at 36 C and used for analysis. The charged fomites are put into the test room to control the reduction performance (room volume 16 m³). Fomites are placed horizontally in the test room, the germcovered site points downwards. DIOSOL-method is adopted. Specimen are packed under sterile conditions and used for determination of bacterial count after disinfection. The decade logarithm of the bacterial count before disinfection (initial germ number) and the decade logaritm of bacterial count after disinfection is composed. By subtracting logarithm of germ numbers logarithm reduction factor has to be generated, which illustrates germ reduction performance. 2. Methodology: Test-Methodology according to DGHM-requirements in the context of a quantitative suspension test. For determining colony count fomites are floated off with sterile common salt solution 0,9% which still contains 0,01% TWEEN 80. From the sweeping off-suspension a decade serial dilution is made; from each dilution level a streaking of 0,1ml is applied on Caso-Agar. The nutrient solution that reveals 10 to 100 colonies is enumerated. Thus number of germs per test body can be calculated in consideration of the dilution level. The sweeping off suspension includes 200ppm sodium thiosulphate to inactivate arrears of the oxidative disinfectant (hydrogen peroxide as active ingredient of the DIOSOL process). Per experimental germ there are applied three test bodies parallel. One test body is used for determination of the initial germ number before disinfection, the second test body provides determination after disinfection and the third test body is used for identification of germ number after disinfection with organic load referred to point 1. 2
3. Results: 3.1 Staphylococcus aureus: Initial germ number: 3,8 * 10 7 KbE/test body = log 7,58 Germ number after disinfection 1,1 * 10 2 KbE/test body = log 2,04 Germ number after disinfection + org. weight 1,9 * 10 2 KbE/test body = log 2,28 Reduction factor without org. weight: 5,54 Reduction factor with org. weight: 5,30 3.2 Enterococcus faecalis: Initial germ number: 5,9 * 10 7 KbE/test body = log 7,78 Germ number after disinfection: 2,2 * 10 2 KbE/test body = log 2,34 Germ number after disinfection + org. weight: 2,9 * 10 2 KbE/test body = log 2,46 Reduction factor without org. weight: 5,44 Reduction factor with org. weight: 5,32 3.3 Pseudomonas aeruginosa: Initial germ number: 1,4 * 10 8 KbE/test body = log 8,15 Germ number after disinfection: 1,3 * 10 3 KbE/test body = log 3,11 Germ number after disinfection + org. weight: 6,7 * 10 3 KbE/test body = log 3,83 Reduction factor without org. weight: 5,04 Reduction factor with org. weight: 4,32 3.4 Escherichia coli: Initial germ number: 7,7 * 10 7 KbE/test body = log 7,89 Germ number after disinfection: 2,4 * 10 2 KbE/test body = log 2,38 Germ number after disinfection + org. weight: 8,3 * 10 2 KbE/test body = log 2,92 Reduction factor without org. weight: 5,51 Reduction factor with org. weight: 4,97 3
3.5 Candida albicans: Initial germ number: 3,9* 10 7 KbE/test body = log 7,51 Germ number after disinfection: 2,3 * 10 2 KbE/test body = log 2,36 Germ number after disinfection + org. weight: 2,9 * 10 2 KbE/test body = log 2,46 Reduction factor without org. weight: 5,15 Reduction factor with org. weight: 5,05 3.6 Trichophyton rubrum Initial germ number: 1,3 * 10 7 KbE/test body = log 7,11 Germ number after disinfection: 4,5 * 10 2 KbE/test body = log 2,65 Germ number after disinfection + org. weight: 8,8 * 10 2 KbE/test body = log 2,94 Reduction factor without org. weight: 4,46 Reduction factor with org. weight: 4,17 3.7 Aspergillus niger Initial germ number: 4,9 * 10 7 KbE/test body = log 7,69 Germ number after disinfection: 3,3 * 10 2 KbE/test body = log 2,52 Germ number after disinfection + org. weight: 6,1 * 10 2 KbE/test body = log 2,79 Reduction factor without org. weight: 5,17 Reduction factor with org. weight: 4,90 4. Interpretation: The initial humidity amounts to 50% at a temperature of 22 C; after disinfection a humidity of ca. 70% has been assessed. The proved reduction performances of the DIOSOL process in terms of the logarithm reduction factor consistently report a sufficient microbiological inactivation performance in the meaning of a demand of disinfection (reduction power > 4 log-steps). An organic load (3,0% of beef-serumalbulin and 0,3% of sheep-erythrocyte) undermines the disinfection product only on a moderate way. Even with the use of organic load there is still reached enough reduction performances. The method is a passive and residue-free disinfection procedure while being gentle to surfaces. In consideration of the reduction performances that can be achieved this 4
method reveals to be excellent for room- and surface-disinfection in fields of human medicine, dentistry and veterinary medicine. Especially those surfaces which can t be reached by usual wipe-disinfection-methods (for example nooks, re-entrant angles or stud holes) can be disinfected in this way. You may contact us for any queries under 0049-551- 39-4973 or 0049-175-9150334. Yours sincerely Dr.med. Dipl.-Chem. Dipl.-Ing.(FH) Ulrich F. Schmelz Medical Doctor, Dipl.-Food Chemist, Dipl.-Ing.(FH) Process- and Plant Technology Medical Director of the Drinking Water- and Hygiene Laboratory of the University Medical Center, Goettingen Many thanks to Mr. Malte Pietsch helping to translate this report 5