Tests Summary
These are abbreviated versions of reports from the various laboratories.
Full testing procedures and results are available on request.
Most laboratories were only asked to test the product's continued efficiency for between 7 and 10 days. Latest tests (see last sheet) show it remains 100% effective for 28 days
MICROBIOLOGY SERVICES AT THE 'HEALTH PROTECTION AGENCY' AT SOUTHAMPTON GENERAL HOSPITAL
Conducted by Senior Microbiologist, Melody Greenwood, BSc, Mphil, CBiol. FIBiol, FIFST, MRCS HC
PROCEDURE
Two porcelain tiles were submitted along with a spray bottle of Steri-X. The tiles were subdivided into 5 x 5cm squares in the laboratory.
Overnight cultures of methicillin-resistant Staphyloccocus aureus (MRSA) NCTC 11940 and Echerichia coli NCTC9001 were grown in a nutrient medium at 37° C. After incubation, serial decimal dilutions of the two cultures were prepared in peptone saline diluent and the level of bacteria in each culture ascertained by the surface drop technique on blood agar. Plates were incubated for 20-24 hours at 30° , colonies counted and the count of the inoculating suspension calculated. A 20 ìl drop of overnight culture was applied to the centre of each tile square ... and the whole tile was sprayed with Steri-X. At intervals of 1, 2, 5, 10, 30, 60 120 and 180 minutes... .20 ìl of sterile distilled water was added to a dried drop using a separate square for each time interval. The water was mixed into the organism drop using a sterile plastic stick, the liquid transferred aseptically to a blood agar and then spread over the surface of the agar using a sterile spreader. Inoculated blood agar plates were incubated at 30° C for 20 hours and then colonies counted if present.
The tiles were then stored at ambient temperature and the remaining drops examined as described above after 10 days.
In a separate parallel experiment the same organism suspensions were used to inoculate 20 ìl into separate 1ml volumes of liquid Steri-X. After the same time intervals described above, 20 ìl volumes were withdrawn and plated to duplicate blood agar plates. The inocula were spread over the surface of the agar using steile spreaders, the plates incubated as before and the colonies counted after incubation.
RESULTS
For the MRSA strain, no count was discernible at any time of testing. This indicates that a 6-7 log reduction of MRSA was obtained within 1 minute of applying Steri-X or 100% kill within 1 minute. For E.coli, counts were detected after 2 minutes but not after 5 minutes. The results indicate a 4-log reduction of E.coli within 2 minutes of applying Steri-X which increased to a 6-7 log reduction within 5 minutes. This is equivalent to a 99.9997% kill within 2 minutes and a 100% kill within 5 minutes.
In a separate parallel experiment, for MRSA a 99.986% kill was obtained within 1 minute and for E.coli a >99.9997% kill was obtained within 1 minute.
Examination of drops after 10 days storage at ambient temperature all failed to recover any colonies of either MRSA or E.coli, indicating that the Steri-X was fully bactericidal.
GLASGOW CALEDONIAN UNIVERSITY
TESTING STERI-X AGAINST E.coli (ATCC 10536)
PROTOCOL
An 0.1 ml volume of Steri-X was applied and evenly distributed across the surface of the wells of a polystyrene 6-well plate. A control plate was set up with an equal volume of sterile distilled water. These were allowed to dry with the lids off in a class II laminar flow cabinet for 1 hour at room temperature. The lids were replaced and the plates were maintained in a temperature controlled room for the 'incubation period' at 20° C +/-2° C. The cultures were prepared by inoculation onto a tryptone soya agar (TSA) slope, grown up overnight at 36° C+/-1° C and the next day, the day before the experiment, re-inoculated onto a fresh TSA slope and grown up overnight as before. The bacterial lawn was gently washed from the slope with a 2.0 ml volume of Tryptone-NaCI and disaggregated by repeated pepetting with a 10ml serological pipette. The absorbance of the culture at 620 nm was adjusted to 1.0 by dilution in tryptone NaCI. The cell suspension density was estimated to be 2.0 x 108 cfu/ml. This was later confirmed by a viable count by diluting the cell suspension to 10-6 and 10-7, then inoculating 1.0 ml into a TSA pour plate (molten 13.5 ml TSA deeps had been previously prepared and maintained at 50° C) A 0.1 ml volume of culture 20 x 107 cfu was then applied to the surface of the wells treated and a well of the mock treated plates for each incubation time point. These were evenly distributed over the surface of the plate. In one well, bacteria were maintained in contact with the surface at 20° C for 1 minute and the other well for 5 minutes for the Steri-X treated wells and 5 minuets for the mock treated plates. The contact was terminated from the Steri-X treated plates by removal of 0.1 ml into duplicate filter funnels containing 50 ml of universal neutraliser. Bacteria were trapped onto 0.22ìm filters and washed with 150 mls of tryptone NaCI (see EN1276). The filters were transferred onto TSA plates and incubated overnight at 36° C +/-1° C. For mock treated plates, the mixture was first diluted to 10-6 and then a k0.1 ml volume applied to a filter funnel containing 50 ms of diluent. These plates were incubated at 36° C +/- 1° C overnight and counted the next day.
RESULTS: Steri-X challenged with E.coli
CONCLUSION
These data indicate that upon a minimum of 1 minutes exposure, seven days after the application and desiccation onto a polystyrene surface, Steri-X reduced the viability of 2.0 x 10(7) cfu of E.coli (ATCC 10536) inoculated onto the surface to below detectable levels.
TESTING STERI-X AGAINST M.R.S.A
GLASGOW CALEDONIAN UNIVERSITY
Note from Steri-X International Ltd.
There is not just one strain of M.R.S.A. In fact the majority of worldwide infections fall within five clonal complexes AND ALL FIVE WERE USED IN THESE TESTS. Any other company who claim they can deal with 'M.R.S.A' therefore, are almost certainly talking about tests on just one strain which may not necessarily be the one affecting your interests.
Remember, WE'VE covered everyone – and further more our product continues to kill bacteria 100% for long after it's been applied – as the Glasgow University tests show.
GLASGOW UNIVERSITY TESTS - BACKGROUND
Although each country has their own names for MRSA clones, MLST Typing data which groups MRSA into Clonal Complexes (CC) made up of related Sequence Types (ST) is the widely adopted international nomenclature. The majority are caused by clones which fall within five complexes.
A representative of each of these Clonal Complexes were used in these tests.
EMRSA 15 is the dominant MRSA in the UK, followed by EMRSA 16. In Scotland these two clones account for72% and 20% respectively. Of all MRSA referred to, the Scottish MRSA reference Laboratory EMRSA-16 and other clones within Clonal Complex 30 are found worldwide. They are the second commonest MRSA in the USA and are a common cause of community acquired MRSA in both the USA and Australia.
The New York/Tokyo clone is the commonest clone in the USA (44%) and Japan and is found in many other countries worldwide.
The Iberian clone was common in the UK in the early 1990's but is now rarely reported. It is, common in many European countries and in South America and is a multi-reistant clone. Clonal Complex 45 is found in several European countries and accounts for 3% of MRSA in USA.
AnaCon Laboratories, California, U.S.A
This is the laboratory specified by airlines to carry out tests to ensure products are safe for use on aircraft. At the request of one major airline, they tested Steri-X with the following results.
Full report available on request, but the following is a summary:
| Flash Point: | Shall not have a flash point lower than 100° C | Conforms |
| Stability: | Shall be subjected to 120 hours storage at 50° C | Conforms |
| Cold Temperature | Shall be subjected to 120 hours storage at –10° C | Conforms |
| Total Immersion Corrosion | Shall not cause staining, pitting, corrosion, weight change Corrosion greater than 0.3 kg/cm2/24hours on Clad Aluminium | Conforms |
| Sandwich Corrosion | When tested per ASTM F 1110, the panels exposed to Corrosion product shall not show …..pitting or etching. | Conforms |
| Effect on Transparent Plastics | Shall not craze, stain or discolor Mil-P-25690 stretched Transparent acrylic plastic. Mil-P-83310 Polycarbonate/Polysulfone Conforms Plastics shall also be tested at 3000 psi outer fiber stress for 30 min | Conforms |
| Results of above tests | Mil P 25690 - No Effect Mil P 83310 - No Effect Polysulfone - No Effect |
Conforms |
| Effect on Painted Surfaces | Shall not cause streaking, discoloration, blistering or Painted Surfaces decrease the pencil hardness of the paint by more than 2. | Conforms |
| Result of above No effect. | Hardness = 2H | Conforms |
The Lord Zuckerman Research Centre, Reading, RG6 6LA, United Kingdom
Test Report of Microbiological Analysis
Testing for conformity with BS EN 1276
RESULTS
S.aureus (Clean) >7.0 PASS
S.aureus (Dirty) >7.0 PASS
P.aeruginosa (Clean) >7.0 PASS
P.aeruginosa (Dirty) >7.0 PASS
E.coli (Clean) >7.1 PASS
E.coli (Dirty) >7.1 PASS
E.hirae (Clean) >6.9 PASS
E.hirae (Dirty) >6.9 PASS
Please note: A 'Pass' requires a minimum reduction of 5
FOLLOWING A REQUEST FROM THE BRITISH NATIONAL HEALTH SERVICE (N.H.S) WE HAD 'STERI-X' TESTED FOR 'SUBSTANTIVITY' (against EMRSA 15.)
IT PROVED TO BE EFFECTIVE FOR AT LEAST 28 DAYS.
Here is a summary of the results of those tests
PROTOCOL
A volume of 200ƒÊ of Steri-X was applied to each of a series of replicate wells in a sterile polystyrene 6-well plate. Steri-X was allowed to dry in a class II safety cabinet and the wells retained for 28 days at a temperature of 20 ¤} 1oC. On the 28th day following treatment, four wells; A,B,C & D were challenged with a bacterial suspension in diluent (0.85% w/v NaCl, 0.1% w/v Tryptone) taken from the second of two consecutive overnight sub-cultures of EMRSA 15: CC22 (SMRL No. 00.9521.M) The cell density of the suspension was approximately measured by the optical density at 620 nm and the density adjusted to be between 1.0 . 2.0 x 108 cfu/ml. A viable count was made by dilution of the suspension to 10-5 and inoculating 1.0 ml into a tryptone soya agar pour plate that was incubated at 36 ¤} 1o C overnight and counted the next day. To reduce the effect of dilution on the efficacy of Steri-X on subsequent challenge, a small volume of 50fÊls of suspension was applied to well A (approx. 5.0 x 106 to 1.0 x 107 cfu.) This was evenly spread over the treated surface with a sterile silicon cell scraper and incubated for 1 minute at 20 ¤} 1o C. Two 10 fÊl volumes were then removed from the well and respectively applied to two sterile filter funnels containing 50 mls of universal neutraliser (1/5000 dilution.) This was then immediately filtered through a 0.22 f&Êm filter, and subsequently further washed with 150 mls of sterile diulent. Filters were aseptically removed and each placed on a separate tryptone soya agar plate. These were incubated at 36 ¤} 1o C overnight and counted the next day. This was repeated for a replicate well B. Replicate wells C and D were treated in exactly the same way with the exception that the contact time for both these replicates was 5 minutes. A control, untreated well was inoculated with 50 ƒÊl of suspension, incubated for 5 minutes at 20 ¤} 1o C. A 10 fÊl volume was removed into 990 ƒÊl of sterile diulent and then serially diluted to 10-4. A 1.0 ml volume was then applied to each of two 50 mls volumes of diluent in a sterile filter funnel and the bacteria collected onto a 0.22 f&Êm filter. Filters were aseptically removed and each placed on a separate tryptone soya agar plate. These were incubated at 36 ¤} 1o C overnight and counted the next day. The residual bacterial suspension was allowed to dry onto the surface of the plate. The plate was then incubated at 20 ¤} 1o C. The same wells were subsequently challenged in the same way using the second of two subcultures of EMRSA 15 on each challenge 1 day and 5 days later.
Result of applying Steri-X to 4 individual wells, incubating for 28 days, the repeatedly challenging the same wells with consecutive inocula of EMRSA 15a after 0, 1 and 5 days for either 1 minute (plates A&B) or 5 minutes (plates C&D)