Saturday 7 October 2023

Disinfection: Carrier Surface Studies


Carrier Surface Studies are performed to provide a verification of the ability of the antimicrobial chemical agent to reduce the microorganism levels that may be present on the facility surfaces.


A variety of representative facility surfaces that are commonly found and represent a worst case porosity should be considered, with number of facility surfaces selected should be based on the criticality of the surface and the risk of such surfaces to harbour contamination.


Test for disinfectant wipes


The methodology is based on USP <1072>, which in turn is based on the U.S. AOAC.


The carrier surface used for testing should be made of the material surfaces selected. The carrier’s measurements will be approximately 1.5 inches x 1.5 inches (around 4cm x 4cm) (this is so as to avoid false positives during handling of the carrier).


A panel of microorganisms should be used. The organisms chosen should be typical and based on the type of isolates that might be found in the area. Type cultures from ATCC will be used.


Total Kill Method


 (1) A fresh culture of each organism is prepared to a known CFU/ml concentration.

 (2) For each organism a small volume of the culture is transferred onto the surface of the selected carrier. Sufficient microorganisms should be placed on the carrier to demonstrate a sufficient reduction of these organisms (103-105).

 (3) The inoculum applied to the carrier should be allowed to thoroughly air dry after which the antimicrobial agent is either generously applied on the carrier wiping. The chemical agent is then allowed to stay in contact with the carrier for a defined period of time (e.g., one minute).

 (4) After being in contact with the chemical agent for the specified time, the carrier is then submerged in a vessel containing a neutralizing agent and appropriate growth medium such as TSB to neutralize the chemical agent.

(5) After a set time, the carrier is then placed within a second vessel containing the same growth medium without the neutralizing agent.

(6) Both of the vessels are then incubation at the appropriate temperature for a suitable time.

(7) Using this method a result of no growth is required in both containers to demonstrate the required log reduction has been achieved. This level of reduction should be assessed against a set of pre-established criteria to determine if the chemical agent provided the level of reduction required.

(8) A positive control to verify the inoculum concentration for each organism should be performed as part of each test. For each positive control, carriers that have been prepared with those to be exposed to the chemical agent should be submerged in a vessel containing a known concentration of saline and gently sonicated or mechanically scrubbed to remove the microorganisms from the carrier. A serial dilution should then be performed from the vessel and plated using the same media type used in the test to allow the recovery of between 10 and 100 CFU per plate.

(9) A negative control should also be used to verify that appropriate aseptic technique was used during the performance of the method. For the negative control the method used in the study should be followed with the exception that a sterile carrier should be used.  No CFUs should be recovered from the negative control.

(10) After the completion of the study the log reduction achieved against each organism should be determined based on the CFUs present in the inoculum (as determined in the controls). The level of reduction should be assessed against a set of pre-established criteria to determine if the chemical agent provided the level of reduction required.


The method should be validated to ensure that the neutralizing agent selected does not prevent growth of the various organisms chosen for the studies and that the neutralizing agent is effective in neutralizing the chemical agent.


(1) To validate the ability of the test organisms to grow in the presence of the neutralizing agent each of the test organism (typically at a concentration of <100 microorganisms) and the neutralizing agent should be plated together (using a standard pour plate technique) utilizing the same media type that will be used in the studies. After appropriate incubation the number of CFUs recovered should be comparable to a positive control to which the neutralizing agent is not added.

(2) To validate the ability of the neutralizing agent to neutralize the chemical agent as used in the study, the study method should be performed as written with the exception that the inoculums (typically at a concentration of <100 microorganisms) should be added after the neutralization step has occurred to the vessel containing the neutralizing agent and growth medium. Both vessels must show growth.


10 discs should be used with each microorganism and for each surface. Each disc must show an effective biocidal reduction of 2-logs.


Test for disinfectant solutions (sprays): enumeration method

This method is based on standard BS EN 13697 is the CEN standard for the surface test method for establishing whether a chemical disinfectant has bactericidal and /or fungicidal activity on non-porous surfaces.


With the method:


1)      Subculture each challenge microorganism from a stock culture to an agar plate and incubate for 24-48hrs.

2)      Wash with 3ml of diluent and aspirate the suspension into a sterile container . Adjust the number of cells in the suspension to 1.5 x 108 – 5 x 108 using sterile diluent and a spectrophotometer.

3)      Hold the suspension at 20+/-1oC and use within two hours.

4)      Add 1mL of microbial test suspension to 1mL of interfering substance and mix.

5)      Use duplicate discs of the material to be examined into a Petri dish. Inoculate with 50┬Ál of microbial suspension to give counts in the desired range of 50 - 300 CFU. Dry the surfaces.

6)      Add 0.1mL of disinfectant onto one of the discs, ensuring that all of the dried inoculum is covered.

7)      Add the same volume of WFI to the other disc ensuring that the dried inoculum is covered. This is the water control.


8)      After the specified contact time each of the discs is transferred (inoculated side down) into culture tubes containing neutraliser and 5g of glass beads.

9)      After a neutralisation time of 5 mins + 10 secs 10-1 and 10-2 dilutions in diluent should be prepared by adding 1ml of solution to 9ml of diluent.

10)  Prepare duplicate 1ml pour plates.

11)  The disc is to be removed, drained, rinsed with 10ml of WFI and placed on a 9cm agar with the tested side facing up.

12)   Add 0.1ml of water to the disc and the surface scraped with a sterile pipette tip to remove any residual dried inoculum for 1 minute. Add molten agar and pour over the plate surface mixed and allowed to set.

13)  Verify the neutralisation control in parallel.

14)  Incubation microbial test plates and count post-incubation.


In terms of acceptance criteria, a 103 log reduction in viability or greater is required for a disinfectant to be classed as having bactericidal activity within 5 minutes 20+/-1oC; a 102 log reduction in viability or greater is required for a disinfectant to be classed as having fungicidal activity on non-porous surfaces within 20 minutes at 20+/-1oC.


Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (

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