Antimicrobial Testing of Dry Surfaces through Large Droplet Inoculation

Alexander Caschera Ryerson https://orcid.org/0000-0003-4301-2522 Evan Ronan Ryerson https://orcid.org/0000-0003-0108-8289 Farhan Yusuf Ryerson https://orcid.org/0000-0002-4161-8718 Amir Tehrani Ryerson https://orcid.org/0000-0001-7833-8675 Lukasz Porosa Ryerson Otini Kroukamp Ryerson https://orcid.org/0000-0003-1084-113X Gideon Wolfaardt Ryerson https://orcid.org/0000-0002-2816-490X Daniel Foucher (  daniel.foucher@ryerson.ca ) Ryerson https://orcid.org/0000-0002-5918-5391


Introduction
Prior to its use in determining the e cacy of antimicrobial-treated surfaces, the LDI protocol was developed to study the survival and persistence of desiccation-tolerant pathogenic and non-pathogenic bacteria. [1][2][3][4][5] For example, in the work done by Ronan et al., 6 the method was used to accurately inoculate glass slides with predeterminable loads of the opportunistic pathogen Pseudomonas aeruginosa (PA01gfp) cells 7 as well as six other isolates from indoor air. Of those six isolates, a notably desiccationtolerant strain of Arthrobacter sp. (IAI-3) was subsequently used in a range of experiments to study the effect of interspecies interaction on bacterial survival during desiccation. Since the completion of that study, this desiccation-tolerant bacterial strain was reclassi ed as Glutamicibacter soli (IAI-3). 8,9 It has been cryopreserved for use as a model organism in the LDI protocol, as it is representative of desiccationtolerant bacteria that may persist for extended periods in air and on dry surfaces.
The LDI protocol was subsequently used by Porosa et al. 10 as a method of testing the antimicrobial properties of a surface-bound treatment. These trials involved determining bacterial survival on benzophenone-anchored long-chain aliphatic quaternary ammonium compound (QAC) coatings, using the previously mentioned G. soli and P. aeruginosa strains, as well as the food pathogen Listeria monocytogenes (Scott A). The ability for the latter to persist on food preparation surfaces is a major cause of contamination in fresh produce and processed meats, and has lead to many disease outbreaks and product recalls. 11 The LDI protocol was able to show a clear difference in bacterial survival on untreated control samples versus those treated with the QAC coatings; a biosafety cabinet-assisted drying step was found to impact the survival of desiccation-sensitive species.
The protocol has since been successfully employed to test the antimicrobial properties of sulfonamide QACs, as well as QAC polymeric systems and related phosphonium derivatives. [12][13][14] These studies allowed for optimization of the procedure which improved the reproducibility of the results. The drying process was adjusted to allow the droplet to air dry unassisted rather than under laminar air ow in a biosafety cabinet, which extended the drying time from 3 to 24 hours. This reduced desiccation stress on the cells and lead to improved survival on all untreated control surface samples regardless of the test microorganism. A variation of the protocol, named the Liquid Reservoir Inoculum (LRI) method, was also developed to examine the adhesion of microbial bio lms to antimicrobial-treated surfaces under aqueous conditions. 12 The LDI protocol reliably demonstrates the antimicrobial effects of ionic coatings and has been developed with adaptability in mind. 10,[12][13][14] While the simplest application of the protocol examines the survival of pure cultures on antimicrobial-treated and control surfaces, co-cultures and microbial communities could also be used as inocula, as demonstrated in the development work that led to this method. 6 The protocol could also incorporate swabbing to collect surviving cells from the surface samples, particularly when the surface material could be damaged by the prescribed saline vortex collection method. Any changes to the procedure, such as the microorganisms used, the concentration and volume of deposited cells, the cell collection method, the choice of control, and drying time must be reported.
Antimicrobial e cacy testing of treated surfaces has previously been caried out using established standard methods 15-17 and published protocols 18,19 which have been examined at length in critical reviews. 20,21 A major deviation of the LDI protocol compared to the ISO 22196/JIS Z 2801 standard methods is the exclusion of a plastic cover lm used to sandwich the inoculum droplet to the sample surface. Though omitting the cover lm does increase desiccation stress on the test microorganism, it more closely mimics a natural contamination event by avoiding the entrapment of liquid inoculum on the surface while also reducing experimental error caused by the adhesion of cells to the lm rather than the test material. This shares similarity to carrier-style disinfection standards, except the treatment is applied to the test surface before the inoculation step of the LDI procedure. 22 As for some of the protocols that have been published, many use an aerosol chamber to deposit bacteria onto their test materials. 23,24 During development of the LDI procedure, Ronan et al. 6 also attempted to use an aerosol chamber to deposit P. aeruginosa and bacterial isolates from indoor air onto glass slides. The results obtained from aerosol deposition were highly variable in the quantity and survival of deposited cells, which negatively affected overall reproducibility.
The LDI method offers simplicity in its application at a low cost while providing reproducible results. A 100 µL inoculating droplet placed directly onto antimicrobial-treated and untreated control surfaces dries naturally without using a cover lm. This allows all cells to encounter the sample surface due to the coffee ring phenomenon 25 and is a good approximation of how surfaces in the built environment are commonly contaminated. All the equipment and tools necessary for the protocol can be found in most basic microbiology laboratories, and the required consumables are generally inexpensive and readily available from lab suppliers. This test is a comparison of bacterial survival on the surface of non-porous antimicrobial-treated and untreated materials. Therefore, the untreated control samples should be made from the same material as the antimicrobial-treated samples and absent of artifacts that may impact the survival of microorganisms during the trial period. If an untreated material is unavailable for use as a control sample, a non-degradable material such as polypropylene or stainless steel can be used as an alternative.
For textiles and other porous materials, it is recommended that the inoculum absorbs completely within the sample during the drying period. This is to prevent cells from being deposited elsewhere instead of the sample. To accomplish this with thin fabrics, a larger piece of material should be folded multiple times to increase its thickness, or multiple swatches of material should be stacked together as used in the AATCC 100 standard method. 26 One of the critical components of the LDI protocol is the choice of medium for the inoculum suspension. Use of a saline solution leads to the formation of salt crystals during drying, which can impact the survival of non-halotolerant organisms. It is therefore recommended that the inoculum be suspended in sterile, potable fresh water prior to inoculation. 6,27 The purpose of this is to avoid the formation of solid materials on the sample surface while providing enough solutes to help withstand hypotonic stress. Alternatively, a soil load medium can be used to stabilize the inoculum and mimic dirty conditions. 22 After the cells have been suspended, the concentration of cells in the inoculum is enumerated to determine the initial microbial load added to the antimicrobial-treated and untreated control surface samples. The inoculum can be diluted if necessary, based on optical density measurements, to achieve any desired concentration prior to inoculation.
Once the antimicrobial-treated and untreated control surface samples are inoculated, they are dried in a covered container to minimize external in uence. An example of a simple, yet highly replicable environment is to keep the samples in empty Petri dishes on a workbench at room temperature for 24 hours to allow the inoculum droplets to completely dry. If the object the samples represent is expected to be used under unique conditions (e.g., at altitude) or if the test microorganism is incapable of surviving ambient temperatures and humidity, an environment-controlled incubator must be used. This change must be factored in when considering the effect on drying time as it could increase desiccation stress on the inoculum.
Collection of the surviving cells after drying should be performed by vortexing the air-dried samples at max speed for one minute within a conical tube containing 5 mL of phosphate buffered saline (PBS) solution. If the test microorganisms are not sensitive to changes in pH, simple isotonic saline could be used as a substitute for PBS. If there is a chance that antimicrobial leachate could be present, it is recommended that an appropriate neutralizing agent be used during the cell collection.
Due to the simplicity of the LDI protocol, most of the required equipment and expertise are available and taught in entry-level microbiology courses. A senior undergraduate biology student should be able to perform this method when given the appropriate materials and access to a containment level 1 facility. After appropriate instruction and safety training, the protocol was successfully employed in a national high-school science fair project (CWSF 2019).
There are a few limitations that can affect the utility of the results obtained with this protocol, though many of these can be easily overcome. The most likely source of error is the environmental contamination of sample materials. These natural contaminates can be vastly outnumbered by depositing high colony forming unit (CFU) counts within the 10 7 range, derived from the enumeration of undiluted overnight cultures of G. soli and Escherichia coli. The bene t of using a high microbial load is that it increases cell survival on control samples and it simpli es the inoculum preparation by removing a cell reduction step, although the starting inoculum can be adjusted using optical density measurements. There is also a risk of antimicrobial leachates during the cell collection step, which can be mitigated by exhaustively rinsing the samples with distilled water and testing the rinse solution prior to inoculation. If this is not possible, it is recommended that an appropriate neutralizing medium should be used during the cell collection. Conversely, the leachate could be retained and tested to determine if the leachate has antimicrobial properties.
Some of the limitations that are inherent with this protocol are also common in the real-world and need to be considered beforehand. Samples that are soiled or pre-contaminated prior to inoculation will likely prevent the test microorganisms from encountering the treated or control material surface, which can affect the reliability of the results. The same is also true if the samples are re-tested using the LDI protocol without cleaning the material since the surface will remain covered in extracellular polymeric substances deposited during the rst trial. Incomplete drying would also affect the reliability of the results since the remaining liquid could act as a safe reservoir for the inoculated species. The test microorganism used in the LDI protocol also needs to be su ciently desiccation-tolerant to allow for comparison of survival on antimicrobial-treated versus untreated control samples. Critical

Reagents
Step: Prepare an aseptic environment to avoid contamination of bacterial cultures and samples.
2. Remove supernatant uid, resuspend each bacterial pellet in 1 mL sterilized potable water and repeat centrifugation to rinse cells.
3. Remove supernatant uid and resuspend each bacterial pellet in 1 mL sterilized potable water. Combine resuspended bacterial pellets to form inoculum. 4. Using a dilution series consisting of 7 microcentrifuge tubes lled with 900 µL of PBS, successively dilute 100 µL of the inoculum by a factor of 10 0 -10 7 .
5. Individually dispense ten 10 µL drops of each of the 10 5 , 10 6 , and 10 7 dilutions of the inoculum onto nutrient agar plates and incubate at optimal solid growth parameters (inverted when dried, 30 °C for G. soli).  Possible Reason: Overnight culture did not grow.
Solution: Repeat Day 1, Step 1 and adjust incubation temperature, shaking speed and time. Make sure that the bacteria used has been freshly streaked and try to transfer cells from multiple colonies.
Problem: Solution appears foamy when vortexing samples in the rinse solution.
Possible reason: Antimicrobial treatment is leaching into the solution and acting as a surfactant.
Solution: Test an aliquot of rinse solution for the presence of a leachate. Make note if the samples are leaching and repeat the experiment with non-leaching antimicrobials. If leaching is unavoidable, use an appropriate neutralizing media during collection (e.g., D/E broth, Sigma-Aldrich, cat. no. D3435).).
Problem: There is uniform microbial growth on all plates between the different sample dilutions.
Possible reason: The saline solution is contaminated.
Solution: Repeat the experiment using freshly autoclaved saline.
Problem: There is no growth on the untreated control samples.
Possible reason: The microbial species used during the trial did not survive desiccation.
Solution: Repeat the experiment using a different microbial species or try to ease desiccation stress by lengthening the drying period using a humidi ed incubator. Note that G. soli has been successfully tested using a 3 h drying period.

Time Taken
Anticipated Results