The Effectiveness of DXR vs. Stachybotrys chartarum
Applied Biosafety Research Program, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Canada
Stachybotrys chartarum (ATCC MYA-3310) is a common indoor mold associated with sick building syndrome. Some allergies and upper respiratory tract disorders are thought to be a result of exposure to the mold spores, however, there is little scientific evidence that correlates the ill health effects to mold exposure1. Current decontamination methods for S. chartarum include using 1 cup of bleach in 1 gallon of water to wash affected surfaces. Porous surfaces pose an additional decontamination challenge in that the surface of the mold is killed while the roots embedded in the material are left untouched. These materials would have to be thrown away as water is absorbed into the material resulting in new mold growth2.

Materials and Methods
Spore growth Kinetics To determine the optimum time to harvest the spores for DXR effectiveness studies, Corn Meal agar plates spread with S. chartarum and incubated at 25C at 100% humidity for 0 to 10 days. Two plates from each of days 3, 6, 8, and 10 were harvested and spores dislodged by adding 5mls of cold 0.85% saline agitating with cell spreader. Resulting solution was enumerated by adding 50ul of agitated spores to 95ul of saline and 10 fold serially diluting in 0.85% saline. All diluted material was added to fresh corn meal plates and incubated for 7 days at 25C at 100% humidity or 7 days. Resulting colonies were enumerated and colony forming units /ml were determined.

Spore Prep
Commercial S. chartarum was spread and grown on cornmeal agar plates at 25°C in 100% humidity for approximately 6-7 days. Plates were placed in sealed Tupperware containers and humidity attained by cohousing with a sterile cloth saturated with sterile water. A lawn of mold was evident after 6-7 days of growth consistent with previous description of colony morphology and appearance. The organism was harvested after 6 or 7 days, by adding 5mls of cold sterile saline (0.85%) and agitating the lawn with cell spreader for several minutes. Resulting suspension was stored at 4°C for 1 week with sampling to determine viability of the organism during this time. Subsequent spore preps were re-inoculated by spreading 100ul of previous spore prep on fresh corn meal plates and harvesting after 6-7 days in the same manner.

Spore Viability
To determine the viability of the S. chartarum culture over time, spore prep was prepared by eluting spores in 5mls of saline and concentrating via centrifugation at 5000xg for 5 minutes, aspirating supernatant and re-suspending into 500ul of fresh 0.85% saline.Two replicates of 50ul were removed at days 0,1,3,7 and added to 950ul saline and 10-fold serially diluted. Resulting dilutions were added to corn meal agar plates and incubated for 7 days at 25C at 100% humidity for 7 days and enumerated.

A neutralization assay was conducted to determine the toxicity of four neutralizers; DE, Saline, Letheen broth, and 1% sodium thiosulfate on the S. chartarum and its effectiveness against DXR. Briefly, 50µL of DXR was mixed with 9.9mls of the various neutralizers and 10µL of fungal spores in soil load were added after the combination. Neutralized product was 10 fold serially diluted in saline and enumerated by passing dilutions through a filter membrane and placing filter pad on corn meal agar plates for 7 days at 25°C at 100% humidity. One assay of two replicates of each neutralizer was used to determine effectiveness.

QCT 2 against DXR
The QCT2 spore suspension was prepared from a 6-day growth culture as described in the spore prep section and stored in the refrigerator at 4°C between experiments. Soil load was prepared by adding 170 µL spore suspension of with 50 µL of mucin, 17.5 µL of trypsin and 12.5 µL of BSA. A 10µL soil load spore prep was added by positive displacement pipetting and dried onto brushed stainless steel coupons. DXR was agitated and 50ul of product added to each coupon for contact times of 1 minute 5 minute and 10 minutes. After subsequent time points, treatments were neutralized by addition of 9.95mls of 1% sodium saline. These were 10-fold serial diluted in saline and filtered. The filter pads were placed on the cornmeal agar plates and incubated under the growth conditions described in spore prep.

DXR Spot Treatment
In order to assess the minimum effective concentration of DXR, 50µl of DXR at varying dilutions was spot treated onto a day 9 S. chartarum lawn. DXR was 2 fold serially diluted in saline from 100% DXR to 0.2% DXR plates. Plates were blotted with 50ul of saline as a positive control and 50ul diluted DXR blotted beside the control and the plates were incubated overnight at 25°C in 100% humidity. Overnight treatments were removed from incubator and the spots (saline and DXR treated) were then agitated with a bacterial picking loop and streaked onto a fresh cornmeal agar plate. These were again incubated according to the spore prep conditions for 6 days. Resulting plates were looked at 7 days later and scored for presence or absence of colonies.

Results and Discussion
Enumeration of Spores and Refrigerator Storage Viability Plates were grown for 6-days at 25°C in 100% humidity with 5 ml of saline was added to the cornmeal agar plate and then agitated with a cell spreader to dislodge the spores from the hyphae on the plate as described in previous section. An average of 4.8 logs/ml of spore was achieved after 6 days of growth (Figure 1). The spores were stored in saline in the fridge at 4°C and were enumerated every few days to assess their viability throughout the week. The spores were found to remain viable for 7 days in the refrigerator at 4°C with approximately a half-log reduction throughout that time (Figure 2).

Figure 1. Growth of S. chartarum over time. Seeded plates were harvested at said time points and spores eluted and enumerated S. chartarum was found to achieve a concentration of 4.8 logs/ml after 6 days of growth and remains relatively constant after that point.
Figure 2. Refrigerator Viability.
S. chartarum spores were sampled form previously prepared spore prep in saline. Briefly prep was found to remain viable for 7 days in the refrigerator.

Neutralization assay
Of the 5 neutralizers chosen for the evaluation of their effect on Stachybotrys chartarum, 0.85% saline (figure 3) had the least effect on the viability of the spores, but all neutralizers performed equally as well.

Figure 3. Neutralization Assay. 50µL of DXR was neutralized by all four neutralizers. None of the neutralizers had negative effects on the spore growth. Saline was chosen.

QCT-2 DXR vs. Stachybotrys chartarum Spore preps mixed with a soil load were dried on stainless steel disks and full strength product applied at 1, 5, and 10 minute durations. An average initial count of 3.1 log/ml of spore was attained within 1 minute with several colonies evident in one experiment. In all treatments of 5 and 10 minute durations, no colonies were evident. A 2.5-3.5 log reduction of spores in a 10ul spot treatment with 50ul of product following a 1 minute contact time (Figure 4). The majority of the time a 1 minute contact time resulted in no detectable levels of spores after treatment. After 5 minutes of treatment no detectable spores were observed in any treatment.

Figure 4. QCT2: DXR vs S. chartarum in a soil load. Fresh spore preps were dislodged from 7 day corn meal plates and mixed with a soil load. 10ul of spores and soil load were added to brushed stainless steel coupons and air dried. Full strength DXR added to coupons and neutralised at said contact times. Materials were serially diluted and enumerated via filtration

Spot Blot DXR vs. S.chartarum Briefly, 50ul of diluted of DXR product were diluted onto lawns of S.chartarum, incubated overnight and harvested the next day by picking affected areas and streaking on fresh plates. Plates were incubated for 7 days and scored in a presence-absence basis. Results indicate that a 100% to 50% DXR concentration is effective in removing viable mold spores from an area (Table 1). Further dilution of DXR resulted in more abundant colonies being eluted from the colony treatment pick. Although this assay is a subjective test due to sampling error and no quantitative measurement made, this does show the effectiveness. Results were scored on the basis of – and + to ++++ indicating: + represents 1 colony, ++ represents less than 10, +++ represents less than 100, while ++++ was over a 100 colonies estimated.

Precentage DXR Spot Treatment Plate 1 Plate 2 100.00% - - 50.00% - + 25.00% ++ ++ 12.50% +++ +++ 6.25% ++++ ++++ 3.13% ++++ ++++ 1.56% ++++ ++++ 0.78% ++++ ++++ 0.39% ++++ ++++ 0.20% ++++ ++++

Figure 5.
Effect of spot treating S. chartarum. Corn meal agar plate were incubated 7 days to achieve lawn. Full strength DXR and two-fold diluted product in hard water added to added in 50ul amounts beside a saline control and incubated overnight. Treated spots were picked and spread onto fresh plates. Following plates were evaluated on a plus minus basis based on observable amount of colonies. + represents 1 colony, ++ represents less than 10, +++ represents less than 100, while ++++ was over a 100 colonies estimated.

Full strength DXR was effective in remediating 3.1 logs of material containing a soil load in approximately 1 minute. Spot testing of full strength DXR to 2-fold diluted DXR (50% concentration) further indicated that spores from an overnight incubation with DXR did not grow. Even 50% of diluting of DXR only had 1 colony present within one replicate. Further dilution of product with hard water produced more observable spores after spot picking indicating that it is less effective the further diluted it is.

1.Lichtenstein, Jamie H. Rosenblum, Ramon M. Molina, Thomas C. Donaghey, Chidozie J. Amuzie, James J. Pestka, Brent A. Coull, and Joseph D. Brain. "Pulmonary Responses to Stachybotrys chartarum and Its Toxins: Mouse Strain Affects Clearance and Macrophage Cytotoxicity." Toxicological Sciences 116.1 (2010): 113-121. NCBI. Web. 15 July 2014.

"Facts about Stachybotrys chartarum and Other Molds." Centers for Disease Control and Prevention. Centers for Disease Control and Prevention, 18 Sept. 2012. Web. 2 Sept. 2014. .