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.
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.
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.