Preparation of MS2 virus, plaque assay, and droplet test solution. (i) Preparation of MS2 virus.
Tryptone yeast extract glucose broth 271 was prepared using the method of the American Type Culture Collection (ATCC) (www.atcc.org
). This culture medium was designated 271B and used for growth of Escherichia coli
, storage of MS2, a plaque assay, and the MS2 extraction and recovery process. These procedures have been used previously in our lab (12
ATCC 15597 and bacteriophage MS2 (ATCC 15597-B1) were obtained from the ATCC. MS2 virus was replicated using E. coli
as the host. 271B was inoculated with an aliquot of frozen E. coli
(20 μl of E. coli
in 10 ml of 271B) and incubated overnight at 37°C. Fresh 271B (100 ml) was inoculated with 1 ml of the overnight culture of E. coli
and then incubated at 37°C with shaking at 100 rpm for 3 to 4 h. An MS2 stock (1.5 ml; ATCC 15597-B1) was added to the overnight culture of E. coli
at a concentration of 109
PFU/ml and incubated with shaking overnight at 37°C (multiplicity of infection, ~20). Lysozyme (0.05 mg/ml) was added to the MS2 overnight culture to liberate the MS2, and then the mixture was shaken vigorously for 5 to 10 min. The lysis solution containing MS2 was then centrifuged at 7,100 × g
for 30 min at 4°C (IEC Multi RF; Thermo Electron Corporation), and the supernatant containing MS2 was filtered through a sterile 0.22-μm-pore-size filter (Whatman International Ltd., Maidstone, United Kingdom) into a sterile container. MS2 coliphage were enumerated using a standard assay method (1
) as described below, and the final MS2 suspension (1011
PFU/ml) was stored at 4°C and used within 1 month after production. This MS2 suspension was designated a stock MS2 suspension. MS2 was selected for the study based on its moderate resistance to decontamination, survivability, ease of preparation and assay, and nonpathogenicity (19
(ii) Plaque assay.
A standard overlay agar assay method was used to enumerate the viruses (1
). Sterile glass tubes containing 100 μl of the overnight bacterial host (E. coli
) and 100 μl of the diluted MS2 phage were warmed in a water bath at 45°C. Three milliliters of melted soft agar (0.5% agar) was added to each tube and mixed thoroughly. The mixture was then poured into a labeled petri dish containing hard agar (1.5% agar). The dishes were covered, and the agar was allowed to gel. The plates were then inverted and incubated at 37°C overnight. The plaques were counted, multiplied by the dilution factor, and divided by the sample volume (in milliliters) to obtain the titer expressed in PFU/ml.
(iii) Droplet test solution.
271B was used as a droplet test solution (nebulizer fluid). All MS2 droplet test solutions were prepared by diluting the stock MS2 suspension in 271B to obtain a final concentration of approximately 107 PFU/ml. This MS2 concentration was chosen to ensure a loading level of ≥1 × 103 PFU/ml so that there was an adequate detection limit for the bioassay described below.
DPARTS. (i) Droplet test system.
Viral droplet size is affected by atmospheric humidity. To generate droplets from which there was not significant evaporation that decreased their original size, a droplet-phase aerosol respirator test system (DPARTS) was designed and constructed (Fig. ) so that the relative humidity (RH) in it was higher than that of the surrounding air. The DPARTS used for loading the FFRs with MS2 droplets consisted of a compressed air supply, high-efficiency particulate air (HEPA) filters, an airflow regulator (Ashcroft, Costa Mesa, CA), a six-jet Collison nebulizer (BGI, Inc., Waltham, MA) with a short tube (length, 3 cm; diameter, 1.5 cm) connecting the nebulizer outlet to the wall of an exposure chamber, a 43-liter exposure chamber (acrylic chamber with a hinged front door; Vandiver Enterprises, Zelienople, PA), a test respirator holder containing an FFR, an exhaust port, and an aerodynamic particle sizer (APS) (model 3321; TSI Inc., Shoreview, MN) with a full-size distribution range of 0.5 to 20 μm.
FIG. 1. Schematic diagram of the droplet chamber test system. 1, compressed air supply; 2, HEPA filter; 3, airflow regulator; 4, nebulizer air inlet; 5, nebulizer; 6, test FFR; 6B, sample coupons on the respirator (T, top; C, center; B, bottom, L, left; R, right); (more ...)
The exposure chamber (35 by 35 by 35 cm) had an internal volume of approximately 43 liters. An airflow regulator was used to control the air pressure in the nebulizer at 20 lb/in2 and produced an airflow rate of approximately 12 liters/min. The chamber was maintained at a slightly positive pressure (less than 0.1-in. water column pressure) to ensure that particles did not leak into the chamber. The exhaust port (diameter, 2.5 cm) was left in the open position to remove excess air during droplet sampling. An FFR mounted in the respirator holder was located 15 cm from the droplet outlet inside the chamber. MS2 coliphage was suspended in 271B, and MS2 droplets were generated using the six-jet Collison nebulizer. The chamber was also equipped with a 0.8-cm-diameter port for droplet sampling with the APS.
(ii) Characterization of MS2 droplets in the DPARTS. (a) Size distribution of MS2 droplets.
The concentration and size distribution of the MS2 droplets were measured in the front center area of a respirator using an APS with a probe that was 15 cm from the droplet outlet inside the chamber (Fig. ). The probe connected to the APS was used only in these size distribution experiments and in the experiments described below to determine the uniformity of the aerosol concentrations in the locations used for loading. The APS measured airborne droplet sizes ranging from 0.5 to 15 μm. A TESTO 635-1 humidity- and temperature-measuring instrument was utilized to measure the RH and temperature in the exposure chamber.
(b) Characterization of uniform loading of MS2 droplets.
To further investigate the concentrations and size distributions of MS2 droplets, the APS was used to analyze the viral droplet data obtained at different respirator locations (top, center, bottom, left, and right areas of respirator samples) (Fig. ).
N95 test respirator and viral droplet loading onto respirators. (i) N95 respirator.
The N95 FFR (model N1105; Willson, Santa Ana, CA) used in this study is a National Institute for Occupational Safety and Health (NIOSH)-approved FFR that can be used by healthcare workers for protection against particulate hazards. This FFR model is comprised of three layers. The outermost and innermost layers are made from hydrophilic materials. The hydrophobic middle layer is composed of melt-blown polypropylene fibers with an electrical charge designed to enhance the efficiency of capture of submicron particles.
(ii) Viral droplet loading onto respirators.
During development of the experimental procedure for loading, the need for sufficient viral droplet particles was considered in order to establish appropriate loading levels to permit adequate detection. Test bioassay samples were diluted appropriately and plated so that there were 30 to 300 PFU/plate to ensure acceptable data quality (1
). The number of PFU/ml was determined by multiplying the average number of countable plaques (30 to 300 PFU) by the dilution factor. Based on the data quality objective of the bioassay technique and the decontamination method (as described below for decontamination experiments), the minimum detection limit for the loading level had to be at least 1 × 103
PFU/ml for an adequate detection limit.
An FFR mounted in the respirator holder was located 15 cm from the droplet outlet inside the DPARTS chamber (Fig. ). All MS2 nebulizer samples were prepared by diluting the stock MS2 suspension with 271B to obtain a final concentration of approximately 107 PFU/ml. Each MS2 solution (45 ml) was added to the nebulizer glass jar for loading. After the chamber was sealed (with the exception of the exhaust port, which was in the open position during loading), the compressed air valve was opened (rate of nebulizer airflow, 12 liters/min; 20 lb/in2), and the air passed through the nebulizer to generate MS2 droplets (the rate for the volumetric MS2 suspension leaving the nebulizer was approximately 0.22 ml/min) and into the exposure chamber for subsequent loading onto the FFR. A stopwatch was used to measure the duration of loading. MS2 droplets were generated continuously, and the RH in the area of the respirator holder containing the FFR was monitored. After 10 s, the RH reached 95% and then stabilized at 95 to 99% for the duration of the exposure period (a high RH was used to maintain droplet particle size without significant evaporation; it was not intended to reflect real workplace conditions). Once the MS2 droplet load reached the desired value (loading time, 5 min; desired load = [MS2 nebulizer suspension concentration in PFU/ml] × [rate of MS2 suspension leaving the nebulizer in ml/min] × [loading time in min] = 107 PFU/ml × 0.22 ml/min × 5 min = approximately 107 PFU), the airflow was stopped. Then the exposed FFR was retrieved and saved for use in decontamination experiments.
Decontamination experiments. (i) Sodium hypochlorite decontamination experiment. (a) Sodium hypochlorite.
Sodium hypochlorite (NaOCl) solutions (stock solution, 6% NaOCl), commonly known as bleach, were used for chemical decontamination. The stock sodium hypochlorite solution (Clorox regular bleach; Environmental Protection Agency registration no. 5813-50) was obtained from a commercial supplier. In the chemical decontamination experiments, all sodium hypochlorite working solutions (0.005, 0.01, 0.05, 0.1, 0.25, 0.5, and 0.75% sodium hypochlorite) were freshly prepared by diluting the 6% sodium hypochlorite stock solution with purified water; these solutions contained 0.06, 0.11, 0.55, 1.10, 2.75, 5.50, and 8.25 mg/liter of sodium hypochlorite, respectively, and were mixed gently at room temperature with continuous stirring for 15 min.
Each FFR loaded with MS2 was submerged in 1 liter of a sodium hypochlorite solution or purified water. Treatment with water (with no NaOCl) was used as a baseline treatment to determine losses due to handling of FFR samples during the chemical decontamination process. In the chemical decontamination experiments control samples of FFRs loaded with MS2 were not submerged in either purified water or a sodium hypochlorite solution. If the efficiency of viral droplet loading onto FFRs is 100%, the number of viruses recovered from the controls and the number of viruses loaded into droplets are the same. For the sodium hypochlorite decontamination experiments, both sides of a complete FFR were decontaminated by submerging the FFR in a sodium hypochlorite solution. After 10 min of treatment, the respirator was removed from the purified water or sodium hypochlorite solution and air dried for 2 min. A toxicity control to determine if there was any interference by residual sodium hypochlorite with the chemical inactivation process under these conditions (10 min of sodium hypochlorite treatment and 2 min of air drying) was examined in a previous study (12
). The results showed that no residual sodium hypochlorite interfered with subsequent bioassays (12
). Three replicate tests (n
= 3) were carried out for each sodium hypochlorite concentration. Each respirator was cut into coupons (2 cm by 2 cm), and each coupon was then placed in 10 ml of 271B in a 50-ml conical tube for extraction.
(b) Virus recovery.
MS2 was extracted from the coupons by vortexing them for 2 min. When extraction procedure was complete, the coupons were discarded, and the supernatant was assayed for viable MS2 as previously described.
(c) Efficacy of the sodium hypochlorite decontamination.
The number of viable MS2 phage was determined by a plaque assay. The efficacy of decontamination (ED) for MS2 was calculated by determining the log reduction as follows: ED = log (N°/N), where N° is the mean number of viable MS2 phage applied to the control coupons (i.e., coupons not subjected to decontamination) and N is the number of viable MS2 phage recovered from test coupons after decontamination.
(ii) UV decontamination experiment. (a) UV decontamination procedures.
UV decontamination was carried out using a UV germicidal lamp in a biological safety cabinet (SterilGARD III model SG403A; Baker Company, Sanford, ME). A low-pressure mercury arc lamp (5.5 mg Hg; lamp type, TUV 36TS 4P SE; lamp voltage, 94 V; lamp wattage, 40 W; wavelength, 253.7 nm) was used as the UV source. The UV intensity on the sample surface was measured using a UVX-25 digital radiometer (model E28457; Cole-Parmer, Vernon Hills, IL). After exposure to MS2, an FFR was treated with UV irradiation using a UV source intensity of 0.4 mW/cm2 at the FFR surface (distance from lamp to sample surface, 42 cm). The UV doses (J/cm2) used in the decontamination treatments were calculated by multiplying the average UV intensity at the FFR surface by the irradiation time (in seconds). In these experiments, the UV treatment was applied only to the side of the FFR closest to the nebulizer (Fig. ). To eliminate the possibility that MS2 contaminated the inside surface of the FFR by traveling directly from the back of the FFR holder during the droplet loading step, a second FFR was placed behind the FFR sample being tested to act as a protective cover.
Because of the relatively low intensity of the UV that reached the FFR, long irradiation times (1, 2, 3, 4, and 5 h; control, 0 h) were used to obtain appropriate UV doses. The survival of the MS2 virus on the FFRs at different times was also examined by storing the MS2-contaminated FFR samples for 1, 2, 3, 4, and 5 h (at the same temperature and RH) but without UV irradiation (control experiments). Three replicate tests were carried out for each irradiation time and each control experiment. To ensure that the temperature and RH did not adversely affect the efficacy of UV decontamination for inactivation of MS2 virus (20
), all UV decontamination and control experiments were carried out at 25 ± 1°C and 53% ± 1% RH. After the decontamination and control treatments, each FFR was cut into square coupons (2 cm by 2 cm), and each coupon was placed in 10 ml of 271B in a 50-ml conical tube for extraction.
(b) Virus recovery.
MS2 recovery was determined as described above for the sodium hypochlorite decontamination experiment.
(c) Efficacy of UV decontamination.
The efficacy of UV decontamination for viable MS2 was calculated as described above for the efficacy of sodium hypochlorite decontamination.