Katia E. Sutyak et al. Following the designated exposure times, the MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay was used to determine overall cell viability. The data were used to approximate an eﬀective time (ET) that would reduce cell viability to 50% (ET-50). 2.5. MTT viability assay The MTT assay was carried out according to the protocol outlined by Dover et al. [29]. Brieﬂy, the viability of ectocervical cells after exposure to subtilosin was measured as a direct proportion of the breakdown of the yellow compound tetrazolium to the purple compound formazan, since only living cells can cause this reaction to occur [32]. Tissues were exposed to subtilosin and the two controls for several designated time points; at the conclusion of each, the liquid in the plate wells was combined with the liquid from the tissue inserts. This mixture was then assayed spectrophotometrically using a 96 well-plate reader (MRX revelation, Dynex Technologies, Va, USA) to determine the level of tetrazolium degradation. The viability (%) of the treated tissue inserts was calculated according to an equation provided by the man- ufacturer: % viability = OD570 (treated tissue)/OD570 (negative control tissue). The exposure time that reduced tissue viability by 50% (ET-50) was calculated according to [V = a + b∗ log(t)] described by Ayehunie et al. [30], where V = % viability, t = time in minutes, and “a” and “b” are constants representing the viability data from the time points preceding and following 50% viability. On the whole, there is a direct relationship between the length of the ET-50 and the toxicity of the tested application (i.e., a shorter ET-50 corresponds to a more harmful compound). 2.6. Semen sample collection and analysis The CFS gathered from a B. amyloliquefaciens culture was used to test the eﬀect of subtilosin exposure on the motility of human spermatozoa. Initially, the CFS was diluted with normal saline (0.9%) so that 200 μL of the ﬁnal material was equivalent to 50 μL, 100 μL, or 200 μL of undiluted CFS. Two semen samples were collected on the day of experi- mentation. Each sample was collected by self-masturbation in a polypropylene specimen container (Fisher) prior to transport to the laboratory. Within 1 hour of collection, the samples were pooled. Total sperm count was calculated using bright ﬁeld light microscopy (Olympus BX50; 400x) after dilution (1:50) of the semen in normal saline. The initial percentage of motile sperm was calculated prior to testing with a neubauer hemacytometer [33]. The determination of motile sperm % was made using randomly selected ﬁeld views (400x) from a count of between 104–201 cells. Any visibly moving spermatozoa (directional or stationary) were counted as motile cells. The percentage of forward progressing spermatozoa was subjectively determined based on the assumption that 70% of the sperm in a normal sample would behave in such a manner. The samples used in this experimentation fell into such a “normal” category. 3 2.7. Treatment of spermatozoa with subtilosin A modiﬁed Sander-Cramer test was used to determine the eﬀect of column-puriﬁed subtilosin on human spermatozoa motility [34]. This measured the eﬀect of subtilosin after 30- second exposure times of 5 volumes (200 μL) of the solution at each dilution (25% and 50% in normal saline, and 100%) with one volume (40 μL) of whole semen. The motilities of cells from random high-magniﬁcation ﬁelds (400x) of the sample were determined in duplicate as described above. 2.8. Data analysis The % motility data were arcsine transformed [35] prior to further examination. StatMost32 (version 4.1) statistical software (DataMost Corporation, Sandy, Utah, USA) was used to calculate all statistical parameters. The % values of motility were presented as averages and 90% conﬁdence lim- its. Any diﬀerences between treatment groups were assessed by the Newman-Keuls multiple range test. Diﬀerences were deemed signiﬁcant at the 0.05 level of conﬁdence. 3. RESULTS 3.1. Determination of protein concentration The concentration of subtilosin in the column-puriﬁed sample was estimated at 135.7 μg/mL. The CFS and column- puriﬁed sample produced identical zones of inhibition at each dilution (data not shown); therefore, the concentrations of protein in both solutions were assumed to be equivalent. While it is improbable that a 100% yield would be attained from column chromatography, previous work has shown that protein concentrations can be precisely calculated based on the comparisons we conducted [36]. Due to the diﬃculty in measuring the CFS protein concentration via other assays, the chosen method was deemed the most accurate and reproducible. 3.2. Cell viability % and ET-50 values After 48 hours of exposure to subtilosin, the EpiVaginal ectocervical tissues retained a high level of viability compared to the positive control, Nonoxynol-9, and the negative control, miconazole nitrate (Table 1). Due to the lack of toxicity of the antimicrobial, the ET-50 value for subtilosin could not be established since the total cell viability did not drop below 50% at any of the given time points. However, a projection of the ET-50 value is possible by an extrapolation of the data. Data presented in Table 1 can be ﬁt to a curve described by Ln(V ) = a + bt2, where a = 4.605995356 and b = −0.00014151 (coeﬃcient of determination, or r2, = 0.9998), from which the ET-50 is estimated at 70 hours. 3.3. Quantitative observations of motile spermatozoa Subtilosin reduces human sperm motility in a dose- dependent manner (Figure 1). The motility of the treated spermatozoa ranged from 0 to 88% of control motility levels over the four-fold range of subtilosin concentrations.