Effect of Black seed oil on E. coli. ● control, ▫ 90% black seed oil. 

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... 6538) and Escherichia coli (ATCC C600) strains were maintained in long term preserve system. This was prepared into the lab using 10% (v/v) glycerol in nutrient broth media. The stock was dispensed into 1 ml aliquots o and stored at - 80 C till required. Fresh cultures were prepared from frozen stocks prior to every experimental procedure. This was performed by streaking, using sterile loop, freshly prepared nutrient agar plates which were then incubated at 37° C for 18hrs, from which a colony was used to run the experiments. iments: All antimicrobial susceptibility experiments were conducted by transferring 5ml of the tested strains grown at late exponential phases (O.D. of A 470nm A0.9 and 1.2, respectively) for S. aureus and E. coli strains. This 8 will give bacterial suspension of 1x10 cfu per ml. Absorbance was measured using the spectrophotometer (PU 8675 Vis spectrophotometer), Philips, Germany. The Black seed oil was added, to the bacterial suspension, to give final concentrations (v/v) of 10%, 30%, 50%, 70% and 90%. A sample of volume 1 ml was taken every 1 hr intervals up to 6 hrs and then at 24 hrs where the serial dilutions were performed and the viable count calculated using the spread plate technique. Viable count was performed by transferring 100 μl of each sample taken onto predried nutrient agar plates where then incubated for 24 hrs and counted for viable cfu per ml. Data were expressed as the log of survivors against exposure time. These were used to set up the survival curve (dose response curve). All experiments were performed in triplicates and the mean was used throughout this study. Experiments were also designed to understand the effect of the Black seed oil on which part of the bacterial cell. This was performed by collecting samples of the exposed populations every 1hr up to 6 hrs and then at 24 hrs to measure the absorbance at the wave length 350 nm. Data were expressed as the absorbance reading A 350nm against the exposure time. This will indicate, based on the absorbance reading, which part of the bacterial cell was targeted by the Black seed oil. In preliminary evaluation of the antimicrobial activity of the Black seed oil, cup cut agar method was performed. A 5% (v/v) phenol was used and data were compared to that of Black seed oil. The cup-cut agar method was used as a preliminary step for the qualitative evaluation of the antimicrobial activity of the Black seed oil at the concentrations of 10%, 30%, 50% and 70% (v/v). In the case of E. coli , activity was not clearly observed. Black seed oil concentrations were, therefore, elevated to 90%, whereas a clear zone of inhibition on E. coli was then determined. On the other hand, inactivating activity of black seed oil was clearly observed at 10%, 30%, 50% and 70% (v/v) on S. aureus . The zone of inhibition was related to the extract concentration (Table 1). It should be mentioned that the S. aureus showed a remarkable susceptibility over that shown by the E. coli . This was clearly noticed on the diam- eter of the zone of inhibition. In gener- al both bacteria were susceptible to the effect of the Black seed oil at different concentrations, where the E. coli showed susceptibility only at the 90% concentration (Table 1). As the Black seed oil has proved to display a significant antimicrobial activity, it is very important therefore to reference this activity to one of the most potent agents. In this context the phenol 5% (v/v) was the reference antimicrobial agent used (Table 1). In the case of S. aureus , the zone of inhibition of 70% oil represented about 1.5 fold of the phenol one. This was 38mm for the extract and 26mm for the phenol. The zone of inhibition for 50%, 30% and 10% concentrations were 25 mm, 24 mm and 18 mm, respectively. In the case of E. coli, its susceptibility was also observed. This was 13 mm for the oil (90%) compared to that of the 5% phenol (25 mm). This represents about half fold of the phenol activity (Table 1). No activity was detected for the concentrations 10, 30, 50 and 70%. This study was also designed to evaluate the antimicrobial activity of Black seed oil quantitatively by determine the remaining of survival cells after exposure to different concentrations over period of times. Results in Figures 1 and 2 showed antibacterial activity of the Black seed oil 70% and 90% towards S. aureus and E. coli , respectively. In the case of S. aureus , the level of bacterial kill was clearly demonstrated. This was about 1.7 log reduction compared to the control over 24 hrs exposure (Fig. 1). The level of reduction in the cfu per ml was from 9 7 1.76 x 10 to 4.6 x 10 cfu per ml. This displayed that about 97% of the population were killed. Similarly, E. coli populations were also susceptible but with lesser extent. This was demonstrated in the rate of bacterial survivals over 24 hrs exposure to 90% of the agent concentration. The reduction in the E. coli survivals was measured by about 0.9 log reductions (Fig. 2). This indicated that about 80% of the population was killed and 20% survived. It is therefore clear that the S. aureus population is more susceptible to the effect of the black seed oil than that of the E. coli one. It is very important to notice that the bacterial population used to investigate the antibacterial activity of the Black seed oil was collected specifically at the late exponential phase for both E. coli and S. aureus (Figs. 3 and 4). This was intended to avoid both the exponential phase that characterized by its susceptibility to the effect of antimicrobials and the stationary phase where the bacteria are already started to die. This will impact that the obtained results was mostly reflect the action of the oil minimizing the incidence of biological and physio- logical variations of the bacteria. Further investigation in this study was aimed to understand the possible antibacterial action of the Black seed oil on the bacterial cell. An experiment of measuring the absorbance of challenged bacterial populations at low wave length ranged at A 350nm was designed. This was aimed to measure the absorbance of bacterial structure when small bacterial organelles such as RNA, DNA and ribosomes are suspended and released into the medium. In such instances, if reading was detected at such low wave lengths, this would therefore indicate the possibility of antibacterial agents has an effect on the bacterial envelope and bacterial organelles were released and absorbance was detected. In this study, detections of light absorbance at A 350nm were succeeded. This would intuitively displayed that black seed oil managed to distort and interfere with the cell envelop for both S. aureus and E. coli to various extents. All detections were explained in Figure 5 over 24 hrs exposure. All samples were collected alongside with the viable count sampling for colony forming unit measurements. Figure 5 has showed that the absorbance was relatively high at the first 3 hrs exposure compared to those at next exposures up to 6hrs whilst, on the other hand, it was higher than that at 24 hrs exposure. The degree of variation was higher in the case of S. aureus than that of the E. coli by about 10 folds (Figure 5). This would clearly display that Gram positive bacteria are more susceptible to be inactivated by black seed oil, which would be explained in the variation in their cell envelope structure. These data are in consistence with the data collected above (Figs. 1, 2). When comparing the activity of the Black seed oil to the most powerful antimicrobial and biocide, data shown that the oil potency was variable based on the type of bacteria. This was about 1.5 and 0.5 folds for the S. aureus and E. coli respectively, of that of phenol. This would indicate that such potency could be the reason behind its acceptability for community and public use and, in treating various cases of bacterial infections. Similarly, the ability of the black seed oil for observed inactivation of the population over 24 hrs of exposure is another evidence of its antibacterial activity. This was clearly demonstrated in the survival curve method. The survivors fractions recovered on the plates were 2% and 20 % for both S. aureus and E. coli respectively. In this respect, the phenol 5% v/v had eradicated the whole population upon exposure. This was demonstrated at zero time of exposure where the plates showed no recovered colonies (data not shown). Such inactivating activity of the Black seed oil was demonstrated by dramatic decrease in the number of survivors. In such instances, the black seed oil would clearly play a very important role in inactivating bacterial populations. This would be elucidated in understanding its mechanism of action. The authors suggest that the oil was able to interfere with bacterial envelope function. Doing so will lead to the inability of bacterial cell to control molecule movements which might lead to bacterial lysis and burst. This would eventually lead to cell death. In this context, preliminary inform- ation about understanding the possible mechanism of action of the black seed oil was obtained. This was achieved by conducting an experiment of measuring the absorbance of challenged bacterial populations at low wave length (A 250 - 350nm ). This will detect the bacterial cell organelles if they are released into the medium (15, 19-21). Findings of optical density measurements for challenged S. aureus and E. coli populations to 70% and 90% black seed oil respectively at A 350nm over 24hrs exposure had confirmed these findings where detection of absorbance A 350nm was achieved. Such readings would laid the principle of bacterial cell envelope is the primary target for black seed oil. In one hand, S. aureus was more susceptible with very low absorbance reading at 24hrs exposure. On the other hand, E. coli susceptibility was significantly less with a higher absorbance (10 folds) at 24 hrs exposure. In both cases, ...

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... 7 cfu per ml. This displayed that about 97% of the population were killed. Similarly, E. coli populations were also susceptible but with lesser extent. This was demonstrated in the rate of bacterial survivals over 24 hrs exposure to 90% of the agent concentration. The redu- ction in the E. coli survivals was meas- ured by about 0.9 log reductions (Fig. 2). ...