Consumables and General Methods - Essay Example

2. Materials and Methods 2 Materials 2 1 Consumables Consumables were purchased from different suppliers. Petri dishes were from Sarsedt, Falcon tubes were bought from VWR International, and ethanol came from Fisher Scientific. 2.1.2 Equipment The equipments used throughout the duration of this experiment are listed in Table 1. Table 1. List of equipment used in the performance of the study. Item Model Supplier WM/250/sclp Rsons Mixture? Water bath OLS200 Grant Centrifuge 3-18 Sigma Incubator-shaker Excella E25 New Brunswick Scientific Balance S1-64 Denver Autoclave Rodwell 2.2 Solutions 2.2.1 Buffers 2.2.1.1 Phosphate buffer saline Phosphate buffer saline tablets (Oxoid Limited) were dissolved in distilled water at the ratio of 1 tablet for every 100 mL water to produce phosphate buffer saline (PBS) solution. 2.2.1.2 Citrate buffer Citrate buffer (0.5 M) was prepared by mixing 250 mL 0.5 M citric acid (Sigma-Aldrich) and 250 mL of sodium citrate (Fisher Scientific). The resulting solution was autoclaved. Fifty mL of 0.5 M citrate buffer was added to 450 mL water to make 0.05 M citrate buffer. 2.2.1.3 Carbonate-bicarbonate buffer Five capsules of carbonate-bicarbonate buffer (Sigma-Aldrich) were dissolved in 500 mL distilled water to produce 0.05 M carbonate-bicarbonate buffer. 2.2.1.4 Human plasma Four percent human plasma was prepared by mixing 4 mL of human plasma (normal pooled) (Sera Laboratories International) with 96 mL 0.05 M carbonate-bicarbonate buffer. The preparation was stored at -20°C. 2.2.2. Cellulase solution Cellulase of the BioChemika product line was purchased from Sigma-Aldrich. Cellulase solution was prepared by dissolving 1 mg cellulase in 1 mL of 0.05 M citrate buffer. 2.2.3 Stock solutions 2.2.3.1 Antibiotics Pliva Pharma donated Mupirocin (Sigma-Aldrich). Mupirocin was dissolved in a solution of 50% water and 50% ethanol. The stock solution of rifampicin (Sigma-Aldrich) was in 50% water and 50% dimethyl sulfoxide (VWR International). 2.2.3.2 Antioxidants The antioxidants used were tannic acid (Alfa Aesar), epigallocatechin (Sigma-Aldrich), and L-ascorbic acid (Fisher Scientific). The antioxidants stock solutions were prepared separately. Four hundred milligrams of L-ascorbic acid and tannic acid were dissolved in 5 mL of water to make a stock solution of 80 mg/mL. Forty milligrams of epigallocatechin were dissolved in 5 mL water for a concentration of stock solution of 8 mg/mL. 2.2.4 Culture Media Mueller-Hinton Broth (MHB), Mueller-Hinton Agar (MHA), and Brain Heart Infusion (BHI) Agar were purchased from Oxoid Limited and were prepared according to the manufacturer’s recommendations. 2.3 General Methods 2.3.1 Preparation of overnight bacterial cultures Cells of Staphylococcus aureus strains SH1000 and UAMS-1 were streaked on Mueller-Hinton Agar (MHA) plates and incubated 37°C for 24 hours. After this period, single colonies were picked, and transferred to tubes with Mueller-Hinton Broth (MHB). The tubes were placed in an incubator-shaker at 37 °C for another 24 hours. The overnight cultures in MHB, after appropriate dilution, served as the inocula for the experimental determination of minimum inhibitory concentration (MIC) of antibiotics and antioxidants, and mutational frequencies (MF). Overnight cultures were always used fresh, and not after storage. 2.3.2 Dilution of antibiotics The desired antibiotic starting concentrations were obtained by diluting the desired volume from the stock solutions. For the determination of the mupirocin MIC, the starting concentration was 16 µg/mL. This amount was diluted doubly using sterile saline to produce decreasing concentrations of 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, and 0.03125 µg/mL. The starting concentration of rifampicin was 2 µg/mL. Again, double dilution was performed. The following concentrations were used to determine the rifampicin MIC: 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.01563, 0.0078, and 0.0039 µg/mL. 2.3.3 Dilution of antioxidants To determine the MICs of the antioxidants that were to be used in the experiment, the antioxidants solutions were also serially diluted similar to what was done for the antimicrobials mupirocin and rifampicin. The starting concentration of all the antioxidants was 8 mg/mL. After double dilutions, the concentrations used were 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, and 0.01563 mg/mL. 2.3.4 Preparation of cellulose disks for biofilm culture Cellulose ester disks with 0.22 µm diameter were purchased from Millipore. Before the disks were used for biofilm culture, they were first sterilized in an autoclave. After cooling, the disks were soaked overnight in buffer. The disks are ready for inoculation with overnight cultures of S. aureus after these treatments. 2.4 Experimental approach 2.4.1 Determination of MIC of the antibiotics rifampicin and muporicin 2.4.1.1 Broth dilution method Twenty µL of each antibiotic dilution were pipetted to duplicate wells of a 96-well microtiter plate. Overnight cultures of S. aureus strains SH1000 and UAMS-1 were diluted to 1:100 by adding 9 µL of culture to 9 mL MHB. One hundred eighty microliters of the cultures were added to the wells with the antibiotics. Wells on columns 11 and 12 of the microtiter plate contained the positive control (sterile saline only), while the negative control wells contained 20 µL of antibiotic but was not inoculated with the bacterial cultures (Figure 1). The microtiter plates were incubated with shaking at 37°C. After 24 hours, the wells were observed for cloudiness, which indicated bacterial growth. Minimum inhibitory concentration (MIC) was the lowest antibiotic concentration where no bacterial growth is observed. 2.4.1.2 Agar dilution method One hundred-eighty microliters of the antibiotic dilution solutions were plated to clearly labelled agar plates. Then 18 mL of pre-cooled MHA was added to the plates. After the plates have set and dried, they were inoculated, using an automatic inoculating machine, with overnight bacterial cultures. The final concentration of each bacterial spots was 1:1000. The plates were placed in the incubator shaker at 37°C. The presence of bacterial spots was observed after the 24- hour incubation period. 2.4.2 Determination of the MIC of the antioxidants tannic acid, epigallocatechin, and L-ascorbic acid using broth dilution method Following the described procedure for the determination of antibiotics, the MIC of the three antioxidants were also determined using the broth dilution method. Twenty µL of the ten dilutions of each antioxidant were added to wells in a microtiter plate. This was followed by 180 µL diluted (1:100) overnight cultures of SH1000 and UAMS-1. After incubation at 37°C for 24 hours, the wells were observed for bacterial growth. 2.4.3 Mutational frequencies of planktonic S. aureus strains SH1000 and UAMS-1 cultures Antibiotic-free plates, and plates with 4x MIC of rifampicin and mupirocin were prepared and labelled. 2.4.3.1 Mutational frequencies in the presence of antioxidants Overnight bacterial cultures of each strain were prepared. Each tube contained 9 mL MHB and 14 µL of the antioxidant stock solutions to achieve the antioxidant MIC. All three antioxidants have the same volume needed to attain their MIC. The MHB bacterial cultures were serially diluted by mixing 45 µL of the culture with 4.5 mL of saline to make a 1:100 dilution. This step was repeated until a final dilution of 10-7 was attained. One hundred microliters of the 10-7 dilution were spread on antibiotic-free plates. The neat MHB, representing 10-1 dilution was plated to the antibiotic plates. Duplicate plates were made for each dilution. The plates were then incubated at 37°C. After 48 hours, the bacterial colonies formed were counted. A few colonies were also picked from the culture plates and streaked into a selection plate to test their mutant status for confirmation purposes. 2.4.3.2 Mutational frequencies in the presence of an oxidant Overnight bacterial cultures of each strain (S. aureus SH1000 and UAMS-1) were prepared. The cultures contained 9 mL MHB and hydrogen peroxide, an oxidant. Similar to the procedure followed in 2.4.3.1, the MHB cultures were serially diluted until a final dilution of 10-7 was attained. One hundred microliters of the last dilution were spread on antibiotic-free plates, while 100 µL of the neat MHB were spread on the antibiotic plates. After incubation at 37°C for 48 hours, bacterial colonies were counted, with a few colonies picked and streaked on a selection plate to test for mutant status. 2.4.4 Mutational frequency of S. aureus biofilms 2.4.4.1 Mutational frequency of S. aureus biofilms without antioxidants The bacterial biofilms were allowed to develop on cellulose ester disks that were prepared as described section 2.3.4. After removal of the disks from overnight soaking, they were inoculated with overnight cultures of S. aureus SH1000 and UAMS-1. The cellulose disks were then carefully placed on BHI agar plates. Three disks were put in 1 plate. The plates were then incubated at 37°C for 48 hours. After forty-eight hours, sterile forceps were used to carefully remove the disks from the agar plate and to dip these disks in 4% human plasma. Then the disks were placed in a new BHI plate for further incubation for 48 hours, and to allow for the establishment of biofilms. The UAMS-1 strain was given another 48 hours in the agar plate to allow it to establish and grow. The cellulose disks with bacterial biofilm growth were removed from the plates after prescribed period. To prevent damaging the biofilm, the disks were carefully and gently washed with 10 mL of sterile saline. Bacterial cells that were adherent to the cellulose disk were removed by incubating the disks in cellulase solution for 30 min at 37°C. This was followed by vortexing for 30 secs after which the disks were discarded. The remaining bacterial suspension was centrifuged at 5000 g for 10 minutes to pellet the cells. The resulting the pellet was resuspended in 10 mL saline while the supernatant was discarded. Following the procedure in 2.4.3.1, the bacterial suspensions of S. aureus SH1000 and UAMS-1 were serially diluted with sterile saline solutions to a final dilution of 10-7. One hundred microliters the neat MHB were plated to antibiotic plates with 4x MIC, while 100 µL of 10-7 dilution were plated to antibiotic-free plates. After 48 h incubation at 37°C, colonies were counted. The bacterial colonies counted from the 10-7 dilution gave the viable count data. Some colonies were picked and streaked to another selection plate to determine the mutational frequency. 2.4.4.2 Mutational frequency of S. aureus biofilms with antioxidants This experiment followed the protocol in section 2.4.4.1 except that the BHI media that was used for growing the bacterial cultures contained the volume of antioxidants L-ascorbic acid, tannic acid, and epigallocatechin corresponding to their respective MIC. Read More