Chapter 3: MATERIALS AND METHODS
3.4 Photoinactivation of E. coli and E. hirae in aqueous solution using
ml eppendorf tube and centrifuging the biomass at 10,000 × g for 10 minutes. The pellets obtained were washed twice with phosphate buffer saline (PBS) of respective pH. The washed pellets were resuspended in phosphate buffer saline (PBS) set at different initial pH 7.5, 8.25, 9.0 followed by serial dilution up to 1000 times. 10 μl of the suspension from 10, 100 and 1000 dilutions were spread on agar plates for initial viable colony counts. The PBS suspended cultures were then added with MB from its 1mM stock solution to ensure initial concentration in the range of 0.73µmol/l - 1.25µmol/l. Two set of mixtures were kept under dark condition on a gel rocker platform for three different incubation periods of 5, 15 and 30 minutes with constant shaking. After dark incubation one set was used as dark controls by spreading 10 μl of suspension on agar plates and the other set was exposed to a light intensity of 2700 lux for 10 minutes using 11W compact fluorescent light (CFL) in a closed chamber.
Photo sensitized bacterial suspensions (10µl) were then spreaded on brain heart infusion agar for E. hirae and nutritive agar plates for E. coli, set in duplicates. All the plates were incubated at 37°C for 24 hours. Viable cells in the culture plates were enumerated by colony counting method (Vilela et al. 2012), which involves counting of distinct viable colonies using a colony counter.
The average results of percentage inactivation of microorganisms from each duplicate runs in the study were calculated as per the following equation
Where, Ci and Cf are the initial and final viable cell counts.
3.4.1 Effect of concentration of photosensitizer, pH of solution and dilution
Photo-inactivation experiments were carried out to first study the combined effect of methylene blue concentration, cell suspension pH and initial viable cell count (dilution) employing the statistically valid full factorial design of experiment. The range and levels of these variables used in this study are presented in Table 3.1. Table 3.2 presents the experimental variables and their levels chosen in each of the experimental runs.
Table 3.1 Range and level of the variables used in the photo-inactivation experiment using Methylene blue as the photo-inactivating compound
Factors Low Level
(-1)
Centre Point (0)
High Level (+1)
Concentration of
methylene blue 0.73 µmol/l 0.99 µmol/l 1.25 µmol/l
pH 7.50 8.25 9.00
Dilution 10 100 1000
Table 3.2 Combination of parameters and their levels used in the photo-inactivation experiments with Methylene Blue as the photo inactivating compound
The initial concentration of photosensitizer was chosen as 0.73 μmol/l based on a report by Ergaieg and Seux, (2009) and Methylene Blue and water suspension was kept in an alkaline range to mimic natural water environment. For ensuring viable cell count, suspension containing the bacterial culture was diluted up to 1000 times as dilution beyond this value did not yield any viable colonies upon culture. For diluting the bacterial culture, 1 ml of freshly grown bacterial culture was centrifuged at 10, 000 ×g for 10 minutes. The pellet obtained is then washed and re-suspended in phosphate buffered saline (PBS). The serial dilution method was followed using Phosphate Buffer Saline (pH 7.2-7.4) up to 10-6 by transferring 1 ml of suspension from the previous test tube into the next, as shown in Fig. 3.2. The serially diluted suspension (10 μl) was then spread on agar plates and incubated at 37°C for 24 hours. Later, viable colonies growing on the agar plates were counted using a colony counter.
Experimental run no.
Coded levels of the variables MB initial
concentration (µmol/L)
pH Dilution
1 0.73 7.5 10
2 0.73 7.5 1000
3 0.73 9.0 10
4 0.73 9.0 1000
5 0.99 8.25 100
6 1.25 7.5 10
7 1.25 7.5 1000
8 1.25 9.0 10
9 1.25 9.0 1000
Fig. 3.2 Schematic representation of serial dilution method 3.4.2 Effect of dark incubation on photo inactivation efficiency
For photo-inactivation of micro-organisms, dark incubation with constant shaking is required to get a homogeneous mixture of bacterial suspension and the photosensitive dye (Ergaieg and Seux, 2009). Fig 3.3 shows initial colony count for E. hirae and E. coli with 10, 100 and 1000 dilution with both MB and SAQS.
(a)
Dilution
10 100 1000
No. of distinct colonies
0 100 200 300 400 500
E. hirae E. coli
(b)
Dilution
10 100 1000
No. of distinct colonies
0 100 200 300 400 500 600
E. hirae E. coli
Fig. 3.3 Initial colony count with 10, 100 and 1000 dilution for E. hirae and E. coli (a) with MB (b) with SAQS
Hence in order to investigate the effect of dark incubation period on photo-inactivation using Methylene Blue, three different periods of dark incubation 5, 15, 30 minutes with constant shaking on a gel rocker has been investigated. For each set of experiments the Methylene Blue concentration was in the range 0.73 μmol/l to 1.25 μmol/l, dilution was upto thousand times and pH was in the range 7.5 to 9.00.
3.4.3 Studies on cell death using Flow cytometry
In order to confirm the results of microbial inactivation obtained from the photo inactivation experiments using the colony counting method flow cytometry method was used.
Photosensitized bacterial cells from the previous experiments as explained in section 3.4 were obtained by centrifuging the cell suspension at 10000 × g for 10 minutes and the pellet was washed with PBS of a suitable pH. Later, the pellet was suspended in 1 ml of PBS followed by addition of 20 µl of propidium iodide (PI) solution (obtained by dissolving in distilled water in the ratio 1:1) and incubation in dark for 15 minutes. 1 ml sample was then analysed
for 100000 cells using BD FACS CaliburTM, USA, flow cytometer equipped with an argon laser (L1) (wavelength: 488nm; Fluorescence channel: FL-2 yellow). PI fluorescence was measured to distinguish between the live and dead bacterial cells.
3.4.4 Mechanism of photo inactivation of bacteria
To gain further insight into the mechanism of photo inactivation analysis, reactive oxygen species (ROS), lipid peroxidation and protein carbonyl index were carried out.
3.4.4.1 Reactive Oxygen Species (ROS) determination
Sample from each photo inactivation experimental run as presented in Table 2 were added with 20μl of 20μM dihydrochlorofluorescin diacetate (DCFDA) and incubated for 30 minutes at 37°C. The suspension was then added with MB and kept in the dark on a gel rocker for 30 minutes. Later, it was exposed to visible light. Following the light exposure period of 10 mins, the suspension was spread on agar plates to check for viable cell count. The suspension was checked for 2, 7 dichlorofluorescin (DCF) fluorescence by excitation at 488nm and emission spectra was analysed in the range 510- 540 nm using Fluoromax 4.
3.4.4.2 Lipid peroxidation assay
Bacterial cell suspension was prepared and inactivation experiments were carried out as described in section 3.4. Experiments were carried out only for 10 time dilutions because higher dilutions didn’t have enough precipitate. The treated bacterial cells were obtained in the form of pellet and lipid peroxidation products of cell lysate were determined as thiobarbituric acid reactive substances (Trivedi, 2005). Bacterial pellet was resuspended in PBS of respective pH (7.5 and 9.0) and sonicated by keeping on ice (4°C) using probe sonicator. An aliquot (100 μl) of bacterial lysate was allowed to react with 10%, trichloroacetic acid (200 μl) for 15 minutes on ice (4°C). Later, centrifuged at 3000 ×g for 15 minutes at 4°C to get the supernatant. The supernatant obtained is made to react with
thiobarbituric acid in ratio 1:1 by placing in boiling water bath for 10 minutes. The mixture is then cooled and absorbance is taken at 532 nm using Tecan plate reader to determine thiobarbituric acid reactive substances using 1, 1, 3, 3 - tetraethoxy propane as the standard.
The equation obtained by fitting the linear trend line is used to calculate the value of lipid peroxidation in different experimental runs.
Fig. 3.4 Standard Curve for Lipid peroxidation assay 3.4.4.3 Estimation of Protein carbonyl
Higher dilutions didn’t have enough precipitate so experiments were carried out only for 10 time dilutions. The pellets of treated bacterial cells were washed with PBS and resuspended in PBS of respective pH (7.5 and 9.0). Cell suspension was lysed by using probe sonicator by keeping the samples on ice (4°C). Lysate was divided equally in two equal portions and added with an equal volume of 10% trichloroacetic acid at 4°C. The mixture was incubated for 15 minutes at 4°C and then centrifuged at 3000 × g for 15 minutes. The precipitate obtained in one of the two portion was added with 500 μl of 0.2% 2,4-dinitrophenylhydrazine
y = 0.0198x + 3.5824 R² = 0.8254
3.5 3.55 3.6 3.65 3.7 3.75 3.8
0 100 200 300 400 500 600
Absorbance
1,1,3,3-Tetraethoxy propane conc. (µM)
(DNPH) in 2N HCl and the other portion was added with 500 μl of 2N HCl. The mixtures were then incubated at 37°C for one hour with continuous vortexing followed by addition of 55μl of 100% TCA for precipitating the protein. Samples were centrifuged and the pellet obtained was washed with a mixture of ethanol and ethylacetate. The pellets were then suspended in 600 μl of 6M guanidine hydrochloride followed by incubation for 30 minutes.
Absorbance of the final mixture was recorded at 370 nm (Castegna et al., 2003) using Tecan elisa plate reader.
3.5 Photoinactivation of E. coli and E. hirae in aqueous solution using Sodium