Materials and Methods bacteria extracted from kitchen sponges

2. Materials and Methods

2.1 Cultures

The microorganisms used in this experiment were vegetative Bacillus subtilis, Escherichia coli, Salmonella typhimurium and Staphylococcus aureus. The microorganisms were provided already grown on nutrient agar plates and were each subcultured onto additional nutrient agar plates to ensure their viability.

2.2 Sponges

A pack of four plain cellulose sponges, (with no scrubbing pads) measuring 160 mm x 110 mm x 10 mm each were purchased from Sainsbury's.

2.3 Microwave

The 900 W domestic microwave used throughout the experiment was from the brand Sharp, (model R-353). The power setting to which it was set was unknown as the screen of the microwave wasn't working. So to resolve this, the microwave was calibrated using an equation designed by Thomas and Webb, which is described below, (Webb, et al., and Mima, et al.). This revealed that the microwave was working at 455 W.

2.4 Calibration of the Microwave

To calibrate the microwave, 1 L of water, (at room temperature), was heated for 60 seconds in the microwave. The difference between the final temperature and the initial temperature was calculated and multiplied by 70. This reveals the wattage output of the microwave, (Webb, et al., 1998; Mima, et al., 2008).

2.5 Preparation of Cultures

A culture for each microorganism was prepared by removing colonies from its nutrient agar plate using a sterile inoculating loop. This loop was then suspended into a Sterilin® containing 15 ml of nutrient broth. The cultures were incubated for two hours at room temperature before being used in the experiment.

2.6 Preparation of Sponges

A sponge was cut, while still in its original packaging to keep it as sterile as possible, with a sterile razor blade. The sponge was cut into 80 mm x 20 mm x 10 mm pieces and these pieces were then wrapped in aluminium foil.

2.7 Inoculation of Sponges

1 ml of the culture was pipetted using a P1000 Gilson pipette onto a piece of sponge, which was taken from the tin foil and held using sterile forceps. Once inoculated the piece of sponge was then put into a screw-capped bottle containing 100 ml of nutrient broth, using the sterile forceps.

2.8 Exposing Sponges to Microwave Radiation

Once all of the sponge pieces were inoculated and put into screw-capped bottles, these bottles were then labelled either ‘control', ‘15 seconds', '30 seconds' or ‘1 minute' and were then put into the microwave and heated for their designated time periods.

2.9 Extracting Microorganisms from Sponges

After the exposure to microwave radiation, a ten-fold serial dilution was prepared using 1% peptone water. For each piece of sponge, 4.5 ml of 1% peptone water was put into four Sterilins® designated either '10-1', '10-2', '10-3' or '10-4' using a P5000 Gilson pipette. Then 0.5 ml of the nutrient broth inside a screw-capped bottle was taken and pipetted into the '10-1' Sterilin® using a P1000 Gilson pipette. This Sterilin® was mixed and then 0.5 ml of the solution was removed from it and pipetted into the '10-2' Sterilin® and this was repeated for the '10-3' and '10-4' Sterilins®.

2.10 Selective Plating

100 µl was taken from each Sterilin® and pipetted onto an agar plate, using a P200 Gilson pipette. The inoculum was spread using the spread plate method with a glass rod which was sterilized using alcohol and a Bunsen burner. The plates were then incubated at 37 oC for 48 hours. After 48 hours the number of colonies on the plates were counted and recorded. The experiment was repeated three times for each microorganism.

2.11 Statistical Analysis

A one-way ANOVA statistically test was conducted using Microsoft Excel 2003 to compare the mean number of bacteria extracted from the kitchen sponges, (log10 CFU/mL per sponge) and see if they were significantly different. The test assumes that the data is continuous as well as approximately distributed and that the data sets have variances which are homogenous. The null hypothesis was that the data sets had the same mean and the samples were considered significantly different if P < 0.05.

As the ANOVA shows that at least one of the pairs in question is significantly different but doesn't indicate which pair(s) then a post hoc test is needed in order to do so. The post hoc test chosen was the Tukey's HSD, (honestly significant differences) post hoc test and it identifies which of the pairs are significantly different and those which are not. This test could only be conducted if the null hypothesis for the one-way ANOVA could be rejected. The difference between the pairs of means were calculated and then compared to the HSD value. If the differences were larger than the HSD value, they were significantly different. The Tukey's post hoc test will be explained in further detail in the appendix section on page 37.

Also a measure of effect size was conducted in order to calculate the proportion of variance that the independent variable, (the microwave treatment) could account for in the dependent variable, (cell survival). The results showed the percentage of variance that was due to the microwave treatment in which the microorganisms had received.

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