Effects of Graphene Oxide and Partially Reduced Graphene Oxide on Escherichia Coli and Staphylococcus Aureus

Escherichia coli (E.Coli) and Staphylococcus aureus (S.A.) are common bacterial species and leading causes of infectious diseases, especially dangerous among the elderly. Previous literature has shown that graphene oxide (GO), composed of strongly oxidized carbon nanosheets; and partially reduced graphene oxide (pRGO), made by partially reducing GO - display antibacterial properties. The effectiveness of GO and pRGO to combat these two bacteria was examined in this study.<br/>E.Coli and S.A. were diluted in a 1:10 ratio with water (control group) or GO or pRGO (experimental groups) and the mixtures incubated while shaken for three days and then examined for cell viability; experimental results demonstrated that neither GO nor pRGO possessed antibacterial properties against these two strains. When GO or pRGO solutions were added into E. Coli and S.A. solutions in a 1:1 ratio and incubated, no obvious increase in the number of dead cells was observed in a live/dead stain using an EVOS Microscope. However, it was noted that in the mixture of E. Coli with either GO, pRGO, or both GO and pRGO together, large clumps of E. Coli were observed under transmitted light microscopy.<br/>Thus, it was speculated that GO and pRGO may promote the formation of biofilms in these bacteria. To measure the extent of biofilm production, a crystal violet assay was conducted. The violet absorbance intensity of four identical samples of each of the two bacteria incubated with GO and pRGO was measured, with a higher absorbance indicating greater formation of biofilm. In the E. Coli assays, the optical density (OD) of the control was 0.22 at 592 nm, while the absorbance of E. Coli incubated with GO was 0.47; incubation with pRGO had an OD of 0.54, and E. Coli incubated in a mixture of GO and pRGO had an OD of 0.43, indicating that all three test mixtures more than doubled the biofilm production as compared to the control. In contrast, GO seems to inhibit biofilm formation in S. aureus by 40%; pRGO does not exhibit a significant effect on biofilm formation, having an optical density similar to that of the control, 0.30; and a mixture of GO and pRGO decreased the OD by 20%. These findings suggest there may be a difference in the response to GO and pRGO between gram negative bacteria such as E. Coli and gram positive bacteria such as S.A., with both GO and pRGO stimulating biofilm production in the gram negative but not the gram positive bacteria. Other gram negative and gram positive bacteria will be tested in the future to see if this is the trend.<br/>To determine whether GO and pRGO are actually pro-bacterial by inducing the formation of biofilms to protect the bacteria from external threats, E. coli was cultured in a 6-well tissue culture plate for two days: two samples in phosphate-buffered saline (PBS), two in a 1:1 mixture of GO and pRGO, one in GO, and one in pRGO. Each sample was divided and treated with either PBS or ampicillin to discern whether the biofilm provides resistance to ampicillin. Results will be discussed.