Transmission electron microscopy (TEM) can provide high resolution imaging of biological specimens. The study is to establish the effects of a modified glutaraldehyde (GA) compare to the standard GA fixation on Acanthamoeba castellanii from TEM perspectives and thus provide precise and accurate information on the ultrastructure studies of the parasite. By increasing the contrast, the ultrastructures of the parasite were more evident. The TEM images were obtained from parasites fixed with the modified GA and the standard GA and then the area of the nucleus and the gray values of the image of the nucleus of the parasites were measured. The mean areas of the nucleus were found to be significantly reduced in the standard GA fixed parasites (12210.4 nm2) compared to the modified GA fixed parasites (8676.3 nm2) (p < 0.05). The mean gray values of the image were significantly reduced from 2024 in the standard GA fixed parasites (2024) to the modified GA fixed parasites (1636) (p < 0.05). The study shows that the modified GA produced significantly better contrast on TEM images of the A. castellanii compared to the standard GA. This was because the modified GA generated more free water molecules during fixation and the uptake of modified GA by the nucleus of the parasite organizing all protein constituents in the cell into a more closely packed configuration than that of the standard GA. With such properties, the modified GA is a better fixative providing better images for ultrastructures of the parasite.
Bacterial infections have remained significant despite our advances in the development of a plethora of disinfectants as well as antimicrobial chemotherapy. This is in part due to our incomplete understanding of the prevalence of bacterial pathogens in the environmental and clinical settings. Several lines of evidence suggest that Acanthamoeba is one of the most ubiquitous/resilient protists that also acts as a host/reservoir for pathogenic microbes. Thus targeting the hardy host, which harbour microbial pathogens, offer a potential avenue to counter infection transmission, particularly hospital/community-acquired infections. This will complement existing approach of applying disinfectants that are targeted against bacterial pathogens directly.
Crude methanol extracts of a marine sponge, Aaptos aaptos, collected from three different localities namely Kapas, Perhentian and Redang Islands, Terengganu, Malaysia, were tested in vitro on a pathogenic Acanthamoeba castellanii (IMR isolate) to examine their anti-amoebic potential. The examination of anti-Acanthamoebic activity of the extracts was conducted in 24 well plates for 72 h at 30 °C. All extracts possessed anti-amoebic activity with their IC(50) values ranging from 0.615 to 0.876 mg/mL. The effect of the methanol extracts on the surface morphology of A. castellanii was analysed under scanning electron microscopy. The ability of the extracts to disrupt the amoeba cell membrane was indicated by extensive cell's blebbing, changes in the surface morphology, reduced in cell size and with cystic appearance of extract-treated Acanthamoeba. Number of acanthapodia and food cup was also reduced in this Acanthamoeba. Morphological criteria of apoptosis in Acanthamoeba following treatment with the sponge's extracts was determined by acridine orange-propidium iodide staining and observed by fluorescence microscopy. By this technique, apoptotic and necrotic cells can be visualized and quantified. The genotoxic potential of the methanol extracts was performed by the alkaline comet assay. All methanol extracts used were significantly induced DNA damage compared to untreated Acanthamoeba by having high percentage of scores 1, 2, and 3 of the DNA damage. Results from cytotoxicity and genotoxicity studies carried out in the present study suggest that all methanol extracts of A. aaptos have anti-amoebic properties against A. castellanii.