ABSTRACT
Objective : This study was to investigate the morphological changes and ultrastructural damages of the Acanthamoeba cysts after treated with the minimal cysticydal concentration (MCC) of Pouzolzia indica methanolic extract fraction 4 solution by light and electron microscopies. Materials and Methods : Acanthamoeba cyst which prepared followed the method of Roongruangchai K15,16 were adjusted to the final concentration of 104 cysts/ml and treated with 1:4 soltion of fraction 4 Pouzolzia indica methanolic extract, the Thai medicinal plant. which was the minimal cysticidal concentration (MCC) 16. The cysts then, were centrifuged and the pellets were prepared for light, scanning and transmission electron microscopies. Result : Light micrographs showed the cytoplasmic clumping and some empty cyst wall. Transmission electron microscopy showed the ruptures of both ectocyst and the endocyst wall with some opercula damages. The cytoplasm aggregated and clumped. Scanning electron microscopy show steps of damage which started from shrinkage and collapsed of the cyst walls, then the cysts began to bulge and swell resulted in decreasing in the wrinkles of the cyst walls. The cytoplasmic contents drain out from the cyst wall and finally the cyst walls were ripped and torn into small pieces. Conclusion : Pouzolzia indica methanolic extract fraction 4 at the MCC of 1:4 caused structural damages to the Acanthamoeba cyst including shrinking of the cyst walls with the ruptured of the ectocyst, endocyst and operculum, edema and break out into pieces. The active ingredients of this Thai medicinal plant should be further studied as this can eventually be one of the regimen for the treatment of the Acanthamoeba keratitis or as a solution for cleaning the contact lens or contact lens case solution.
ABSTRACT
Objective: The purpose of this study was to compare the least concentrations of 5 fractions of Pouzolzia indica methanolic extract which can be lethal to the cyst form of the Acanthamoeba spp. Methods: Acanthamoeba spp. was isolated from a keratitis patient and was cultured using nonnutrient agar plates enriched with heat-killed E.coli for seven days at room temperature for the production of mature cysts. The cysts were harvested, washed in normal saline solution and adjusted to the final concentration of 104 cysts/ml. They were mixed with several dilutions of each fraction of Pouzolzia indica methanolic extract. After incubation for 1 hour, they were washed and centrifuged to remove the herbal extract supernatants. The cysts were recultured in the same medium for 7 days to confirm that they were all dead. Results: Pouzolzia indica methanolic extract fraction No. 1 which was eluted by water could not kill the cyst, while the crude extract (Fraction C) could at the concentration of 1:2. The fraction No.2 which was eluted by water: methanol had the minimal cysticidal concentration of 1:4, fraction No.3 which was eluted by methanol had the minimal cysticidal concentration of 1:8 and the fraction No.4 which was eluted by ethyl acetone had the minimal cysticidal concentration of 1:4. Concluison: Our results demonstrated that the Pouzolzia indica methanolic extract of several fractions can be cysticidal to an Acanthamoeba cyst, and this can modified to be a better disinfecting solution for contact lens cases.
ABSTRACT
Objective: This study was to investigate morphological changes and fine structures of the cyst form of Acanthamoeba after treatment with various concentrations of povidone – iodine (BetadineTM) using transmission electron microscopy. Methods: Acanthamoeba spp. were isolated from patients with amebic karatitis and obtained from the cultures on 3% non-nutrient agar (NNA) plates seeded with heat killed Escherichia coli (NNA-E coli) with incubation at 30°C for 7 days. The cysts were harvested and washed in ameba saline solution and adjusted to a final concentration of 104 cysts/ml. Various concentrations of povidone-iodine were put in the microtiter plate wells. The minimum cysticidal concentration was the lowest concentration that there was no excystment after 1 week of incubation. The cysts were prepared for routine transmission electron microscopy to determine the structural and organelle damages. Results: Structural damages were observed in the cysts treated with povidone-iodine of 0.04% dilution. Many cysts showed shrinkage of amoeba from the cyst wall and there was a slight withdrawal of the cytoplasm from the cyst wall. Many cysts were ruptured and broken into small pieces. Conclusion: Structural damages were observed in the cysts treated with 0.04% dilution of povidone-iodine solution or more than that. The damage started with pores produced in the cyst wall and the loss of water, shrinkage and loss of the cytoplasm of the inside cell from the cyst wall, followed by breaking of the cyst wall and the inside cell into small pieces.