Memorias do Instituto Oswaldo Cruz · 2025
Breaking the ice - simplified freeze-fracture of parasitic protists: a cost-effective approach without highly specialised equipment.
СтатьяНазвание и аннотация: Перевод в очереди
Обработка статьи · этапы, ошибки и управление
Загрузка состояния…
Ошибки и ограничения
История · было → стало
Состояние обновляется каждые 5 секунд, пока блок открыт. Процент показывается только там, где известен объём работы. Пауза останавливает продолжение этапа; текущий запрос к источнику может завершиться.
Attias Marcia, Pacheco Luis Otávio da Silva, Ortiz Sharmila Fiama das Neves, Verdan Raphael, Miranda Kildare, Gadelha Ana Paula, de Souza Wanderley, Benchimol Marlene
Аннотация
BACKGROUND: We present a simplified freeze-fracture method to reveal the internal structure of protozoan parasites and their host cells, without the need for costly transmission electron microscopy (TEM) equipment. In a traditional procedure, the four key steps in making a freeze-fracture replica are (1) freezing, (2) fracturing, (3) replication, and (4) replica cleaning. In conventional protocols, visualisation is performed using a TEM.
OBJECTIVES: This study aimed to adapt the traditional freeze-fracture protocol to a more accessible method, eliminating the need for vacuum equipment and TEM, while preserving the capacity to visualise cellular structures.
METHODS: In our adapted method, vacuum equipment was not required for sample fracturing, shadowing, or replica preparation. Cells were fixed with glutaraldehyde, embedded in a gelatin-chitosan matrix, cryoprotected with dimethyl sulfoxide (DMSO), and subsequently frozen in Freon, followed by immersion in liquid nitrogen. We manually break the cells with a previously frozen razor blade and macerate them with osmium tetroxide (OsO4). The samples are visualised using scanning electron microscopy (SEM).
FINDINGS: We present various structures of Toxoplasma gondii, Trypanosoma cruzi, Trichomonas vaginalis, Giardia intestinalis, and cultured cells observed using our adapted freeze-fracture method.
MAIN CONCLUSIONS: This is a rapid and low-cost technique that reveals cell structures comparable to those observed using traditional freeze-fracture methods, although with reduced resolution.
OBJECTIVES: This study aimed to adapt the traditional freeze-fracture protocol to a more accessible method, eliminating the need for vacuum equipment and TEM, while preserving the capacity to visualise cellular structures.
METHODS: In our adapted method, vacuum equipment was not required for sample fracturing, shadowing, or replica preparation. Cells were fixed with glutaraldehyde, embedded in a gelatin-chitosan matrix, cryoprotected with dimethyl sulfoxide (DMSO), and subsequently frozen in Freon, followed by immersion in liquid nitrogen. We manually break the cells with a previously frozen razor blade and macerate them with osmium tetroxide (OsO4). The samples are visualised using scanning electron microscopy (SEM).
FINDINGS: We present various structures of Toxoplasma gondii, Trypanosoma cruzi, Trichomonas vaginalis, Giardia intestinalis, and cultured cells observed using our adapted freeze-fracture method.
MAIN CONCLUSIONS: This is a rapid and low-cost technique that reveals cell structures comparable to those observed using traditional freeze-fracture methods, although with reduced resolution.