acanthamoeba culture and preparation for microscopic motility analysis

<meta charset="utf8"></head><body>棘 阿米巴 物种运动的长期跟踪和定量

Acanthamoeba research has increased dramatically in recent years due to the increased prevalence of Acanthamoeba keratitis (AK), which is a parasitic infection following the attachment of Acanthamoeba to the cornea1. While outbreaks of AK can be attributed to inappropriate contact lens care or ineffective contact lens care solutions2,3,4,5, there are currently no requirements to demonstrate Acanthamoeba disinfection efficacy for any product on the market. However, there is an ongoing effort in the scientific community and within standards organizations to examine the protocols necessary for quantifying the disinfection efficacy of contact lens care products6,7. Further, due to its resemblance in cellular and functional aspects to human macrophages, Acanthamoeba has been noted as having a significant role in hosting and disseminating other human pathogens8, in addition to the pathogenicity the amoeba itself brings.
Recent techniques have been described that have been able to quantify Acanthamoeba motility - which is generally not highly subject to Brownian movement9,10 - in regard to cytopathic effects or under-exposure to the electric field9,11, as well as advancements in motility analysis in giant virus research using Acanthamoeba as the trackable viral vector12,13. There have also been constant and significant improvements in cell and particle tracking occurring over the last 20 years using new software programs such as the imaging software used here, and new algorithms and deep learning technologies14. However, this is a relatively new and growing field of science regarding benchtop research, clinical application, and industrial standards, and there has been a paucity of data published regarding methods to visualize and track this amoeba, particularly in order to quantify behavioral changes following adherence to contact lenses or during or after contact lens disinfection. Other fields expanding into long-term visual monitoring have supported this effort15,16,17. Due to Acanthamoeba&#39;s inherently challenging nature - including a general resistance to plasmids (which could confer fluorescence), the ability of the amoeba to consume and destroy standard cell dyes, and a unique outer protein makeup - making antibody tagging difficult - methods available to other cells which make them visible in settings other than brightfield imaging have been unusable in this organism. Thus, quantifying the motility of this amoeba has demonstrated a significant addition to the field. Using the method described here, we have been able to ascertain that amoeba remain motile for at least 12 h without nutrients18 and that amoeba that is challenged with a disinfection process and ceases motility during disinfection can recover their motility post-disinfection if they are not fully lysed19.
This protocol details how to visually track and quantify the motility of amoeba microscopically. The primary steps are to record amoeba in brightfield using the appropriate focus and timing between images, transform the images into binary using imaging software, and then use an imaging software&#39;s tracking plugin to set the experimental parameters and follow each amoeba to determine the required measurements such as speed, distance, and confinement. Following this, it is possible to quantify the chemotaxis of an amoeba or an amoeba population in order to define directionality. The key contribution of this method is to visualize and quantify amoeba behavior during different states of nutritional support, surface adherence, disinfection challenge, or other environmental alterations such as cohabitation with mammalian cell culture.

<meta charset="utf8"></head><body>作者聚焦：研究 棘阿米巴 的行为以制定预防 棘阿米巴 角膜炎的针对性策略

棘阿米巴属的定量运动性

棘阿米巴 显微镜运动分析的培养和制备

acanthamoeba-culture-and-preparation-for-microscopic-motility-analysis

Research

JoVE Journal

Biology

Acanthamoeba Culture and Preparation for Microscopic Motility Analysis

680 次观看. 首先，用 30 ml AC6 培养基填充 T75 培养瓶。在无菌条件下将含有棘阿米巴的样品瓶的内容物加入培养瓶中。在 26 至 30 摄氏度下孵育培养物 3 至 5 天，直到培养瓶达到 50% 至 80% 汇合。在需要细胞的前一天，摇动并轻快地敲击主培养瓶两次，以去除贴壁滋养体。用 30 mL AC6 培养基填充新的 T75 培养瓶。然后将 2 毫升主培养物转移到培养瓶中。将...

视频: 棘阿米巴 显微镜运动分析的培养和制备

Quantification of Acanthamoeba spp. Motility

Acanthamoeba keratitis is a severe ocular infection that poses treatment challenges and can lead to blindness. Despite its ubiquity and potential contamination of contact lenses after water exposure, the natural behavior of this pathogen remains elusive. Understanding Acanthamoeba's movement patterns can inform us about how it colonizes contact lenses and contaminates the patient's cornea. It is fortunate that Acanthamoeba spp. are visible via brightfield microscopy starting at 4x magnification. Previous techniques have been developed to quantify Acanthamoeba motility in regard to cytopathic effects or under-exposure to the electric field. Here, we describe a method to track and quantify Acanthamoeba spp. motility long-term (hours to days), which is a protocol applicable to multiple amoeba strains, surfaces, and nutritional status of amoeba. This procedure is germane to determining many core motility quantifications, such as distance, speed, confinement, and directionality, which are necessary to monitor different stages of infection, proliferation, or behavioral change.

This procedure describes how to visualize, track, and quantify Acanthamoeba spp. motility.

棘阿米巴 显微镜运动分析的培养和制备

棘阿米巴显微镜运动分析的培养和制备