Name: Micro-Colony Forming Unit Assay to Quantify Mycobacteria
Uploaded: 2023-07-28T13:10:00
Description: Watch this Scientific Journal Video about Micro-Colony Forming Unit Assay to Quantify Mycobacteria at JoVE.com

micro-colony forming unit assay to quantify mycobacteria

使用 Micro-CFU 方法加速结核病测定

Tuberculosis (TB) is the leading cause of death worldwide by a single infectious agent, bacterium Mycobacterium tuberculosis (M.tb), killing more people than any other pathogen. In 2021, TB was responsible for 1.6 million deaths and was surpassed by COVID-19 during the 2019-2021 pandemic1. Moreover, according to the World Health Organization&#180;s global TB report of 2022, the COVID-19 pandemic was responsible for an increase in new TB cases. The WHO also reports large drops in the number of people diagnosed with TB during this period, which could increase further the number of TB cases1.
The Bacillus Calmette-Gu&#233;rin (BCG) is a live-attenuated strain of the pathogenic Mycobacterium bovis, used for the first time as a vaccine more than 100 years ago. This is the only vaccine against TB and is the oldest licensed vaccine in the world still in use2,3. Currently, there are 12 vaccines in different phases of clinical trials4, and dozens of vaccines are under pre-clinical development5,6. Pre-clinical assessment of vaccines against TB includes the evaluation of the safety and immunogenicity7, which can be obtained in diverse animal models such as zebrafish, mice, guinea pigs, rabbits, cattle, and non-human primates8,9,10. Additionally, assessing the capacity of a vaccine to induce protection against M.tb infection and/or transmission, i.e., the vaccine efficacy, requires an M.tb challenge in vivo5,11. Interestingly, BCG vaccination induces non-specific effects that affect the survival of other bacterial and viral pathogens12,13&#160;through the mechanism of trained immunity14. To quantify the viable bacterial burden in an infected animal, the method of choice is the enumeration of bacterial colonies through the colony-forming units (CFU) assay5,15. CFU is a unit that estimates the number of microorganisms (bacteria or fungi) that form colonies under specific growth conditions. CFUs originate from viable and replicative microorganisms, and the absolute number of living microorganisms within each colony is difficult to estimate. It is uncertain whether a colony has originated from one or more microorganisms. The CFU unit reflects this uncertainty, hence a great variability can be observed in replicates of the same sample. This time-consuming assay requires specialized technicians trained to work in a biosafety level 3 (BSL3) facility, substantial laboratory and incubator space, takes from 4 to 6 weeks to conclude, and is prone to contamination.
In this study, we describe an optimized method for colony enumeration, the micro-CFU (mCFU), and offer a simple and rapid solution to analyze the results15,16,17,18,19,20. The mCFU assay requires tenfold fewer reagents, reduces the incubation period threefold, taking 1 to 2 weeks to conclude, reduces lab space and reagent cost, and minimizes the health and safety risks associated with working with large numbers of M.tb. We propose that this method should be universally adopted for pre-clinical studies of vaccine efficacy determination, ultimately leading to time reduction in the development of vaccines against TB. Finally, this optimized method of CFU enumeration has been used to quantify not only Mycobacteria but also other bacteria, such as Escherichia coli and Ralstonia solanacearum21.

作者聚焦：优化 CFU 测定以有效评估结核病疫苗功效和抗原呈报分析 

用于结核病疫苗功效评估的微集落形成单位测定

CFU determination is necessary to measure the efficacy of the immune response against bacteria. However, it is very low throughput and becomes cumbersome and expensive when applied to pathogenic bacteria such as Mycobacterium tuberculosis. This is a significant limitation when we need to test large panels of treatments.The research focuses on tuberculosis and on the assessments of TB vaccines. It explores safety, immunogenicity, and efficacy of vaccines against Mycobacterium tuberculosis. The study presents an optimized method to micro-CFU for enumerating bacterial colonies to assess vaccine efficacy in preclinical studies.This protocol aims to optimize CFU determination by generating results from a small droplet similar to those from a regular Petri dish. Quantifying bacteria viability from such a small sample allows us to analyze thousands of data points using a fraction of the materials and time necessary for the classical method. Our research aims to dissect the antigen presentation machinery and to identify the most relevant antigens to optimize antigen presentation.This will inform us to design and develop better vaccines to improve or replace the current TB vaccine, which is BCG.

Watch this Scientific Journal Video about Micro-Colony Forming Unit Assay to Quantify Mycobacteria at JoVE.com

Micro-Colony Forming Unit Assay to Quantify Mycobacteria  

用于定量分枝杆菌的微菌落形成单位测定

要使用孔板进行连续稀释，首先将分枝杆菌悬浮液放在 96 孔板的 A 排和 E 排上。将 180 微升去离子水加入剩余的孔中，进行连续稀释过程。使用12通道移液器，将悬浮液重悬于A行中，并将20微升转移到B行中，重复B排和C行的悬浮液转移，直到D行达到最后的稀释度。让液滴轻轻接触琼脂，确保正确转移并最大限度地减少尖端内的液体滞留。让液滴干燥并关闭琼脂平板。在 37 摄氏度下孵育，同时监测细菌生长。或者，将琼脂平板放入密封的塑料袋中以防止干燥。孵育 6 至 10 天后，检查肉眼可见的单个菌落。使用最低放大倍率物镜将板置于倒置显微镜下。或者，使用相机拍摄液滴的照片，并使用 ImageJ 在计算机上手动计数菌落进行自动菌落计数。使用微菌落形成单元或微量CFU测定从少量琼脂平板中产生大量数据。使用脂质体进行沙奎那韦递送可提高对具有不同耐药性的微杆菌菌株的治疗效果。

Research

JoVE Journal

Medicine

Micro-Colony Forming Unit Assay to Quantify Mycobacteria

Micro-Colony Forming Unit Assay for Efficacy Evaluation of Vaccines Against Tuberculosis

Tuberculosis (TB), the leading cause of death worldwide by an infectious agent, killed 1.6 million people in 2022, only being surpassed by COVID-19 during ...

科研

医学

Bacillus Calmette-Gu&#233;rin (BCG) Vaccine Production

卡介苗（BCG）疫苗生产

Micro-Colony Forming Unit Counting of Mycobacteria Using Fiji &#40;Image J&#41;

Micro-Colony Forming Unit Counting of Mycobacteria Using Fiji (Image J)  

使用斐济分 枝杆 菌的微菌落形成单位计数（图像J）