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

Accelerazione della determinazione della tubercolosi con il metodo Micro-CFU

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.

Autore in primo piano: Ottimizzazione della determinazione delle CFU per una valutazione efficiente dell'efficacia del vaccino contro la tubercolosi e dell'analisi della presentazione dell'antigene 

Saggio dell'unità formante microcolonie per la valutazione dell'efficacia dei vaccini contro la tubercolosi

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  

Saggio dell'unità formante microcolonie per quantificare i micobatteri

Per eseguire la diluizione seriale utilizzando una piastra a pozzetti, posizionare prima le sospensioni di Mycobacterium sulle file A ed E di una piastra a 96 pozzetti. Aggiungere 180 microlitri di acqua deionizzata nei pozzi rimanenti per il processo di diluizione seriale. Utilizzando una pipetta a 12 canali, risospendere le sospensioni nella fila A e trasferire 20 microlitri nella fila B.Ripetere il trasferimento della sospensione per le file B e C fino a raggiungere l'ultima diluizione nella riga D.Per la placcatura di microgocce, utilizzare una pipetta multicanale da 0,5 a 10 microlitri con puntali sottili per trasferire cinque microlitri da ciascuna fila della piastra a 96 pozzetti alla piastra quadrata media solida.Lasciare che le goccioline tocchino leggermente l'agar, garantendo un corretto trasferimento e riducendo al minimo la ritenzione di liquidi all'interno della punta. Lasciare asciugare le goccioline e chiudere la piastra di agar. Incubarlo a 37 gradi Celsius monitorando la crescita batterica.Facoltativamente, mettere le piastre di agar in un sacchetto di plastica sigillato per evitare che si secchino. Dopo 6-10 giorni di incubazione, verificare la presenza di singole colonie visibili ad occhio nudo. Posizionare la lastra sotto un microscopio invertito utilizzando l'obiettivo con l'ingrandimento più basso.In alternativa, utilizzare una fotocamera per scattare una foto della gocciolina e contare manualmente le colonie sul computer utilizzando ImageJ per il conteggio automatico delle colonie. L'unità formante micro-colonia o il saggio micro-CFU è stato utilizzato per produrre elevate quantità di dati da una piccola quantità di piastre di agar. L'uso di liposomi per la somministrazione di saquinavir ha aumentato l'efficacia del trattamento contro i ceppi di microbatteri con diverse resistenze ai farmaci.

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 ...

Ricerca

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

Produzione di vaccini contro il Bacillus Calmette-Guérin (BCG)

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

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

Conteggio delle unità formanti microcolonie di micobatteri utilizzando le Figi (Immagine J)