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

Versnelling van de bepaling van tuberculose met behulp van de micro-CFU-methode

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.

Auteur in de schijnwerpers: Optimalisatie van CFU-bepaling voor efficiënte beoordeling van de werkzaamheid van tbc-vaccins en analyse van antigeenpresentatie 

Microkolonievormende eenheidstest voor evaluatie van de werkzaamheid van vaccins tegen tuberculose

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  

Microkolonievormende eenheidstest om mycobacteriën te kwantificeren

Om seriële verdunning uit te voeren met behulp van een putplaat, plaatst u eerst Mycobacterium-suspensies op rijen A en E van een plaat met 96 putjes. Voeg 180 microliter gedeïoniseerd water toe aan de resterende putjes voor het seriële verdunningsproces. Gebruik een 12-kanaals pipet om de suspensies in rij A te suspenderen en 20 microliter over te brengen naar rij B.Herhaal de suspensieoverdracht voor rijen B en C totdat de laatste verdunning is bereikt in rij D.Gebruik voor microdruppelplating een multikanaalspipet van 0,5 tot 10 microliter met dunne punten om vijf microliter van elke rij van de plaat met 96 putjes over te brengen naar de massieve middelgrote vierkante plaat.Laat de druppeltjes de agar lichtjes raken, zorg voor een goede overdracht en minimaliseer het vasthouden van vloeistof in de punt. Laat de druppels drogen en sluit de agarplaat. Incubeer het bij 37 graden Celsius terwijl je de bacteriegroei in de gaten houdt.Plaats de agarplaten eventueel in een afgesloten plastic zak om uitdroging te voorkomen. Controleer na zes tot 10 dagen incubatie op individuele kolonies die met het blote oog zichtbaar zijn. Plaats de plaat onder een omgekeerde microscoop met behulp van het objectief met de laagste vergroting.U kunt ook een camera gebruiken om een foto van de druppel te maken en handmatig kolonies tellen op de computer met behulp van ImageJ voor het automatisch tellen van kolonies. De microkolonievormende eenheid of micro-CFU-assay werd gebruikt om grote hoeveelheden gegevens te produceren uit een kleine hoeveelheid agarplaten. Het gebruik van liposomen voor de toediening van saquinavir verhoogde de werkzaamheid van de behandeling tegen de microbacteriestammen met verschillende resistenties tegen geneesmiddelen.

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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 ...