This work was supported by internal funding from the Faculty of Medicine, Universidade Cato&#769;lica Portuguesa, and external funding from Funda&#231;&#227;o para a Ci&#234;ncia e a Tecnologia (FCT), under the grants UIDP/04279/2020, UIDB/04279/2020, and EXPL/SAU-INF/0742/2021.

NOTE: The protocol described here is for BCG but can be applied to any Mycobacteria. BCG can be used as a surrogate bacterium for TB experiments when BSL3 facilities are not available22. The following procedures using BCG should be performed under a biosafety level 2 (BSL2) laboratory and follow the appropriate biosafety guidelines and good laboratory practices for the manipulation of hazard group 2 microorganisms.
1. Culture media preparation
<ol>
	<...

Accelerating Tuberculosis Determination Using the Micro-CFU Method

<ol>
	<li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Global Tuberculosis Report 2022. , (2022).</li><li>Bettencourt, P. J. G., Joosten, S. A., Lindestam Arlehamn, C. S., Behr, M. A., Locht, C., Neyrolles, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=100+years+of+the+Bacillus+Calmette-Guérin+vaccine.">100 years of the Bacillus Calmette-Guérin vaccine.</a> Vaccine. 39 (50), 7221-7222 (2021).</li><li>Bettencourt, P. J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+100th+anniversary+of+bacille+Calmette-Gu&#233;rin+(BCG)+and+the+latest+vaccines+against+COVID-19.">The 100th anniversary of bacille Calmette-Gu&#233;rin (BCG) and the latest vaccines against COVID-19.</a> The International Journal of Tuberculosis and Lung Disease. 25 (8), 611-613 (2021).</li><li>Scriba, T. J., Netea, M. G., Ginsberg, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Key+recent+advances+in+TB+vaccine+development+and+understanding+of+protective+immune+responses+against+Mycobacterium+tuberculosis.">Key recent advances in TB vaccine development and understanding of protective immune responses against Mycobacterium tuberculosis.</a> Seminars in Immunology. 50, 101431 (2020).</li><li>McShane, H., Williams, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+preclinical+animal+models+utilised+for+TB+vaccine+evaluation+in+the+context+of+recent+human+efficacy+data.">A review of preclinical animal models utilised for TB vaccine evaluation in the context of recent human efficacy data.</a> Tuberculosis. 94 (2), 105-110 (2014).</li><li>Voss, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+and+challenges+in+TB+vaccine+development.">Progress and challenges in TB vaccine development.</a> F1000Research. 7, 199 (2018).</li><li>Satti, I., McShane, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+approaches+toward+identifying+a+correlate+of+immune+protection+from+tuberculosis.">Current approaches toward identifying a correlate of immune protection from tuberculosis.</a> Expert Review of Vaccines. 18 (1), 43-59 (2019).</li><li>Young, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+tuberculosis.">Animal models of tuberculosis.</a> European Journal of Immunology. 39 (8), 2011-2014 (2009).</li><li>Pedroza-Rold&#225;n, C., Flores-Valdez, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+mouse+models+and+vaccine+candidates+for+preventing+chronic/latent+tuberculosis+infection+and+its+reactivation.">Recent mouse models and vaccine candidates for preventing chronic/latent tuberculosis infection and its reactivation.</a> Pathogens and disease. 75 (6), (2017).</li><li>Gong, W., Liang, Y., Wu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+Models+of+Tuberculosis+Vaccine+Research:+An+Important+Component+in+the+Fight+against+Tuberculosis.">Animal Models of Tuberculosis Vaccine Research: An Important Component in the Fight against Tuberculosis.</a> BioMed Research International. 2020, 1-21 (2020).</li><li>Bettencourt, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+antigens+presented+by+MHC+for+vaccines+against+tuberculosis.">Identification of antigens presented by MHC for vaccines against tuberculosis.</a> NPJ vaccines. 5 (1), 2 (2020).</li><li>Moorlag, S. J. C. F. M., Arts, R. J. W., van Crevel, R., Netea, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-specific+effects+of+BCG+vaccine+on+viral+infections.">Non-specific effects of BCG vaccine on viral infections.</a> Clinical Microbiology and Infection. 25 (12), 1473-1478 (2019).</li><li>Wilkie, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+in-vitro+evaluation+of+the+non-specific+effects+of+BCG+vaccination+in+a+randomised+controlled+clinical+study.">Functional in-vitro evaluation of the non-specific effects of BCG vaccination in a randomised controlled clinical study.</a> Scientific Reports. 12 (1), 7808 (2022).</li><li>Netea, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trained+immunity:+A+program+of+innate+immune+memory+in+health+and+disease.">Trained immunity: A program of innate immune memory in health and disease.</a> Science. 352 (6284), aaf1098 (2016).</li><li>Bettencourt, P., Pires, D., Carmo, N., Anes, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+Confocal+Microscopy+for+Quantification+of+Intracellular+Mycobacteria+in+Macrophages.">Application of Confocal Microscopy for Quantification of Intracellular Mycobacteria in Macrophages.</a> Microscopy: Science, Technology, Applications and Education. 1, 614-621 (2010).</li><li>Bettencourt, P., Carmo, N., Pires, D., Tim&#243;teo, P., Anes, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycobacterial+infection+of+macrophages:+the+effect+of+the+multiplicity+of+infection.">Mycobacterial infection of macrophages: the effect of the multiplicity of infection.</a> Antimicrobial research: Novel bioknowledge and educational programs. , 651-664 (2017).</li><li>Pires, D., Bettencourt, P., Carmo, N., Niederweis, M., Anes, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Mycobacterium+tuberculosis+outer-membrane+porins+in+bacterial+survival+within+macrophages.">Role of Mycobacterium tuberculosis outer-membrane porins in bacterial survival within macrophages.</a> Drug Discovery Today. 15 (23-24), 1112-1113 (2010).</li><li>Pires, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycobacterium+tuberculosis+Modulates+miR-106b-5p+to+Control+Cathepsin+S+Expression+Resulting+in+Higher+Pathogen+Survival+and+Poor+T-Cell+Activation.">Mycobacterium tuberculosis Modulates miR-106b-5p to Control Cathepsin S Expression Resulting in Higher Pathogen Survival and Poor T-Cell Activation.</a> Frontiers in immunology. 8 (DEC), 1819 (2017).</li><li>Pires, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Cathepsins+in+Mycobacterium+tuberculosis+Survival+in+Human+Macrophages.">Role of Cathepsins in Mycobacterium tuberculosis Survival in Human Macrophages.</a> Scientific reports. 6 (August), 32247 (2016).</li><li>Bettencourt, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin-binding+protein+regulation+by+microRNAs+as+a+novel+microbial+strategy+to+modulate+phagocytosis+by+host+cells:+the+case+of+N-Wasp+and+miR-142-3p.">Actin-binding protein regulation by microRNAs as a novel microbial strategy to modulate phagocytosis by host cells: the case of N-Wasp and miR-142-3p.</a> Frontiers in cellular and infection microbiology. 3 (June), 19 (2013).</li><li>Bhuyan, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microliter+spotting+and+micro-colony+observation:+A+rapid+and+simple+approach+for+counting+bacterial+colony+forming+units.">Microliter spotting and micro-colony observation: A rapid and simple approach for counting bacterial colony forming units.</a> Journal of Microbiological Methods. 207, 106707 (2023).</li><li>Jackson, S., McShane, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+in+Developing+a+Controlled+Human+Tuberculosis+Challenge+Model.">Challenges in Developing a Controlled Human Tuberculosis Challenge Model.</a> Current topics in microbiology and immunology. , 1-27 (2022).</li><li>Darrah, P. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+tuberculosis+in+macaques+after+intravenous+BCG+immunization.">Prevention of tuberculosis in macaques after intravenous BCG immunization.</a> Nature. 577 (7788), 95-102 (2020).</li><li>Madura Larsen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BCG+stimulated+dendritic+cells+induce+an+interleukin-10+producing+T-cell+population+with+no+T++helper+1+or+T+helper+2+bias+in+vitro.">BCG stimulated dendritic cells induce an interleukin-10 producing T-cell population with no T helper 1 or T helper 2 bias in vitro.</a> Immunology. 121 (2), 276-282 (2007).</li><li>Bickett, T. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+the+BCG-Induced+Macrophage+and+Neutrophil+Mechanisms+for+Defense+Against+Mycobacterium+tuberculosis.">Characterizing the BCG-Induced Macrophage and Neutrophil Mechanisms for Defense Against Mycobacterium tuberculosis.</a> Frontiers in immunology. 11, 1202 (2020).</li><li>Pires, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interference+of+Mycobacterium+tuberculosis+with+the+endocytic+pathways+on+macrophages+and+dendritic+cells+from+healthy+donors:+role+of+cathepsins.">Interference of Mycobacterium tuberculosis with the endocytic pathways on macrophages and dendritic cells from healthy donors: role of cathepsins.</a> Drug Discovery Today. 15 (23-24), 1112-1112 (2010).</li><li>Betts, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimising+Immunogenicity+with+Viral+Vectors:+Mixing+MVA+and+HAdV-5+Expressing+the+Mycobacterial+Antigen+Ag85A+in+a+Single+Injection.">Optimising Immunogenicity with Viral Vectors: Mixing MVA and HAdV-5 Expressing the Mycobacterial Antigen Ag85A in a Single Injection.</a> PLoS ONE. 7 (12), e50447 (2012).</li><li>Tanner, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+haemoglobin+and+iron+on+in+vitro+mycobacterial+growth+inhibition+assays.">The influence of haemoglobin and iron on in vitro mycobacterial growth inhibition assays.</a> Scientific reports. 7 (1), 43478 (2017).</li><li>McNeill, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+mycobacterial+infection+by+macrophage+Gch1+and+tetrahydrobiopterin.">Regulation of mycobacterial infection by macrophage Gch1 and tetrahydrobiopterin.</a> Nature communications. 9 (1), 5409 (2018).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Pereira, M., Vale, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saquinavir:+From+HIV+to+COVID-19+and+Cancer+Treatment.">Saquinavir: From HIV to COVID-19 and Cancer Treatment.</a> Biomolecules. 12 (7), 944 (2022).</li><li>Pires, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repurposing+Saquinavir+for+Host-Directed+Therapy+to+Control+Mycobacterium+Tuberculosis+Infection.">Repurposing Saquinavir for Host-Directed Therapy to Control Mycobacterium Tuberculosis Infection.</a> Frontiers in immunology. 12, 647728 (2021).</li><li>Pires, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomal+Delivery+of+Saquinavir+to+Macrophages+Overcomes+Cathepsin+Blockade+by+Mycobacterium+tuberculosis+and+Helps+Control+the+Phagosomal+Replicative+Niches.">Liposomal Delivery of Saquinavir to Macrophages Overcomes Cathepsin Blockade by Mycobacterium tuberculosis and Helps Control the Phagosomal Replicative Niches.</a> International journal of molecular sciences. 24 (2), (2023).</li><li>Maartens, G., Wilkinson, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuberculosis.">Tuberculosis.</a> The Lancet. 370 (9604), 2030-2043 (2007).</li><li>Matarazzo, L., Bettencourt, P. J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mRNA+vaccines:+a+new+opportunity+for+malaria,+tuberculosis+and+HIV.">mRNA vaccines: a new opportunity for malaria, tuberculosis and HIV.</a> Frontiers in Immunology. 14, 1172691 (2023).</li><li>Young, D., Dye, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Development+and+Impact+of+Tuberculosis+Vaccines.">The Development and Impact of Tuberculosis Vaccines.</a> Cell. 124 (4), 683-687 (2006).</li><li>Kommareddi, S., Abramowsky, C. R., Swinehart, G. L., Hrabak, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nontuberculous+mycobacterial+infections:+Comparison+of+the+fluorescent+auramine-o+and+Ziehl-Neelsen+techniques+in+tissue+diagnosis.">Nontuberculous mycobacterial infections: Comparison of the fluorescent auramine-o and Ziehl-Neelsen techniques in tissue diagnosis.</a> Human Pathology. 15 (11), 1085-1089 (1984).</li><li>Sabiiti, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Tuberculosis+Molecular+Bacterial+Load+Assay+(TB-MBLA).">A Tuberculosis Molecular Bacterial Load Assay (TB-MBLA).</a> Journal of visualized experiments: JoVE. (158), e60460 (2020).</li><li>Somosko&#776;vi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Recoveries+of+Mycobacterium+tuberculosis+Using+the+Automated+BACTEC+MGIT+960+System,+the+BACTEC+460+TB+System,+and+Lo&#776;wenstein-Jensen+Medium.">Comparison of Recoveries of Mycobacterium tuberculosis Using the Automated BACTEC MGIT 960 System, the BACTEC 460 TB System, and Lo&#776;wenstein-Jensen Medium.</a> Journal of Clinical Microbiology. 38 (6), 2395-2397 (2000).</li><li>Tanner, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+in+vitro+direct+mycobacterial+growth+inhibition+assay+(MGIA)+for+the+early+evaluation+of+TB+vaccine+candidates+and+assessment+of+protective+immunity:+a+protocol+for+non-human+primate+cells.">The in vitro direct mycobacterial growth inhibition assay (MGIA) for the early evaluation of TB vaccine candidates and assessment of protective immunity: a protocol for non-human primate cells.</a> F1000Research. 10, 257 (2021).</li></ol>

DP and PJGB declare that the study was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

TB is an important public health problem with increasing importance, particularly in low and middle-income countries. The disruption of healthcare settings to diagnose and treat TB during the COVID-19 pandemic caused a negative impact on the incidence of new cases1. In addition, the multi-drug and extensively-drug resistant M.tb strains, and the co-infection of M.tb and HIV must be urgently addressed to control this epidemic1,3...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96-well plates</td>
			<td>VWR</td>
			<td>734-2781</td>
			<td></td>
		</tr>
		<tr>
			<td>DSLR 15-55 mm lens</td>
			<td>Nikon</td>
			<td>AF-P DX NIKKOR 18-55mm f/3.5-5.6G VR</td>
			<td></td>
		</tr>
		<tr>
			<td>DSLR camera</td>
			<td>Nikon</td>
			<td>D3400</td>
			<td></td>
		</tr>
		<tr>
			<td>DSLR macro lens</td>
			<td>Sigma</td>
			<td>MACRO 105mm F2.8 EX DG OS HSM</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal calf serum</td>
			<td>Gibco</td>
			<td>10270106</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji Software</td>
			<td></td>
			<td>https://fiji.sc/</td>
			<td>Fiji is an open-source software supported by several laboratories, institutions, and individuals. All the required plugins are included.</td>
		</tr>
		<tr>
			<td>Igepal CA-630</td>
			<td>Sigma-Aldrich</td>
			<td>18896</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Middlebrook 7H10</td>
			<td>BD</td>
			<td>262710</td>
			<td></td>
		</tr>
		<tr>
			<td>Middlebrook 7H9</td>
			<td>BD</td>
			<td>271310</td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel pipette (0.5 - 10 &#181;l)</td>
			<td>Gilson</td>
			<td>FA10013</td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel pipette (20 - 200 &#181;l)</td>
			<td>Gilson</td>
			<td>FA10011</td>
			<td></td>
		</tr>
		<tr>
			<td>Mycobacterium bovis BCG&#160;</td>
			<td>American Type Culture Collection</td>
			<td>ATCC35734</td>
			<td>strain TMC 1011 [BCG Pasteur]</td>
		</tr>
		<tr>
			<td>OADC enrichment</td>
			<td>BD</td>
			<td>211886</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>NZYTech</td>
			<td>MB25201</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 medium</td>
			<td>Gibco</td>
			<td>21875091</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Gibco</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer UV-6300PC</td>
			<td>VWR</td>
			<td>634-6041</td>
			<td></td>
		</tr>
		<tr>
			<td>Square Petri dish 120 x 120 mm</td>
			<td>Corning</td>
			<td>BP124-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Tyloxapol</td>
			<td>Sigma-Aldrich</td>
			<td>T8761</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound bath Elma P 30 H</td>
			<td>VWR</td>
			<td>142-0051</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 the 2019-2021 pandemic. The disease is caused by the bacterium Mycobacterium tuberculosis (M.tb). The Mycobacterium bovis strain Bacillus Calmette-Gu&#233;rin (BCG), the only TB vaccine, is the oldest licensed vaccine in the world, still in use. Currently, there are 12 vaccines in clinical trials and dozens of vaccines under pre-clinical development. The method of choice used to assess the efficacy of TB vaccines in pre-clinical studies is the enumeration of bacterial colonies by the colony-forming units (CFU) assay. This time-consuming assay takes 4 to 6 weeks to conclude, requires substantial laboratory and incubator space, has high reagent costs, and is prone to contamination. Here we describe an optimized method for colony enumeration, the micro-CFU (mCFU), that offers a simple and rapid solution to analyze M.tb vaccine efficacy results. 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. Moreover, to evaluate the efficacy of a TB vaccine, samples may be obtained from a variety of sources, including tissues from vaccinated animals infected with Mycobacteria. We also describe an optimized method to produce a unicellular, uniform, and high-quality mycobacterial culture for infection studies. Finally, we propose that these methods should be universally adopted for pre-clinical studies of vaccine efficacy determination, ultimately leading to time reduction in the development of vaccines against TB.

Author Spotlight: Optimizing CFU Determination for Efficient Assessment of TB Vaccine Efficacy and Antigen Presentation Analysis 

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

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.

The mCFU assay described here increases the amount of information that can be retrieved from a single Petri dish to at least 96-fold. Figure 5&#160;depicts a comparison of two drug-delivery methods for the repurposed use of saquinavir (SQV)31,32&#160;as a host-directed drug to treat tuberculosis. In this assay, four different strains of Mycobacterium tuberculosis were used to infect primary human macrophages. M. tubercul...

Watch this Scientific Journal Video about Optimizing CFU Determination for Efficient Assessment of TB Vaccine Efficacy and Antigen Presentation Analysis at JoVE.com

The determination of colony-forming units (CFU) is the gold-standard technique for quantifying bacteria, including Mycobacterium tuberculosis which can take weeks to form visible colonies. Here we describe a micro-CFU for CFU determination with increased time efficiency, reduced lab space and reagent cost, and scalability to medium and high throughput experiments.

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 ...

micro-colony-forming-unit-assay-for-efficacy-evaluation-vaccines

Research

JoVE Journal

Medicine

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

Begin by collecting the BCG culture at the mid or late log growth phase. Centrifuge it had 3000 G for 10 minutes, then discard the supernatant. Next, add 10 milliliters of PBS to wash the bacteria.After centrifugation, discard the supernatant. Resuspend the pellet in five milliliters of infection media. Place the tube in an ultrasound bath for 15 minutes at full power with a frequency of 80 hertz.Now centrifuge at 1, 000 G for 10 minutes. Collect the supernatant and discard the pellet. Measure the optical density of the supernatant.

Micro-Colony Forming Unit Assay to Quantify Mycobacteria

Micro-Colony Forming Unit Assay to Quantify Mycobacteria  

To perform serial dilution using a well plate, first place Mycobacterium suspensions on rows A and E of a 96-well plate. Add 180 microliters of deionized water into the remaining wells for the serial dilution process. Utilizing a 12-channel pipette, resuspend the suspensions in row A and transfer 20 microliters to row B.Repeat the suspension transfer for rows B and C until the last dilution is reached in row D.For micro droplet plating, use a 0.5 to 10 microliter multi-channel pipette with thin tips to transfer five microliters from each row of the 96-well plate to the solid medium square plate.Allow the droplets to slightly touch the agar, ensuring proper transfer and minimizing liquid retention inside the tip. Allow the droplets to dry and close the agar plate. Incubate it at 37 degrees Celsius while monitoring bacterial growth.Optionally, place the agar plates in a sealed plastic bag to prevent drying. After six to 10 days of incubation, check for individual colonies visible to the naked eye. Place the plate under an inverted microscope using the lowest magnification objective.Alternatively, use a camera to take a picture of the droplet, and manually count colonies on the computer using ImageJ for automated colony counting. The micro-colony forming unit or micro-CFU assay was used to produce high amounts of data from a small amount of agar plates. Using liposomes for saquinavir delivery increased the efficacy of the treatment against the microbacterium strains with different drug resistances.

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

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

1.4K Views.  Universidade Católica Portuguesa.  The determination of colony-forming units (CFU) is the gold-standard technique for quantifying bacteria, including Mycobacterium tuberculosis which can take weeks to form visible colonies. Here we describe a micro-CFU for CFU determination with increased time efficiency, reduced lab space and reagent cost, and scalability to medium and high throughput experiments.

Accelerating Tuberculosis Determination Using the Micro-CFU Method

Summary