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>
	<li>Prepare Middlebrook 7H9 broth supplemented with 10% (v/v) oleic acid, albumin, dextrose, and catalase (OADC) enrichment, according to the supplier&#39;s instructions. Supplement the broth with 0.05% (v/v) of tyloxapol. 
	NOTE: Tyloxapol is a non-ionic liquid polymer that has been used as a surfactant to prevent bacterial clump formation16.</li>
	<li>Prepare Middlebrook 7H10 solid medium supplemented with 10% (v/v) OADC enrichment according to the supplier&#39;s instructions.</li>
	<li>Distribute 40 mL of medium per square Petri dish (120 mm x 120 mm). Allow the plates to dry to minimize condensation at the surface of the agar. 
	NOTE: This specific size of petri dish is fundamental to allow direct transposal of at least 96 droplets from a 96-well plate. Effective drying of the plates will later facilitate the plating of small droplets of bacterial suspension and prevent the droplets from spreading.</li>
	<li>Prepare either Roswell Park Memorial Institute Medium (RPMI 1640) or Dulbecco&#39;s Modified Eagle Medium (DMEM) to produce the infection medium. In either case, supplement the medium with 10% fetal calf serum, 1% L-glutamine, and 1 mM sodium pyruvate. Do not add penicillin and streptomycin to the medium.</li>
</ol>
2. Sample preparation
<ol>
	<li>Obtain samples from a variety of sources. Typically, to quantify CFU to evaluate the efficacy of a TB vaccine, acquire samples from vaccinated and unvaccinated animal tissues. For example, mouse lung and spleen11&#160;or macaque lung, thoracic and peripheral lymph nodes, spleen, liver, skin, blood, bone marrow, and bronchoalveolar lavage wash23. Alternatively, obtain samples from in vitro cultures of macrophages/dendritic cells/neutrophils infected with BCG18,19,20, 24,25,26.</li>
</ol>
3. Production of BCG culture
NOTE: For in vivo studies of TB vaccines, the aim is to improve the efficacy of BCG. Therefore, BCG-vaccinated groups are usually used as control. BCG strains used for human vaccination are ideal for testing in animal models. In this case, a culture of BCG must be reconstituted according to the supplier&#39;s instructions27. However, a BCG culture for in vivo studies can also be produced in-house11. The production of unicellular, uniform, and high-quality BCG culture for in vitro infection protocols has been produced very successfully in several studies11, 16, 18,19,20, 26, 28, 29, using the following protocol, which can also be used for animal challenge studies.
<ol>
	<li>Culture 50 mL of BCG in 7H9 broth, at 37 &#176;C, with agitation at 200 rpm. Vary the volume according to the needs of the experiment.</li>
	<li>Every day, for 8-10 days, collect 100 &#181;L of the culture and dilute it by adding 900 &#181;L of PBS in a 1 mL cuvette. Then proceed by measuring the optical density of bacteria (OD at &#955;=600 nm; OD600) in a spectrophotometer. Draw a growth curve from those values. Identify the mid-log phase of the culture (when the OD is doubling consistently per unit of time).</li>
	<li>Prepare a subsequent culture and incubate until reaching the mid/late log growth phase as in steps 3.1 and 3.2. Use the values obtained in the previous step as guidance. Ensure that the culture does not reach the stationary growth phase (when the OD starts stabilizing) to maintain a good quality culture of viable bacteria.</li>
	<li>Collect the culture at the mid/late log growth phase. Centrifuge at 3000 x g for 10 min. Remove the supernatant.</li>
	<li>Add 10 mL of PBS to wash the bacteria. Centrifuge at 3000 x g for 10 min. Remove the supernatant.</li>
	<li>Resuspend the bacteria with 5 mL of infection media. Place the tube in an ultrasound bath for 15 min, full power at 80 Hz.</li>
	<li>Centrifuge at 1000 x g for 10 min. Collect the supernatant avoiding the pellet as it is rich in bacterial clumps that should be avoided in a high-quality BCG culture and discard it.</li>
	<li>Measure the OD of the supernatant. Here, cultures at the exponential growth phase, with an OD600 of 0.1, are equivalent to 1 x 107 CFU/mL. 
	NOTE: Each laboratory should produce its own BCG growth curves before starting experiments to establish a linear regression between OD600&#160;and CFU using the spectrophotometer. Please note that spectrophotometers have different light path distances, which can vary the readings obtained for the same sample.</li>
	<li>Carry out simple calculations to establish the number of bacteria to add to each host cell culture. The number of bacteria per host cell is the Multiplicity of Infection (MOI). Use an MOI of 10 bacteria per host cell, which is the most common MOI used for BCG infection experiments.</li>
</ol>
4. Micro-colony forming unit assay
NOTE: After an in vivo or in vitro infection experiment is completed, the enumeration of bacteria can be performed by mCFU. For in vivo studies, samples must be first homogenized in a bead beater or another tissue homogenizer. For in vitro cultures of macrophages/dendritic cells/neutrophils infected with BCG, samples must be lysed using a non-ionic detergent (e.g., 0.05% solution of non-ionic, non-denaturing detergent).
<ol>
	<li>Serial dilutions using a 96-well plate: perform serial 10-fold dilutions of the lysates, in a sterile 96-well plate according to the scheme in Figure 1A. Distribute the lysates on rows A and E. For each plate, the maximum number of samples and/or replicates is 24.</li>
	<li>Add 180 &#181;L of dH2O to the remaining wells to perform the serial dilution.</li>
	<li>Using a 12-channel pipette, resuspend the lysates in row A and transfer 20 &#181;L to row B (20 &#181;L lysate + 180 &#181;L dH2O). Homogenize well. Sequentially repeat this step for rows B and C until reaching the last dilution in row D. 
	NOTE: We usually perform three dilutions (100, 101, 102, 103), thus using 4 rows of the plate (A-D or E-H) for each set of 12 samples and/or replicates.</li>
	<li>Micro droplet plating: use a 0.5-10 &#181;L (thin tips are preferred) multichannel pipette to transfer 5 &#181;L from each row of the 96-well plate to the solid medium square plate, according to Figure 1B.</li>
	<li>While slowly pipetting the 5 &#181;L droplets, allow them to slightly touch the agar. This will help to take off the droplet from the tip towards the agar and reduce the possibility of retention of the liquid inside the tip.</li>
	<li>Allow the droplets to dry, close the agar plate, and incubate it at 37 &#176;C while monitoring bacterial growth. Optionally, incubate the agar plates in a sealed plastic bag to prevent the plates from drying.</li>
	<li>Micro-colony counting: following approximately 6-10 days of incubation, check for individual colonies, visible to the naked eye (Figure 2).</li>
	<li>Count the colonies using the lowest magnification objective (4x or lower) of an inverted optical microscope or magnifying glass. Counts should be performed in the dilutions where the number of colonies is lower than 300 and higher than 30. Alternatively, use a camera to take a picture of the droplet to manually count colonies on the computer or use software such as ImageJ to automate colony counting.</li>
	<li>To express cell numbers in CFU/mL, use the following equation: 
	<img alt="Equation 1" src="/files/ftp_upload/65447/65447eq01.jpg" style="margin: auto;" /> 
	Where C = number of colonies counted, V = volume plated in &#181;L, and Dil = dilution where the colonies were counted (100, 101, 102, 103). For example, if 30 colonies were counted in a 5 &#181;L droplet in dilution 102, then: 
	<img alt="Equation 2" src="/files/ftp_upload/65447/65447eq02.jpg" style="margin: auto;" /></li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65447/65447fig01.jpg" /> 
Figure 1.&#160;Schematic representation of the mCFU protocol. (A) Serial 10-fold dilutions of the BCG-containing lysates in a 96-well plate. (B) Square Petri dish containing solid culture medium and overlayed by 96 droplets of 5 &#181;L each. Droplets are pipetted directly from the 96-well plate using a multichannel pipette. Created with BioRender.com. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65447/65447fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65447/65447fig02.jpg" /> 
Figure 2.&#160;Micro-colony forming units of BCG following 10 days of incubation. On the left, a photo of a square Petri dish overlayed by 96 droplets of 5 &#181;L each, as previously represented in Figure 1B. On the right, individual photos of 3 droplets correspond to an original lysate (100) and two dilutions (101, 102). Photos were taken using a DSRL camera equipped with an 18-55 mm zoom lens (plate) or a 105 mm macro lens (droplets). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65447/65447fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
5. Micro-colony forming unit counting in Fiji (ImageJ)
NOTE: The mCFU method allows CFU quantification of large sets of samples. Pictures of the droplets may be recorded for posterior analysis to facilitate colony counting. Several photographic devices can produce images with sufficient quality for this purpose. These include digital cameras, webcams, camera-attached microscopes and magnifying glasses, and cell phones. Free image analysis software such as ImageJ offers the possibility of manual or automated colony counting in those images. To demonstrate both methods, Fiji will be used, which is a distribution of ImageJ that packages several tools for scientific image analysis30. Fiji can be downloaded from&#160;https://fiji.sc/.
<ol>
	<li>Manual counting method
	<ol>
		<li>Open the image containing the mCFU in Fiji. Select Plugins &#62; Analyze &#62; Cell Counter.</li>
		<li>On the Cell Counter menu, select Initialize and then select a counter (e.g., Type 1).</li>
		<li>Proceed by clicking on each colony. Each click will be shown on the picture and will update the counter (Figure 3). To undo accidental clicks, select Delete.</li>
		<li>Register the value displayed on the counter. Click the Reset button to reset the count and open a new image to count additional samples. 
		NOTE: Further instructions on this plugin can be found at&#160;https://imagej.net/plugins/cell-counter.</li>
	</ol>
	</li>
	<li>Automated counting method
	<ol>
		<li>Open the image containing the mCFU in Fiji. Select Image &#62; Type &#62; 8 -bit. This will convert the image to an 8-bit gray-scale image.</li>
		<li>Select the Oval tool in the tool bar and draw an oval around the area with the colonies (Figure 4A). The oval may be adjusted after being drawn.</li>
		<li>Select Edit &#62; Clear Outside, to remove any interference from the outside area (Figure 4B). Select Image &#62; Adjust &#62; Threshold.</li>
		<li>Move the sliders in the threshold menu until the colonies appear in red and background noise is minimized (Figure 4C).</li>
		<li>Select Apply and exit the threshold window. A black-and-white image is generated (Figure 4D).</li>
		<li>Select Analyze &#62; Analyze Particles. In the analyze particles window, specify the range for colony area (between 1 and infinity, measured in squared pixels) and circularity (between 0 and 1, where 1 is a perfect circle; Figure 4E).</li>
		<li>Select Outlines in the show popup menu. Check Display Results for detailed measurements for each colony in the results window. Check Clear Results to erase any previous measurements. Check the Summarize box to display the summarized results of the measurements (Figure 4E).</li>
		<li>Initiate the analyzer by selecting OK. A new window appears, displaying all the outlined colonies that were detected and counted. The results window displays the details for each colony, and the summarized results window shows the total colonies counted (Figure 4F). 
		&#8203;NOTE: The settings for size and circularity will vary with the image&#39;s resolution and magnification and the colonies&#39; size and shape. Repeat the process several times until the best settings are found that detect all colonies. Further instructions on the analyze particles plugin can be found at&#160;https://imagej.nih.gov/ij/docs/menus/analyze.html#ap.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65447/65447fig03.jpg" /> 
Figure 3.&#160;A manual method for counting mCFU using the cell counter plugin on Fiji software. The blue dots indicate colonies already clicked on by the user. The menu on the right displays the number of colonies counted so far (the count is 41). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65447/65447fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65447/65447fig04.jpg" /> 
Figure 4.&#160;An automated method for counting mCFU using Fiji software. (A, B) The region of interest with the colonies is selected using the oval selection tool, and the outside area is removed using the clear outside command. (C, D) A black-and-white image of the colonies is generated using the threshold tool. (E, F) The number of colonies is quantified using the analyze particles tool. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65447/65447fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Accelerating Tuberculosis Determination Using the Micro-CFU Method

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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 epidemic1,34. New vaccines to replace or improve BCG are currently in clinical trials, and new candidates could hit the market in the coming years35. The development of new vaccine candidates against TB relies on pre-clinical studies using diverse animal models. The vaccine efficacy determination in animal models requires bacterial burden quantification in the infected organs to demonstrate the protective effect of the vaccine5,36. Currently, there are direct and indirect methods to quantify the bacterial burden in infected mice and non-human primates. The direct methods include histological preparations and bacterial staining using fluorescent auramine-o and Ziehl-Neelsen37, the quantification of mycobacterial load in samples based on RT-qPCR38, and others.
The mycobacterial growth inhibition assay (MGIA) is an in vitro assay based on the BACTEC MGIT system and is a sensitive method used to quantify live mycobacterial burden in vaccinated and unvaccinated animals. The system is a fully automated instrument that can test for the presence of Mycobacteria in specifically designed test tubes. The vial contains a fluorescent reporter that reacts to the concentration of oxygen in the sample. The fluorescence is proportional to the amount of oxygen consumed, giving an indication of the number of live bacteria present in the sample, every hour39. In the MGIA method, PBMCs and autologous serum samples are collected from animals and co-cultured with mycobacteria for 96 h. Then, adherent monocytes are lysed to release intracellular mycobacteria. The system will quantify the bacterial burden indirectly by measuring oxygen consumption40. This method is typically used for drug resistance studies but has been applied recently to the TB vaccine efficacy evaluation40.
These methods can be complemented with the standard CFU to determine the total number of viable mycobacteria. For example, the evaluation of vaccine candidates in a murine aerosol M.tb challenge experiment is achieved by enumerating colonies obtained from tissues by CFU11.
The mCFU method described here can be a substantial improvement over the classic CFU method, as it is more rapid to perform and inexpensive in terms of reagents used and in terms of laboratory space required. This method allows the simultaneous comparison of a large set of samples in a single experiment, thus decreasing the effects of assay-to-assay variations. For example, as shown in the experiment described in Figure 5, the CFU was determined for 1536 samples using the mCFU method in 16 square Petri dishes, which represents a 96-fold reduction in the number of plates used. This is more relevant for experiments using primary biological and clinical samples, where availability may be an issue. The compact form in which the colonies are displayed on an agar plate conveniently allows the use of specialized or common image-recording equipment. This, in combination with automated colony counting methods such as the one described here using the Fiji software, helps reduce the reliance on the user&#39;s ability to identify and quantify the CFU. Even so, like the standard CFU technique, this method suffers from the inability to distinguish merged colonies leading to a potential under-representation of the CFU. However, this can be minimized by selecting a higher dilution to perform the quantification or by counting the colonies under the microscope before their overgrowth results in colony merger. This is more relevant if the sample produces colonies with heterogeneous growth rates. Researchers should validate mCFU implementation for their specific experimental conditions in those cases.
The critical steps within the protocol are the following points: the serial dilutions made should be performed using a 96-well plate, and the dilutions must be thoroughly mixed; the microdroplet plating should be performed with a 0.5-10 &#181;L multichannel pipette to transfer 5 &#181;L from each row of the 96-well plate to the solid medium square plate, without touching and damaging the medium; the micro-colony counting must be performed between 6-10 days of incubation. However, at the time of counting, care must be taken to ensure the colonies are not excessively large or too small.
The modifications and troubleshooting of the technique include the utilization of different bacterial strains. Therefore, for each strain, the optimal growth medium should be used. The limitations of the technique are identical to the limitations of the conventional CFU method, which include the difficulty of enumerating bacteria in low dilutions. Additionally, specific limitations of this method include the necessity of using a microscope to enumerate the colonies.
Finally, this method can be used to quantify the bacterial burden in TB vaccination studies and for test drug resistance/susceptibility studies and host-pathogen interaction studies. For example, Figure 5 shows the application of mCFU for the determination of intracellular killing of M.tb strains, including clinical strains, multi-drug resistant and extensively-drug resistant strains treated with different drug formulations, in human macrophages. Importantly, this method can also be applied to any microorganism, provided that the culturing conditions for each organism are met.

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><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 &amp;micro;l)</td><td>Gilson</td><td>FA10013</td><td></td></tr><tr><td>Multichannel pipette (20 - 200 &amp;micro;l)</td><td>Gilson</td><td>FA10011</td><td></td></tr><tr><td>&lt;em&gt;Mycobacterium bovis&lt;/em&gt; BCG&amp;nbsp;</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.

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. tuberculosis H37Rv laboratory strain and three clinical strains isolated from patients with active TB by the Portuguese National Institute of Health&#39;s Dr. Ricardo Jorge (INSA): a drug-susceptible strain (INSA code 33427), a multiple drug-resistant (MDR) strain (INSA code 34192), and an extensively drug-resistant (XDR) strain (INSA code 163761). Each infection by each strain was further multiplied into eight different treatment conditions comparing three different concentrations of free-drug or liposome-loaded drug to the respective non-treated controls. Finally, each condition was analyzed at four different time points post-infection. To quantify intracellular bacterial growth, the total amount of lysates extracted from each time point was 32. This was followed by three serial dilutions to each lysate increasing the number to 128 samples. Multiplied by the four-time points analyzed results in 512 samples. Since the experiment was repeated using macrophages from at least three different blood donors, the total number of analyzed samples increased to 1536. Using the mCFU method described here, this experiment accounted for only 16 square Petri dishes versus 1536 that would be necessary using the standard CFU protocol. As shown in Figure 5, the results obtained with this method can demonstrate statistically significant differences between treatments.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65447/65447fig05.jpg" /> 
Figure 5. The mCFU method can produce high amounts of data from a small number of agar plates. This set of experiments tested the efficacy of the drug saquinavir (SQV) to induce intracellular killing of M.tb laboratory and clinical strains in human macrophages. SQV was administered to the macrophage cultures in its free form or loaded in liposomes (LipSQV). The treatments were performed in three different concentrations: 50, 20, and 10 &#181;g/mL. Macrophages were infected with different M.tb strains for 3 h and then treated with selected concentrations of LipSQV and free SQV. Liposomes without the drug (LipUnloaded) and DMSO were used as controls. To evaluate bacterial intracellular survival, at discrete time points, macrophages were lysed, and serial dilutions of the bacterial suspension were plated on 7H10 agar plates. mCFU units were counted following 2-3 weeks. Lines depict the average mCFU per sample from at least 3 independent experiments. Bars represent the average mCFU percentage calculated relative to the respective controls at day 7 post-infection. Symbols represent each experiment with macrophages from a different donor. Error bars represent the standard error of the mean. Multiple group comparisons were performed using one-way ANOVA followed by a Holm-Sidak post-hoc test. * p &#8804;0.05, ** p &#8804;0.01, *** p &#8804;0.001. This figure has been modified from33. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65447/65447fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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

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.

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Medicine

2.0K 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.

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

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb (BCG lux/M.Tb lux)

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal titers. Studies of serum bactericidal titers in response to childhood vaccines have enabled us to develop and validate cut-off levels for protective immune responses and such cut-offs are in routine use. No such assays have been taken forward into the routine assessment of vaccines that induce primarily cell-mediated immunity in the form of effector T cell responses, such as TB vaccines. In the animal model, the performance of a given vaccine candidate is routinely evaluated in standardized bactericidal assays, and all current novel TB-vaccine candidates have been subjected to this step in their evaluation prior to phase 1 human trials. The assessment of immunogenicity and therefore likelihood of protective efficacy of novel anti-TB vaccines should ideally undergo a similar step-wise evaluation in the human models now, including measurements in bactericidal assays.
Bactericidal assays in the context of tuberculosis vaccine research are already well established in the animal models, where they are applied to screen potentially promising vaccine candidates. Reduction of bacterial load in various organs functions as the main read-out of immunogenicity. However, no such assays have been incorporated into clinical trials for novel anti-TB vaccines to date.
Although there is still uncertainty about the exact mechanisms that lead to killing of mycobacteria inside human macrophages, the interaction of macrophages and T cells with mycobacteria is clearly required. The assay described in this paper represents a novel generation of bactericidal assays that enables studies of such key cellular components with all other cellular and humoral factors present in whole blood without making assumptions about their relative individual contribution. The assay described by our group uses small volumes of whole blood and has already been employed in studies of adults and children in TB-endemic settings. We have shown immunogenicity of the BCG vaccine, increased growth of mycobacteria in HIV-positive patients, as well as the effect of anti-retroviral therapy and Vitamin D on mycobacterial survival in vitro. Here we summarise the methodology, and present our reproducibility data using this relatively simple, low-cost and field-friendly model.
Note: Definitions/Abbreviations
BCG lux = M. bovis BCG, Montreal strain, transformed with shuttle plasmid pSMT1 carrying the luxAB genes from Vibrio harveyi, under the control of the mycobacterial GroEL (hsp60) promoter. 
CFU = Colony Forming Unit (a measure of mycobacterial viability).

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux

We describe an alternative approach to the enumeration of mycobacteria in vitro, which uses reporter-gene tagged mycobacteria instead of colony-forming units (CFU). &#x201C;Survival&#x201D; of organisms as well as host response-markers are measured simultaneously, providing a low-cost, versatile and functional system for studies of host/pathogen interactions in the context of tuberculosis.

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal ...

Immunology and Infection

Rapid Point-of-Care Assay of Enoxaparin Anticoagulant Efficacy in Whole Blood

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the low-molecular-weight-heparin (LMWH), enoxaparin. There are also urgent clinical situations where it would be important if this could be determined rapidly. The present assay is designed to accomplish this. We only assayed human blood samples that were spiked with known concentrations of enoxaparin. The essential feature of the present assay is the quantification of the efficacy of enoxaparin in a patient's blood sample by degrading it to complete inactivity with heparinase. Two blood samples were drawn into Vacutainer tubes (Becton-Dickenson; Franklin Lakes, NJ) that were spiked with enoxaparin; one sample was digested with heparinase for 5 min at 37 &deg;C, the other sample represented the patient's baseline anticoagulated status. The percent shortening of clotting time in the heparinase-treated sample, as compared to the baseline state, yielded the anticoagulant contribution of enoxaparin. We used the portable, battery operated Hemochron 801 apparatus for measurements of clotting times (International Technidyne Corp., Edison, NJ). The apparatus has 2 thermostatically controlled (37 &deg;C) assay tube wells. We conducted the assays in two types of assay cartridges that are available from the manufacturer of the instrument. One cartridge was modified to increase its sensitivity. We removed the kaolin from the FTK-ACT cartridge by extensive rinsing with distilled water, leaving only the glass surface of the tube, and perhaps the detection magnet, as activators. We called this our minimally activated assay (MAA). The use of a minimally activated assay has been studied by us and others. 2-4 The second cartridge that was studied was an activated partial thromboplastin time (aPTT) assay (A104). This was used as supplied from the manufacturer. The thermostated wells of the instrument were used for both the heparinase digestion and coagulation assays. The assay can be completed within 10 min. The MAA assay showed robust changes in clotting time after heparinase digestion of enoxaparin over a typical clinical concentration range. At 0.2 anti-Xa I.U. of enoxaparin per ml of blood sample, heparinase digestion caused an average decrease of 9.8% (20.4 sec) in clotting time; at 1.0 I.U. per ml of enoxaparin there was a 41.4% decrease (148.8 sec). This report only presents the experimental application of the assay; its value in a clinical setting must still be established.

Demonstration of a rapid quantitative assay of the inhibition of blood coagulation by the low-molecular-weight-heparin, enoxaparin. The contribution of enoxaparin is assessed by removing its influence through digestion with heparinase. A fuller description of the assay is detailed in our published paper.1 The assay still requires clinical confirmation.

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the ...

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set&#160;up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host&#160;cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb&#160;into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read&#160;out inferring on the pathogenesis of Mtb&#160;within host cells.

Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host&#160;cell using automated confocal microscopy.

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic Therapy is a novel approach for a more tumor selective treatment.
Photodynamic Therapy (PDT) that makes use of a nontoxic photosensitizer (PS), which, upon activation with light of a specific wavelength in the presence of oxygen, generates oxygen radicals that elicit a cytotoxic response1. Despite its approval almost twenty years ago by the FDA, PDT is nowadays only used to treat a limited number of cancer types (skin, bladder) and nononcological diseases (psoriasis, actinic keratosis)2.
The major advantage of the use of PDT is the ability to perform a local treatment, which prevents systemic side effects. Moreover, it allows the treatment of tumors at delicate sites (e.g. around nerves or blood vessels). Here, an intraoperative application of PDT is considered in osteosarcoma (OS), a tumor of the bone, to target primary tumor satellites left behind in tumor surrounding tissue after surgical tumor resection. The treatment aims at decreasing the number of recurrences and at reducing the risk for (postoperative) metastasis.
In the present study, we present in vitro PDT procedures to establish the optimal PDT settings for effective treatment of widely used OS cell lines that are used to reproduce the human disease in well established intratibial OS mouse models. The uptake of the PS mTHPC was examined with a spectrophotometer and phototoxicity was provoked with laser light excitation of mTHPC at 652 nm to induce cell death assessed with a WST-1 assay and by the counting of surviving cells. The established techniques enable us to define the optimal PDT settings for future studies in animal models. They are an easy and quick tool for the evaluation of the efficacy of PDT in vitro before an application in vivo.

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

The protocol reported here allows the evaluation of the efficacy of photodynamic therapy (PDT) in cell lines and the optimization of the PDT settings before applying the therapy in animal models.

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic ...

Visualization of the Charcoal Agar Resazurin Assay for Semi-quantitative, Medium-throughput Enumeration of Mycobacteria

There is an urgent need to discover and progress anti-infectives that shorten the duration of tuberculosis (TB) treatment. Mycobacterium tuberculosis, the etiological agent of TB, is refractory to rapid and lasting chemotherapy due to the presence of bacilli exhibiting phenotypic drug resistance. The charcoal agar resazurin assay (CARA) was developed as a tool to characterize active molecules discovered by high-throughput screening campaigns against replicating and non-replicating M. tuberculosis. Inclusion of activated charcoal in bacteriologic agar medium helps mitigate the impact of compound carry-over, and eliminates the requirement to pre-dilute cells prior to spotting on CARA microplates. After a 7-10 day incubation period at 37 &#176;C, the reduction of resazurin by mycobacterial microcolonies growing on the surface of CARA microplate wells permits semi-quantitative assessment of bacterial numbers via fluorometry. The CARA detects approximately a 2-3 log10 difference in bacterial numbers and predicts a minimal bactericidal concentration leading to &#8805;99% bacterial kill (MBC&#8805;99). The CARA helps determine whether a molecule is active on bacilli that are replicating, non-replicating, or both. Pilot experiments using the CARA facilitate the identification of which concentration of test agent and time of compound exposure require further evaluation by colony forming unit (CFU) assays. In addition, the CARA can predict if replicating actives are bactericidal or bacteriostatic.

The charcoal agar resazurin assay (CARA) is a semi-quantitative, medium-throughput method to assess activity of test agents against mycobacteria that are replicating, non-replicating, or both. The CARA permits rapid evaluation of time- and concentration-dependent activity and identifies parameters to pursue by colony forming unit (CFU) assays.

There is an urgent need to discover and progress anti-infectives that shorten the duration of tuberculosis (TB) treatment. Mycobacterium tuberculosis, the ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

An Automated Culture System for Use in Preclinical Testing of Host-Directed Therapies for Tuberculosis

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), was the most significant infectious disease killer globally until the advent of COVID-19. Mtb has evolved to persist in its intracellular environment, evade host defenses, and has developed resistance to many anti-tubercular drugs. One approach to solving resistance is identifying existing&#160;approved drugs that will boost the host immune response to Mtb. These drugs could then be repurposed as adjunctive host-directed therapies (HDT) to shorten treatment time and help overcome antibiotic resistance.
Quantification of intracellular Mtb growth in macrophages is a crucial aspect of assessing potential HDT. The gold standard for measuring Mtb growth is counting colony-forming units (CFU) on agar plates. This is a slow, labor-intensive assay that does not lend itself to rapid screening of drugs. In this protocol, an automated, broth-based culture system, which is more commonly used to detect Mtb in clinical specimens, has been adapted for preclinical screening of host-directed therapies. The capacity of the liquid culture assay system to investigate intracellular Mtb growth in macrophages treated with HDT was evaluated. The HDTs tested for their ability to inhibit Mtb growth were all-trans Retinoic acid (AtRA), both in solution and encapsulated in poly(lactic-co-glycolic acid) (PLGA) microparticles and the combination of interferon-gamma and linezolid. The advantages of this automated liquid culture-based technique over the CFU method include simplicity of setup, less labor-intensive preparation, and faster time to results (5-12 days compared to 21 days or more for agar plates).

Rapid and efficient quantification of intracellular M. tuberculosis growth is crucial for pursuing improved therapies against tuberculosis (TB). This protocol describes a broth-based colorimetric detection assay using an automated liquid culture system to quantify Mtb growth in macrophages treated with candidate host-directed therapies.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), was the most significant infectious disease killer globally until the advent ...

Quantification of Bacterial Infection in Host Cells by the Colony-Forming Unit Assay

To harvest, slightly tilt the dish and carefully use a pipette to remove the entire media from the well. Add 0.5 milliliters of distilled sterile water to the well. After 30 seconds, transfer the resulting water lysate to a 1.5-milliliter microcentrifuge tube and vortex vigorously for 10 seconds.
Add 4.5 microliters and 50 microliters of the water lysate to 450 microliters of distilled sterile water to prepare the 10-fold and 100-fold dilutions. Briefly, vortex to ensure thorough mixing. Add 50 microliters of the original 10-fold diluted and 100-fold diluted samples onto lysogeny broth agar plates and spread using a plate spreader.
To assay for emergent Lm, extract 10 microliters of the overlaying media from the dish, and divide it into two 5-microliter aliquots in microcentrifuge tubes. Add 5 microliters of DMEM/FCS to one aliquot and add 5 microliters of DMEM/FCS containing 5 microliters per milliliter gentamicin to the second aliquot as control.
Wait 5 minutes at room temperature. Add 90 microliters of distilled sterile water to each aliquot and vortex the tube vigorously for 10 seconds. Then, add 50 microliters of the sample on LB agar plates, and spread using a plate spreader. Store the plates in 37-degrees-Celsius incubator for two days before enumerating Lm colonies.

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An Assay to Evaluate Drug Efficacy against a Mycobacterium tuberculosis Infection Model

For drug testing, prepare two anti-tuberculosis drugs in triplicate on a new 96-well plate as follows. First, add 125 microliters of 7H9-C medium to wells B2 through G10. Next, prepare the two drugs at double the highest desired final concentration in 1 milliliter of 7H9-C.
Using a pipette, add 250 microliters of each drug to wells B, C, and D-11, and then E, F, and G-11 respectively for triplicate treatments. Next, using a multichannel pipette, serially dilute the test drugs two-fold by moving 125 microliters from B11 through G11 into B10 through G10. Mix by pipetting five times at each step.
Continue to move 125 microliters from column to column, right to left across the plate, and stop after column 4. After mixing column 4, discard 125 microliters into a waste container. Columns 2 and 3 should not contain any drugs. This will allow them to be used as a background and for positive growth controls.
Next, retrieve the 96-well plate containing the Mtb-infected macrophages from the incubator. Without tilting the plate, use a multichannel pipette to remove 150 microliters of medium from wells B2 through G11. Then, tilt the plate as shown here, and insert the pipette tips into the bottom edge of the wells, and remove the remaining medium, about 50 microliters.
As Mtb macrophage aggregates adhere to the bottom of the well, no material should be lost. However, removal of the medium should be as gentle as possible and care must be taken to avoid resuspension during this process.
Gently, add 100 microliters of 7H9-C medium to wells B2 through G11 of the plate, which contain the Mtb macrophage aggregates. Using a multichannel pipette, transfer 100 microliters of the drug-containing 96-well plate to the corresponding wells of the infection plate. Then, place the plate in a sealed bag and incubate it for three days at 37 degrees Celsius.
The resazurin assay relies on the oxidative species produced by metabolically active Mtb to convert the blue resazurin to fluorescent pink resorufin. The change in color and fluorescence can be used as a surrogate marker to determine the amount of bacterial growth.
Prepare a stock solution of resazurin at a final concentration of 0.8 milligrams per milliliter in water. Filter through a 0.22-micrometer pore-size PVDF membrane for sterilization. Prepare a working solution in resazurin by mixing the stock solution, water, and Tween-80 in a 2 to 1 to 1 ratio. Final concentrations are 0.4 milligrams per milliliter resazurin in 5% Tween-80.
Using a plate reader, set up a program to read the fluorescence at 530-nanometer excitation and 590-nanometer emission for 24 hours every 30 minutes at 37 degrees Celsius. Pre-warm the plate reader to 37 degrees Celsius.
Using a multichannel pipette, add 20 microliters of resazurin working solution to wells B2 through G11 of the drug-treated plate. Place the plate on the reader and start the program.

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

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Chapters in this video

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb (BCG lux/M.Tb lux)

Rapid Point-of-Care Assay of Enoxaparin Anticoagulant Efficacy in Whole Blood

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

Visualization of the Charcoal Agar Resazurin Assay for Semi-quantitative, Medium-throughput Enumeration of Mycobacteria

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

An Automated Culture System for Use in Preclinical Testing of Host-Directed Therapies for Tuberculosis

Quantification of Bacterial Infection in Host Cells by the Colony-Forming Unit Assay

An Assay to Evaluate Drug Efficacy against a Mycobacterium tuberculosis Infection Model