Name: Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds (Video) | JoVE Journal
Uploaded: 2018-02-14T15:00:00.000+00:00
Duration: 8 min 54 s
Description: Learn broth microdilution screening for antifungal drug development using Candida and Cryptococcus strains. This rapid method enables efficient evaluation of natural and synthetic compounds with automation.

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5 minutes

We thank CAPES-Brazil, CNPq-Brazil, FAP/DF for financial support. We are grateful to Dr. Hugo Costa Paes for revising the manuscript.

1. Solutions and Media
<ol>
	<li>Prepare 2X Roswell Park Memorial Institute (RPMI) 1640 medium, phosphate buffered saline (PBS), Sabouraud dextrose broth, and Sabouraud dextrose agar as per Table 1.</li>
</ol>
2. Fungal Inoculum Growth Conditions
<ol>
	<li>Store all fungal strains as frozen stocks in 35% glycerol at -80 &#176;C, until needed.</li>
	<li>Perform the following steps before each experiment.
	<ol>
		<li>For Candida albicans strains:
		<ol>
			<li>Thaw a stock vial and transfer 200 &#181;L to 10 mL of Sabouraud dextrose broth in a sterile 50 mL polypropylene tube with a cap and culture overnight at 30 &#176;C with agitation (200 rpm). Remember to incline the tube and leave the cap slightly open for better aeration of the culture.</li>
		</ol>
		</li>
		<li>For Cryptococcus neoformans strains:
		<ol>
			<li>Scrape the frozen surface of a stock vial. Plate the cells onto Sabouraud dextrose agar plates and incubate for 48 h at 30 &#176;C. After visible growth of isolated colonies, keep the plates in the refrigerator (4 &#176;C) sealed with paraffin film for up to 15 days.</li>
			<li>Collect a medium-sized isolated colony from the plate using a sterile toothpick or sterile inoculating loop and inoculate 10 mL of Sabouraud dextrose broth in a sterile 50 mL conical tube. Incubate for approximately 24 h at 30 &#176;C under agitation (200 rpm).</li>
			<li>Do not exceed the 24 h incubation. Remember to incline the tube and leave the cap slightly open for better aeration. It is always good to standardize the growth stage of the cells before each test, since this is an important factor affecting antimicrobial resistance. 
			NOTE: Both fungi were grown at 30 &#176;C for rapid propagation in our experiments, but the temperature can be changed to reflect the aims of the study, for example clinical treatment (37 &#176;C). Most importantly, this initial incubation time should be established beforehand for each fungal isolate and maintained throughout all the tests to ensure reproducibility.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>After fungal growth, collect the cells by centrifuging the conical tubes at 1,200 x g for 5 min at room temperature. Discard the supernatant and add 10 mL of PBS.</li>
	<li>Resuspend the cells and centrifuge again at 1,200 x g for 5 min at room temperature.</li>
	<li>Repeat the PBS wash and centrifugation twice more. After the third wash, resuspend the cells in 5 mL (according to the pellet size) of 2X RPMI-1640 medium.</li>
	<li>Prepare 1 mL of a 1:100 or 1:1,000 dilution in a microcentrifuge tube (depending on the turbidity of the cell suspension).</li>
	<li>Aliquot 10 &#181;L of this dilution, place it in a hemocytometer chamber and count the total number of cells in the four corner quadrants under the microscope. Calculate the concentration by the formula: (total cell number/4) x dilution factor x 104 (chamber dilution constant).
	<ol>
		<li>Consider a 100% viability if viability counts and growth conditions have been systematically correlated for the isolate being studied. 
		NOTE: The standardization and quality control of viability counts can be done by back-plating in accordance to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) antifungal Minimum Inhibitory Concentration (MIC) method for yeasts10.</li>
		<li>If the growth/viability correlation has not been established for the isolate under study, measure the fungal viability by counting live cells in a hemocytometer with the aid of dyes that selectively color dead cells, such as phloxine B11, trypan blue12, or Janus Green13. Use the fungal cells for this protocol only if the viability of the population at this point is 90% or above. 
		NOTE: Dead/alive dyes may not work well with the fungus of choice. Please test them before use. For example, the trypan blue dye does not work well with C. neoformans.</li>
	</ol>
	</li>
	<li>After that, prepare the cell suspensions in 2X RPMI-1640 medium (2X adjusted inoculum in RPMI-1640 medium). For 96-well plates consider a volume of 5 mL for each plate.
	<ol>
		<li>For all C. albicans strains, prepare a stock cell suspension of 4 x 103 cells/mL. This concentration is 2X of the final cell concentration in each well (2 x 103 cells/mL).</li>
		<li>For C. neoformans strains, prepare a stock culture of cells 2 x 104 cells/mL. This concentration is twice the final cell concentration in each well (1 x 104 cells/mL).</li>
		<li>For other fungi, test with a known antifungal which concentration will be ideal for the particular study in relation to the incubation time. Take metabolism and duplication time of the fungus into consideration.</li>
	</ol>
	</li>
</ol>
3. Peptides (Unknown Agent)
<ol>
	<li>Store the lyophilized peptides at -20 &#176;C, and dissolve them in deionized water before each experiment. The maximum storage time once dissolved in water will depend on the nature of each peptide.</li>
	<li>Prepare aliquots of twice the highest final concentration tested in the assay (2X). Ideally, prepare a small number of aliquots with enough peptide for one time use to avoid freeze-thaw cycles. 
	NOTE: The choice of concentration should be based on literature and the properties of the peptide. It is recommended to start the serial dilutions with approximately 100 &#181;M of the peptide, and then decrease or increase this concentration range depending on the obtained results.</li>
</ol>
4. Reference Antifungals (Positive Controls)
<ol>
	<li>For those diluted in water: prepare a 2X solution of the highest concentration of the analysis (see Section 5: Antifungal Assay for the dilution steps).
	<ol>
		<li>For C. albicans strains, prepare a solution of 128 &#181;g/mL of fluconazole or 128 &#181;g/mL of caspofungin. The plate concentration for both will be 64 &#181;g/mL.</li>
		<li>For C. neoformans strains, prepare a solution of 32 &#181;g/mL of amphotericin B (water-soluble solution). The plate concentration will be 16 &#181;g/mL. 
		NOTE: Normally amphotericin B is diluted in dimethyl sulfoxide (DMSO), since this anti-fungal is poorly soluble in water. However, there are water-soluble amphotericin B preparations commercially available.</li>
	</ol>
	</li>
	<li>For antifungals diluted in an organic solvent: prepare a 100X stock solution in DMSO, then dilute it to 10X in water for use. 
	NOTE: Thus, in the well, the final concentration of DMSO will not exceed 1%. A well where the fungus will grow in media containing 1% DMSO as control is required given that some fungi do not tolerate well this concentration of the solvent. Remember that DMSO is photosensitive, so cover the plate with foil or place it in a dark chamber for the duration of the incubation period.</li>
</ol>
5. Antifungal Assay
NOTE: In vitro antifungal assays are performed based on the broth microdilution susceptibility test from Clinical and Laboratory Standards Institute (CLSI) M27-A3 guidelines with some modifications.
<ol>
	<li>Prepare a two-fold serial dilution of each peptide and control antifungal in 96-well polystyrene microplates to a final volume of 50 &#181;L.
	<ol>
		<li>Using a pipette add 100 &#181;L of the antifungal/peptide in the 2X concentration of the highest desired final concentration in columns 1-3, in row A.</li>
		<li>Using a multichannel pipette, add 50 &#181;L of sterile water into the other wells, in rows B to H.</li>
		<li>Remove 50 &#181;L of the wells with the highest concentration (row A), transfer to the next well of the next concentration (row B) and homogenize.</li>
		<li>Repeat the above steps until the well with the lowest concentration and discard 50 &#181;L of this well (row H). Leave Columns 11 and 12 (rows A, B, C) with only water for either blank control or negative/growth control.</li>
		<li>Add 50 &#181;L of the 2X adjusted inoculum in RPMI-1640 medium to each well (columns 1-3 plus column 11 (negative/growth control)); the final concentration for C. albicans will be 2 x 103 cells/mL, and the final concentration for C. neoformans strains will be 104 cells/mL.</li>
		<li>Prepare blank controls (50 &#181;L water + 50 &#181;L 2X RPMI-1640 medium without cells) and negative/growth control (50 &#181;L water + 50 &#181;L adjusted inoculum 2X RPMI-1640 medium without antifungal), and load into the well plate as described above.</li>
	</ol>
	</li>
	<li>Repeat the procedure for each compound to be tested and the chosen antifungal control (columns 4-10). 
	NOTE: The serial dilutions can be done vertically as the experiment below, or horizontally in the plate (i.e., if the number of dilutions of the given compound/extract that needs to be tested is eight or more).
	<ol>
		<li>If the compounds, reference drugs, or any of its components are photosensitive, perform the assay in reduced light, cover the plates with foil, or place in a dark chamber during incubations.</li>
	</ol>
	</li>
	<li>Consider the following optional recommendations.
	<ol>
		<li>Seal the plate with a clear cover plate that permits gas exchange, as this helps reduce evaporation.</li>
		<li>Place the plate in a humid chamber; it will also help reduce evaporation.</li>
	</ol>
	</li>
	<li>Incubate the plates at 37 &#176;C for 24 h or 48 h for all C. albicans strains, and for 48 h with 200 rpm shaking for C. neoformans. The lower final volume (100 &#181;L) ensures that no spillover occurs. 
	NOTE: No significant difference was observed between MIC readings at 24 h and 48 h for C. albicans strains. Nonetheless, readings at 48 h are easier to visualize.</li>
	<li>Observe all wells, with the assistance of an inverted optical microscope, before the incubation period and after to verify changes in morphology as well as to check for signs of contamination. 
	NOTE: Readings can be done visually and photographed at the end of the experiment. Before each reading, homogenize slightly the plate. The reads can also be performed by measuring its OD at 600 nm, if cell clumps or filamentation are not observed.</li>
	<li>Perform the experiments at least three times on separate dates.</li>
</ol>

<ol><li>Armstrong-James, D., Meintjes, G., Brown, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+neglected+epidemic%3A+fungal+infections+in+HIV%2FAIDS%5BTitle%5D)+AND+%22Trends+Microbiol%22%5BJournal%5D)">A neglected epidemic: fungal infections in HIV/AIDS.</a> Trends Microbiol. 22 (3), 120-127 (2014).</li>
<li>Romani, L. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/21394104">Immunity to fungal infections.</a> Nat Rev Immunol. 11 (4), 275-288 (2011).</li>
<li>Pfaller, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antifungal+drug+resistance%3A+mechanisms%2C+epidemiology%2C+and+consequences+for+treatment%5BTitle%5D)+AND+%22Am+J+Med%22%5BJournal%5D)">Antifungal drug resistance: mechanisms, epidemiology, and consequences for treatment.</a> Am J Med. 125 (1 Suppl), S3-S13 (2012).</li>
<li>Hancock, R. E., Diamond, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+cationic+antimicrobial+peptides+in+innate+host+defences%5BTitle%5D)+AND+%22Trends+Microbiol%22%5BJournal%5D)">The role of cationic antimicrobial peptides in innate host defences.</a> Trends Microbiol. 8 (9), 402-410 (2000).</li>
<li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Snake+cathelicidin+from+Bungarus+fasciatus+is+a+potent+peptide+antibiotics%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Snake cathelicidin from Bungarus fasciatus is a potent peptide antibiotics.</a> PLoS One. 3 (9), e3217(2008).</li>
<li>Du, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((AaeAP1+and+AaeAP2%3A+novel+antimicrobial+peptides+from+the+venom+of+the+scorpion%2C+Androctonus+aeneas%3A+structural+characterisation%2C+molecular+cloning+of+biosynthetic+precursor-encoding+cDNAs+and+engineering+of+analogues+with+enhanced+antimicrobial+and+anticancer+activities%5BTitle%5D)+AND+%22Toxins+%28Basel%29%22%5BJournal%5D)">AaeAP1 and AaeAP2: novel antimicrobial peptides from the venom of the scorpion, Androctonus aeneas: structural characterisation, molecular cloning of biosynthetic precursor-encoding cDNAs and engineering of analogues with enhanced antimicrobial and anticancer activities.</a> Toxins (Basel). 7 (2), 219-237 (2015).</li>
<li>Benincasa, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fungicidal+activity+of+five+cathelicidin+peptides+against+clinically+isolated+yeasts%5BTitle%5D)+AND+%22J+Antimicrob+Chemother%22%5BJournal%5D)">Fungicidal activity of five cathelicidin peptides against clinically isolated yeasts.</a> J Antimicrob Chemother. 58 (5), 950-959 (2006).</li>
<li>CLSI. <a target="_blank" href="https://clsi.org/standards/products/microbiology/documents/m27s/">Reference Method for Broth Dilution Antifungal Susceptibiliy Testing of Yeasts; Approved Standard -Third Edition. CLSI document M27-A3</a>. , Clinical and Laboratory Standards Institute. Wayne, PA. (2008).</li>
<li>Guilhelmelli, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Activity+of+Scorpion+Venom-Derived+Antifungal+Peptides+against+Planktonic+Cells+of+Candida+spp.+and+Cryptococcus+neoformans+and+Candida+albicans+Biofilms%5BTitle%5D)+AND+%22Front+Microbiol%22%5BJournal%5D)">Activity of Scorpion Venom-Derived Antifungal Peptides against Planktonic Cells of Candida spp. and Cryptococcus neoformans and Candida albicans Biofilms.</a> Front Microbiol. 7, 1844(2016).</li>
<li>The European Committee on Antimicrobial Susceptibility Testing. Method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for yeasts, version, 7.3.1 2017. , Available from:
 <a target="_blank" href="http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/AFST/Files/EUCAST_E_Def_7_3_1_Yeast_testing__definitive.pdf">http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/AFST/Files/EUCAST_E_Def_7_3_1_Yeast_testing__definitive.pdf</a> (2017).</li>
<li>Roongruangsree, U. T., Kjerulf-Jensen, C., Olson, L. W., Lange, L. <a target="_blank" href="http://onlinelibrary.wiley.com/doi/10.1111/j.1439-0434.1988.tb04474.x/full">Viability Tests for Thick Walled Fungal Spores (ex: Oospores of Peronospora manshurica).</a> Journal of Phytopathology. 123 (3), 244-252 (1988).</li>
<li>Boedijn, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Trypan+blue+as+stain+for+fungi%5BTitle%5D)+AND+%22Stain+Technol%22%5BJournal%5D)">Trypan blue as stain for fungi.</a> Stain Technol. 31 (3), 115-116 (1956).</li>
<li>Goihman-Yahr, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Studies+on+plating+efficiency+and+estimation+of+viability+of+suspensions+of+Paracoccidioides+brasiliensis+yeast+cells%5BTitle%5D)+AND+%22Mycopathologia%22%5BJournal%5D)">Studies on plating efficiency and estimation of viability of suspensions of Paracoccidioides brasiliensis yeast cells.</a> Mycopathologia. 71 (2), 73-83 (1980).</li>
<li>Tati, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Histatin+5-spermidine+conjugates+have+enhanced+fungicidal+activity+and+efficacy+as+a+topical+therapeutic+for+oral+candidiasis%5BTitle%5D)+AND+%22Antimicrob+Agents+Chemother%22%5BJournal%5D)">Histatin 5-spermidine conjugates have enhanced fungicidal activity and efficacy as a topical therapeutic for oral candidiasis.</a> Antimicrob Agents Chemother. 58 (2), 756-766 (2014).</li>
<li>Petrou, M. A., Shanson, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Susceptibility+of+Cryptococcus+neoformans+by+the+NCCLS+microdilution+and+Etest+methods+using+five+defined+media%5BTitle%5D)+AND+%22J+Antimicrob+Chemother%22%5BJournal%5D)">Susceptibility of Cryptococcus neoformans by the NCCLS microdilution and Etest methods using five defined media.</a> J Antimicrob Chemother. 46 (5), 815-818 (2000).</li>
<li>Zaragoza, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Process+analysis+of+variables+for+standardization+of+antifungal+susceptibility+testing+of+nonfermentative+yeasts%5BTitle%5D)+AND+%22Antimicrob+Agents+Chemother%22%5BJournal%5D)">Process analysis of variables for standardization of antifungal susceptibility testing of nonfermentative yeasts.</a> Antimicrob Agents Chemother. 55 (4), 1563-1570 (2011).</li>
<li>Rodriguez-Tudela, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Influence+of+shaking+on+antifungal+susceptibility+testing+of+Cryptococcus+neoformans%3A+a+comparison+of+the+NCCLS+standard+M27A+medium%2C+buffered+yeast+nitrogen+base%2C+and+RPMI-2%25+glucose%5BTitle%5D)+AND+%22Antimicrob+Agents+Chemother%22%5BJournal%5D)">Influence of shaking on antifungal susceptibility testing of Cryptococcus neoformans: a comparison of the NCCLS standard M27A medium, buffered yeast nitrogen base, and RPMI-2% glucose.</a> Antimicrob Agents Chemother. 44 (2), 400-404 (2000).</li>
<li>Beggs, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Growth+phase+in+relation+to+ketoconazole+and+miconazole+susceptibilities+of+Candida+albicans%5BTitle%5D)+AND+%22Antimicrob+Agents+Chemother%22%5BJournal%5D)">Growth phase in relation to ketoconazole and miconazole susceptibilities of Candida albicans.</a> Antimicrob Agents Chemother. 25 (3), 316-318 (1984).</li>
<li>Alcouloumre, M. S., Ghannoum, M. A., Ibrahim, A. S., Selsted, M. E., Edwards, J. E. Jr <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fungicidal+properties+of+defensin+NP-1+and+activity+against+Cryptococcus+neoformans+in+vitro%5BTitle%5D)+AND+%22Antimicrob+Agents+Chemother%22%5BJournal%5D)">Fungicidal properties of defensin NP-1 and activity against Cryptococcus neoformans in vitro.</a> Antimicrob Agents Chemother. 37 (12), 2628-2632 (1993).</li>
</ol>

The authors have nothing to disclose.

Microdilution tests can analyze the potential antifungal activity of a target compound using small quantities of the compound, and at the same time test it in a range of concentrations. Accordingly, this protocol is recommended as a first step in screening for potential new antifungal compounds. The protocol presented here is based on the M27-A3 protocol, initially designed to aid in the selection of antifungal therapy in clinics, and can be adapted to a variety of new antifungal compounds. Overall, this protocol can foc...

Fungal infections have become an important medical concern in recent decades, having considerably increased mainly due to a rise in the number of immunocompromised individuals such as those undergoing cancer treatment and those living with HIV/AIDS or transplanted organs1,2. However, a very limited array of available antifungal drugs and the increasing number of reports on fungal resistance to them contribute to the major problems regarding the therapeutics of systemic mycoses3.
A potential source of new antifungal compounds are antimicrobial peptides (AMPs), small cationic peptides produced by many organisms as part of their innate immune response to infection4. Nevertheless, the screening method to test these compounds against fungal pathogens is not standardized. Different procedures have been used to assess the antifungal activity of AMPs, sometimes for the same model microorganism5,6,7. These differences and the lack of detail in some protocols complicate comparisons between compounds and hampers reproducibility.
One way to standardize the testing of new drug candidates is to follow guidelines used to define antifungal susceptibility in clinical settings, such as the Clinical and Laboratory Standards Institute (CLSI) M27-A3 guidelines. However, these antifungal sensitivity tests are too restrictive, and do not take into consideration variation in metabolism across species, as they were only established for a few select agents. For example, they do not take into account the metabolic needs of non-fermenting yeasts.
This protocol allows the assessment of activity of prospective antifungal compounds, and is implemented here for the search for antifungal peptides. It is based on the broth microdilution susceptibility test from the CLSI M27-A3 guidelines with modifications that optimize the screening of new compounds8,9. These changes allow for the use of small amounts of compound, variations in temperature or initial inoculum, and different media for optimal pre-test growth, while standardizing the results with the use of reference antifungals as controls. This method, with the use of multi-well culture plates, makes it possible to quickly and reliably screen a large number of compounds.
Due to its inherent flexibility, this protocol can be used with different chemical classes of compounds and against other microorganisms, with few adaptations.

<table><tbody><tr><td>&lt;strong&gt;Media and Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>RPMI 1640 medium with l-glutamine, without sodium bicarbonate</td><td>Thermo Fisher</td><td>31800-022</td><td></td></tr><tr><td>3-(&lt;em&gt;N&lt;/em&gt;-morpholino) propane sulfonic acid&lt;em&gt; (MOPS) (o que a gente usa tem um s&amp;oacute;dio, completa o nome dele please)&lt;/em&gt;</td><td>Sigma-Aldrich</td><td></td><td>Use to buffer 2X RPMI medium</td></tr><tr><td>Sodium chloride (NaCl)</td><td>Din&amp;acirc;mica</td><td>1528-1</td><td>137 mM for Phosphate buffered saline (PBS)</td></tr><tr><td>Potassium chloride (KCl)</td><td>J.T.Baker</td><td>3040-01</td><td>2.7 mM for Phosphate buffered saline (PBS)</td></tr><tr><td>Sodium phosphate dibasic (Na2HPO4)</td><td>Sigma-Aldrich</td><td>V000129</td><td>10 mM for Phosphate buffered saline (PBS)</td></tr><tr><td>Potassium dihydrogen phosphate (KH2PO4)</td><td>Sigma-Aldrich</td><td>60230</td><td>2 mM for Phosphate buffered saline (PBS)</td></tr><tr><td>BD Difco Sabouraud dextrose broth</td><td>BD</td><td>238230</td><td></td></tr><tr><td>BD Difco Sabouraud Dextrose Agar</td><td>BD</td><td>210950</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>V000123</td><td>35% for (solu&amp;ccedil;&amp;atilde;o de estoque? Criopreserva&amp;ccedil;&amp;atilde;o?)</td></tr><tr><td>Sterile water</td><td></td><td></td><td>Para dilui&amp;ccedil;&amp;atilde;o das drogas na dilui&amp;ccedil;&amp;atilde;o seriada</td></tr><tr><td>&lt;strong&gt;Antifungal drugs&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Amphotericin B</td><td>Sigma-Aldrich</td><td>A2942</td><td></td></tr><tr><td>Fluconazole</td><td>Sigma-Aldrich</td><td>F8929</td><td></td></tr><tr><td>Caspofungin</td><td>Sigma-Aldrich</td><td>PHR1160</td><td></td></tr><tr><td>&lt;strong&gt;Plastics&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>50 mL conical tube</td><td>Sarstedt</td><td>62.547.254</td><td></td></tr><tr><td>Dish petri</td><td>J.Prolab</td><td>0304-5</td><td></td></tr><tr><td>96 well plate</td><td>Corning</td><td>3595</td><td></td></tr><tr><td>Sterile Solution Reservoir</td><td>KASVI</td><td>K30-208</td><td>Use to pippet the solutions using the multichannel pippet</td></tr><tr><td>&lt;strong&gt;Equipment and other materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Optical microscope</td><td>Nikon</td><td>E200MV</td><td></td></tr><tr><td>Centrifugue</td><td>Thermo Fisher</td><td>MegaFuge 16R</td><td></td></tr><tr><td>Incubator</td><td>Ethik Technology</td><td>403-3D</td><td>Set to 37&amp;deg; C</td></tr><tr><td>Shaker</td><td>New Brunswick Scientific</td><td>Excella E25</td><td>Set to 37&amp;deg; C, 200 RPM</td></tr><tr><td>Cell counting chamber, Neubauer</td><td>BOECO Germany</td><td>BOE 13</td><td></td></tr><tr><td>Multichannel pipette</td><td>HTL</td><td>5123</td><td></td></tr></tbody></table>

broth microdilution in vitro screening: an easy and fast method to detect new antifungal compounds

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

Fungal infections have become an important medical condition in the last decades, but the number of available antifungal drugs is limited. In this scenario, the search for new antifungal drugs is necessary. The protocol reported here details a method to screen peptides for their antifungal properties. It is based on the broth microdilution susceptibility test from the Clinical and Laboratory Standards Institute (CLSI) M27-A3 guidelines with modifications to suit the research of antimicrobial peptides as potential new antifungals. This protocol describes a functional assay to evaluate the activity of antifungal compounds and may be easily modified to suit any particular class of molecules under investigation. Since the assays are performed in 96-well plates using small volumes, a large-scale screening can be completed in a short amount of time, especially if carried out in an automation setting. This procedure illustrates how a standardized and adjustable clinical protocol can help the bench-work pursuit of new molecules to improve the therapy of fungal diseases.

The MIC is defined as the lowest antimicrobial compound concentration that completely inhibits visible fungal growth at the end of the incubation period. Since the objective of this protocol is to have a fast method to screen potential antifungals, any well with clear media similar to the blank wells is considered a positive result, whereas any well with turbidity analogous to the negative/growth control wells is considered negative. However, if there is an interest in knowing whether a g...

An easy and adaptable broth microdilution method for screening antifungal compounds and extracts.

Fungal infections have become an important medical condition in the last decades, but the number of available antifungal drugs is limited. In this ...

broth-microdilution-vitro-screening-an-easy-fast-method-to-detect-new

Research

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We describe a simple, rapid and robust method for the formation of Candida albicans biofilms using 96 well microtiter plates and its utility in antifungal susceptibility testing of cells within biofilms.

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

We have developed a high-density microarray platform consisting of 3D nano-biofilms of C. albicans called CaBChip. The susceptibility profile of drugs tested on a CaBChip is comparable to the conventional 96-well plate model, suggesting that the fungal chip is ideally suited for true high-throughput screening of antifungal drugs.

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

Source: Koeth, L. M., et. al., A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically. J. Vis. Exp. (2015).This video demonstrates the broth microdilution method for determining the minimum inhibitory concentration (MIC) of a lipoglycopeptide antibiotic against Gram-positive bacteria. It covers bacterial colony isolation, dilution, inoculation of microplate wells with varying antibiotic concentrations, and verification of culture...

A Broth Microdilution Method for Antibiotic Susceptibility Testing of Gram-Positive Bacteria

This video demonstrates an in vitro assay to screen antifungal compounds using a broth microdilution assay. Test compounds are serially diluted in a microtiter plate and incubated with a fungal inoculum. Post-incubation, the plate is examined for wells without visible fungal growth to assess the antifungal potential of the test compounds and their minimum inhibitory concentration (MIC).

An In Vitro Broth Microdilution Assay for the Screening of Antifungal Compounds

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 cell using automated confocal microscopy.

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

Here, we present a protocol for determination of dalbavancin susceptibility of clinically relevant Gram-positive bacteria using a broth microdilution reference methodology.

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

A high throughput, real-time assay was developed to simultaneously identify (1) eukaryotic cell-penetrant antimicrobials targeting an intracellular bacterial pathogen, and (2) assess eukaryotic cell cytotoxicity. A variation on the same technology was thereafter combined with digital dispensing technology to enable facile, high-resolution, dose-response, and two- and three-dimensional synergy studies.

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

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.

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

We describe a modified agar-based method designed to quantify the antifungal effects of plant-derived products. Both volatile and non-volatile contributions to the antifungal activity can be assessed through this protocol. In addition, efficacy against fungi can be measured at key developmental stages in a single experimental setup.

Measuring Volatile and Non-volatile Antifungal Activity of Biocontrol Products

A 96-well microtiter plate-based protocol using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) reduction assay is described herein, to study antibodies' effects on biofilms formed by C. tropicalis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

Here, we present a protocol to identify potential anti-tuberculosis drug candidates through phenotypic screening, encompassing the chemical synthesis of a 4-aminoquinoline compound, determination of its minimum inhibitory concentration against Mycobacterium tuberculosis, and assessment of its cytotoxicity in mammalian cell lines.

Identification of Potential Anti-TB Candidates: A Step-by-Step Guide to Synthesis, MIC Determination, and Cytotoxicity Assessment in Mammalian Cells

Learn broth microdilution screening for antifungal drug development using Candida and Cryptococcus strains. This rapid method enables efficient evaluation of natural and synthetic compounds with automation.

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

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