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 st...

<ol>
	<li>Armstrong-James, D., Meintjes, G., Brown, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+neglected+epidemic:+fungal+infections+in+HIV/AIDS.">A neglected epidemic: fungal infections in HIV/AIDS.</a> Trends Microbiol. 22 (3), 120-127 (2014).</li><li>Romani, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunity+to+fungal+infections.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifungal+drug+resistance:+mechanisms,+epidemiology,+and+consequences+for+treatment.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+cationic+antimicrobial+peptides+in+innate+host+defences.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Snake+cathelicidin+from+Bungarus+fasciatus+is+a+potent+peptide+antibiotics.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fungicidal+activity+of+five+cathelicidin+peptides+against+clinically+isolated+yeasts.">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="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Reference Method for Broth Dilution Antifungal Susceptibiliy Testing of Yeasts; Approved Standard -Third Edition. CLSI document M27-A3. , (2008).</li><li>Guilhelmelli, F., 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=Activity+of+Scorpion+Venom-Derived+Antifungal+Peptides+against+Planktonic+Cells+of+Candida+spp.+and+Cryptococcus+neoformans+and+Candida+albicans+Biofilms.">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>Roongruangsree, U. T., Kjerulf-Jensen, C., Olson, L. W., Lange, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viability+Tests+for+Thick+Walled+Fungal+Spores+(ex:+Oospores+of+Peronospora+manshurica).">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypan+blue+as+stain+for+fungi.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+plating+efficiency+and+estimation+of+viability+of+suspensions+of+Paracoccidioides+brasiliensis+yeast+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histatin+5-spermidine+conjugates+have+enhanced+fungicidal+activity+and+efficacy+as+a+topical+therapeutic+for+oral+candidiasis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Susceptibility+of+Cryptococcus+neoformans+by+the+NCCLS+microdilution+and+Etest+methods+using+five+defined+media.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Process+analysis+of+variables+for+standardization+of+antifungal+susceptibility+testing+of+nonfermentative+yeasts.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+phase+in+relation+to+ketoconazole+and+miconazole+susceptibilities+of+Candida+albicans.">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. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fungicidal+properties+of+defensin+NP-1+and+activity+against+Cryptococcus+neoformans+in+vitro.">Fungicidal properties of defensin NP-1 and activity against Cryptococcus neoformans in vitro.</a> Antimicrob Agents Chemother. 37 (12), 2628-2632 (1993).</li></ol>

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>Media and Reagents</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-(N-morpholino) propane sulfonic acid (MOPS) (o que a gente usa tem um s&#243;dio, completa o nome dele please)</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td>Use to buffer 2X RPMI medium</td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Din&#226;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&#231;&#227;o de estoque? Criopreserva&#231;&#227;o?)</td>
		</tr>
		<tr>
			<td>Sterile water</td>
			<td></td>
			<td></td>
			<td>Para dilui&#231;&#227;o das drogas na dilui&#231;&#227;o seriada</td>
		</tr>
		<tr>
			<td>Antifungal drugs</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>Plastics</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>Equipment and other materials</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&#176; C</td>
		</tr>
		<tr>
			<td>Shaker</td>
			<td>New Brunswick Scientific</td>
			<td>Excella E25</td>
			<td>Set to 37&#176; 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

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...

Watch this Scientific Journal Video about Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds at JoVE.com

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

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

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 ...

The overall goal of this method is to evaluate the activity of potential antifungal compounds, using a modification of the broth microdilution susceptibility test, used in clinical laboratories. This method is used to evaluate the fungal activity of natural or synthetic compounds, one of the first tests in the development of anti fungal therapies. We provide instructions for an initial screen for new antifungals using small volumes in a short time, especially, if the experiments are carried out with automation.Demonstrating the procedure will be Daniel Zamith-Miranda, a post doctoral fellow in my laboratory. Begin this procedure by growing the fungal strains for the assay as described in the text protocol. On the following day, collect the cells by centrifuging the conical tubes at 1200 G, for five minutes at room temperature.Discard the supernatant, add ten milliliters of PBS to each tube, and resuspend the cells. Centrifuge again at 1200 G for five minutes at room temperature. Repeat the PBS wash and centrifugation two more times.After the third wash, resuspend the cells in five milliliters of double strength RPMI 1640 medium. Depending on the turbidity of the cell suspension, prepare one milliliter of a one to 100, or one to 1000, dilution of each strain, in a microcentrifuge tube. Aliquat ten microliters of this dilution, place it in a hemocytometer chamber, and count the total number of cells in the four corner quadrants under the microscope.After calculating the density of each cell suspension, prepare stock cell suspensions in double strength RPMI 1640 medium. Prepare 4, 000 cells per milliliter for C.albicans strains, and 20, 000 cells per milliliter for C.neoformans strains. The lypholized peptides are stored in aliquats for one time use at minus 20 degrees Celsius, and only dissolved in deionized water before each experiment.The peptide concentration should be twice the highest final concentration tested in the assay. Reference antifungals are also prepared. The antifungal assay is performed in triplicate and 96 well polystyrene microplates.Using a two fold serial dilution of each peptide and control antifungal, and a final volume of 50 microliters per well. The most crucial step in the protocol is the serial dilutions, so be very careful during the next few steps to avoid any error that will compromise your whole analysis. Using a pipette, add 100 microliters of the antifungal peptide in the double strength concentration of the highest desired final concentration in columns one through three in row A.Using a multichannel pipette, add 50 microliters of sterile water to the other wells in rows B to H.Remove 50 microliters of the wells with the highest concentration.Transfer to the corresponding wells of the next concentration, and homogenize by pipetting up and down several times. Repeat the serial dilution until the wells with the lowest concentration are reached, and discard 50 microliters from these wells. Leave columns 11 and 12 of rows A, B, and C with only water for blank controls, or negative growth controls.Add 50 microliters of the double strength adjusted inoculum in RPMI 1640 medium to each well of columns one to three, plus column 11. The final concentration for C.albicans will be 2, 000 cells per milliliter. The final concentration for C.neoformans will be 10, 000 cells per milliliter.Prepare negative growth controls by mixing 50 microliters of water and 50 microliters of adjusted inoculum without the antifungal. Prepare blank controls by combining 50 microliters of water and 50 microliters of medium without cells. Load the controls into the appropriate wells.Observe all wells using an inverted optical microscope, and then incubate the plates at 37 degrees with shaking at 200 rpm. After the incubation period, observe all wells again to verify changes in morphology, as well as to check for signs of contamination. Acquire an image of each plate using a digital camera.In this representative assay, three antimicrobial peptides, or AMPs, with concentrations ranging from 100 micromolar to point seven eight micromolar, were evaluated for antifungal activity against C.neoformans. A growth control and a blank were included. 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.Since the minimum inhibitory concentration, or MIC, is defined as the lowest antimicrobial compound concentration that completely inhibits visible fungal growth at the end of the incubation period, 25 micromolar is considered the MIC for AMP1. And, 12 point five micromolar, the MIC for AMP2. AMP3 will have to be reevaluated, as the fungus grew at all concentrations tested.In the same manner, three distinct peptides tested against C.albicans, indicted 50 micromolar is the MIC for AMP1. Six micromolar is the MIC for AMP2, and 25 micromolar is the MIC for AMP3. An example of a poor result, likely from improper pipetting during the dilution step, is illustrated by columns five to seven of row C in this plate, where differences in C.neoformans growth are present between replicates.Once mastered, this technique can be done in approximately two hours, plus the time of fungal growth, if it's performed properly. Remember to be aware of the physical chemical features of these tested molecules. Then use appropriate solvents as control in case they are needed, and to always use the cells in the same growth phase.Following this procedure, other methods could be performed such as time kill assay, the use of viability dyes, and ST60 assays to answer additional questions, like whether the compound has a fungicidal or fungistatic effect. After it's development, this technique paved the way for researchers in the field of medical micrology to explore several sources of natural or synthetic compounds in the search for antifungal compounds. After watching this video, you should have a good understanding of how to start a fungal culture, prepare cells for the assay, perform the serial dilutions, and read the results after incubating the fungal strains with our tested compounds.Don't forget, that working with fungal pathogens can be extremely hazard. And preparation such as wearing personal protection equipment, working inside a biological safety hood, and using appropriate safety practice should be taken while performing this procedure.

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

Research

JoVE Journal

Biology

18.5K vistas.  University of Brasília. The overall goal of this method is to evaluate the activity of potential antifungal compounds, using a modification of the broth microdilution susceptibility test, used in clinical laboratories. This method is used to evaluate the fungal activity of natural or synthetic compounds, one of the first tests in the development of anti fungal therapies. We provide instructions for an initial screen for new antifungals using small volumes in a short time, especially, if the experiments are carried o...