The work was supported by grants from Sigrid Juselius Foundation (SP, MP), Finnish Cultural Foundation (AA, MH), Academy of Finland (SP, MP), Orion Farmos Foundation (MH), Tampere Tuberculosis Foundation (SP, MH and MP) and Jane and Aatos Erkko Foundation (SP and MP). We thank our Italian and French collaborators, Prof. Supuran, and Prof. Winum, for providing carbonic anhydrase inhibitors for safety and toxicity evaluation for anti-TB and anti-cancer drug development purposes. We thank Aulikki Lehmus and Marianne Kuuslahti for the technical assistance. We also thank Leena M&#228;kinen and Hannaleena Piippo for their help with the zebrafish breeding and collection of embryos. We sincerely thank Harlan Barker for critical evaluation of the manuscript and insightful comments.

The zebrafish core facility at Tampere University has an establishment authorization granted by the National Animal Experiment Board (ESAVI/7975/04.10.05/2016). All the experiments using zebrafish embryos were performed according to the Provincial Government of Eastern Finland, Social and Health Department of Tampere Regional Service Unit protocol # LSLH-2007-7254/Ym-23.
1. Setting Up of Overnight Zebrafish Mating Tanks
<ol>
	<li>Place 2-5 adult male zebrafish and 3-5 adult female zebrafish ...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+test+chemicals+for+the+ECVAM+international+validation+study+on+in+vitro+embryotoxicity+tests.+European+Centre+for+the+Validation+of+Alternative+Methods.">Selection of test chemicals for the ECVAM international validation study on in vitro embryotoxicity tests. European Centre for the Validation of Alternative Methods.</a> Alternatives to Laboratory Animals. 30 (2), 177-198 (2002).</li><li>Selderslaghs, I. W., Van Rompay, A. R., De Coen, W., Witters, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+screening+assay+to+identify+teratogenic+and+embryotoxic+chemicals+using+the+zebrafish+embryo.">Development of a screening assay to identify teratogenic and embryotoxic chemicals using the zebrafish embryo.</a> Reproductive Toxicology. 28 (3), 308-320 (2009).</li><li>Brannen, K. C., Panzica-Kelly, J. M., Danberry, T. L., Augustine-Rauch, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+zebrafish+embryo+teratogenicity+assay+and+quantitative+prediction+model.">Development of a zebrafish embryo teratogenicity assay and quantitative prediction model.</a> Birth Defects Research Part B Developmental and Reproductive Toxicology. 89 (1), 66-77 (2010).</li><li>Hermsen, S. A., van den Brandhof, E. J., van der Ven, L. 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Part C, Embryo Today: Reviews. 93 (3), 268-280 (2011).</li><li>Lantz-McPeak, 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=Developmental+toxicity+assay+using+high+content+screening+of+zebrafish+embryos.">Developmental toxicity assay using high content screening of zebrafish embryos.</a> Journal of Applied Toxicology. 35 (3), 261-272 (2015).</li><li>Truong, L., Harper, S. L., Tanguay, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+embryotoxicity+using+the+zebrafish+model.">Evaluation of embryotoxicity using the zebrafish model.</a> Methods in Molecular Biology. 691, 271-279 (2011).</li><li>Rodrigues, G. 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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+zebrafish+as+a+model+system+to+study+cardiovascular+development.">The zebrafish as a model system to study cardiovascular development.</a> Trends in Cardiovascular Medicine. 4 (5), 207-212 (1994).</li><li>Aspatwar, 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=Nitroimidazole-based+inhibitors+DTP338+and+DTP348+are+safe+for+zebrafish+embryos+and+efficiently+inhibit+the+activity+of+human+CA+IX+in+Xenopus+oocytes.">Nitroimidazole-based inhibitors DTP338 and DTP348 are safe for zebrafish embryos and efficiently inhibit the activity of human CA IX in Xenopus oocytes.</a> Journal of Enzyme Inhibition and Medicinal Chemistry. 33 (1), 1064-1073 (2018).</li><li>Rami, 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=Hypoxia-targeting+carbonic+anhydrase+IX+inhibitors+by+a+new+series+of+nitroimidazole-sulfonamides/sulfamides/sulfamates.">Hypoxia-targeting carbonic anhydrase IX inhibitors by a new series of nitroimidazole-sulfonamides/sulfamides/sulfamates.</a> Journal of Medicinal Chemistry. 56 (21), 8512-8520 (2013).</li><li>Spitsbergen, J. M., Kent, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+state+of+the+art+of+the+zebrafish+model+for+toxicology+and+toxicologic+pathology+research--advantages+and+current+limitations.">The state of the art of the zebrafish model for toxicology and toxicologic pathology research--advantages and current limitations.</a> Toxicologic Pathology. , 62-87 (2003).</li><li>Teraoka, H., 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=Induction+of+cytochrome+P450+1A+is+required+for+circulation+failure+and+edema+by+2,3,7,8-tetrachlorodibenzo-p-dioxin+in+zebrafish.">Induction of cytochrome P450 1A is required for circulation failure and edema by 2,3,7,8-tetrachlorodibenzo-p-dioxin in zebrafish.</a> Biochemical and Biophysical Research Communications. 304 (2), 223-228 (2003).</li><li>Zon, L. I., Peterson, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+drug+discovery+in+the+zebrafish.">In vivo drug discovery in the zebrafish.</a> Nature Reviews Drug Discovery. 4 (1), 35-44 (2005).</li><li>Hill, A. J., Teraoka, H., Heideman, W., Peterson, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+model+vertebrate+for+investigating+chemical+toxicity.">Zebrafish as a model vertebrate for investigating chemical toxicity.</a> Toxicological Sciences. 86 (1), 6-19 (2005).</li><li>Kari, G., Rodeck, U., Dicker, A. 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In vitro toxicity test using cultured cells can detect survival and morphological studies of the cells providing limited information about the toxicity induced by the test compound. The advantage of toxicity screening of chemical compounds using zebrafish embryos is rapid detection of chemically induced phenotypic changes in a whole animal during embryonic development in a relevant model organism. Approximately 70% of protein-coding human genes have orthologs counterparts in the zebrafish genome<sup class="xref"...

Drug development is an expensive process. Before a single chemical compound is approved by the Food and Drug Administration (FDA) and European Medicines Agency (EMA) several thousand compounds are screened at a cost of over one billion dollars1. During the preclinical development, the largest part of this cost is required for the animal testing2. To limit the costs, researchers in the field of drug development need alternative models for the safety screening of chemical compounds3. Therefore, in the early phase of the drug development, it would be very beneficial to use a method that can rapidly evaluate the safety and toxicity of the compounds in a suitable model. There are several protocols that have been used for the toxicity screening of chemical compounds involving animal and cell culture models but there is not a single protocol that is validated and is in common use4,5. Existing protocols using zebrafish vary in length and have been used by individual researchers who evaluated the toxicity as per their convenience requirement6,7,8,9,10,11,12.
In the recent past, the zebrafish has emerged as a convenient model for the evaluation of the toxicity of chemical compounds during embryonic development6,7. The zebrafish has many in-built advantages for the evaluation of chemical compounds13. Even large-scale experiments are amenable, as a zebrafish female can lay batches of 200-300 eggs, which develop rapidly ex&#160;vivo, do not need external feeding for up to a week and are transparent. The compounds can be added directly into the water, where they can (depending on the nature of the compound) diffuse through the chorion, and after hatching, through the skin, gills and mouth of larvae. The experiments do not require copious amounts of chemical compounds14 due to the small size of the embryo. Developing zebrafish embryos express most of the proteins required to achieve the normal developmental outcome. Therefore, a zebrafish embryo is a sensitive model to assess whether a potential drug can disturb the function of a protein or signaling molecule that is developmentally significant. The organs of the zebrafish become functional between 2-5 dpf15, and compounds that are toxic during this sensitive period of embryonic development induce phenotypic defects in zebrafish larvae. These phenotypic changes can be readily detected using a simple microscope without invasive techniques11. Zebrafish embryos are widely used in toxicological research due to their much greater biological complexity compared to in vitro drug screening using cell culture models16,17. &#160;As a vertebrate, the genetic and physiologic makeup of zebrafish is comparable to humans and hence toxicities of chemical compounds are similar between zebrafish and humans8,18,19,20,21,22. Zebrafish is, thus, a valuable tool in the early phase of drug discovery for the evaluation of toxicity and safety of the chemical compounds.
In the present article, we provide a detailed description of the method used for evaluating the safety and toxicity of carbonic anhydrase (CA) inhibitor compounds using 1-5-day post fertilization (dpf) zebrafish embryos by a single researcher. The protocol involves exposing zebrafish embryos to different concentrations of chemical inhibitor compounds and studying the mortality and phenotypic changes during the embryonic development. At the end of the exposure to the chemical compounds, the LC50 dose of the chemical is determined. The method allows an individual to carry out efficient screening of 1-5 test compounds and takes about 8-10 h depending on the experience of the person with the method (Figure 1). Each of the steps required to assess the toxicity of the compounds is outlined in Figure 2. The evaluation of toxicity of CA inhibitors requires 8 days, and includes setting up of mating pairs (day 1); collection of embryos from breeding tanks, cleaning and transferring them to 28.5 &#176;C incubator (day 2); distribution of the embryos into the wells of a 24-well plate and addition of diluted CA inhibitor compounds (day 3); phenotypic analysis and imaging of larvae (day 4-8), and determination of LC50 dose (day8). &#160;This method is rapid and efficient, requires a small amount of the chemical compound and only basic facilities of the laboratory.

<table border="0" cellpadding="0" cellspacing="0" style="width:972px;" width="972">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">24-well plates</td>
			<td style="width:185px;">Nunc</td>
			<td style="width:260px;">Thermo Scientific</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Balance (Weighing scale)</td>
			<td style="width:185px;">KERN</td>
			<td style="width:260px;">PLJ3000-2CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Balance (Weighing scale)</td>
			<td style="width:185px;">Mettler Toledo</td>
			<td style="width:260px;">AB104-S/PH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">CaCl2</td>
			<td style="width:185px;">JT.Baker</td>
			<td style="width:260px;">RS421910024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Disecting Probe</td>
			<td style="width:185px;">Thermo Scientific</td>
			<td>17-467-604&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">DMSO</td>
			<td style="width:185px;">Sigma Aldrich, Germany</td>
			<td style="width:260px;">D4540</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Falcon tubes 15 mL</td>
			<td style="width:185px;">Greiner bio-one</td>
			<td style="width:260px;">188271</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">High molecular weight methylcellulose</td>
			<td style="width:185px;">Sigma Aldrich, Germany</td>
			<td style="width:260px;">M0262&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Incubator for zebrafish larvae</td>
			<td style="width:185px;">Termaks</td>
			<td style="width:260px;">B8000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">KCL</td>
			<td style="width:185px;">Merck</td>
			<td style="width:260px;">1.04936.0500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Methyl Blue</td>
			<td style="width:185px;">Sigma Aldrich, Germany</td>
			<td style="width:260px;">28983-56-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">MgSO4</td>
			<td style="width:185px;">Sigma Aldrich, Germany</td>
			<td style="width:260px;">M7506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Microcentrifuge tubes</td>
			<td style="width:185px;">Starlab</td>
			<td style="width:260px;">S1615-5500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">NaCl</td>
			<td style="width:185px;">VWR Chemicals</td>
			<td style="width:260px;">27810.295</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Paraffin Histoplast IM</td>
			<td style="width:185px;">Thermo Scientific</td>
			<td style="width:260px;">8331</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Pasteur pipette&#160;</td>
			<td style="width:185px;">Sarstedt</td>
			<td style="width:260px;">86.1171</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Petri dish</td>
			<td style="width:185px;">Thermo Scientific</td>
			<td>101R20&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Petri plates</td>
			<td style="width:185px;">Sarstedt</td>
			<td style="width:260px;">82.1473</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Pipette (1 mL and 200 &#956;L)</td>
			<td style="width:185px;">Thermo Scientific</td>
			<td style="width:260px;">4641230N, 4641210N&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Plates 24-Well</td>
			<td style="width:185px;">Thermo Scientific</td>
			<td style="width:260px;">142485</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Steriomicroscope/Camera</td>
			<td style="width:185px;">Zeiss</td>
			<td style="width:260px;">Stemi 2000-C/Axiocam 105 color</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Vials (1.5 mL)</td>
			<td style="width:185px;">Fisherbrand</td>
			<td style="width:260px;">11569914</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Zebrafish AB strains</td>
			<td style="width:185px;">ZIRC&#160;</td>
			<td style="width:260px;">&#160; ZL1&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

rapid evaluation of toxicity of chemical compounds using zebrafish embryos

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has transparent embryos and rapid external development. Zebrafish embryos can, therefore, also be used for the rapid evaluation of toxicity of the drugs that are precious and available in small quantities. In the present article, a method for the efficient screening of the toxicity of chemical compounds using 1-5-day post fertilization embryos is described. The embryos are monitored by stereomicroscope to investigate the phenotypic defects caused by the exposure to different concentrations of compounds. Half-maximal lethal concentrations (LC50) of the compounds are also determined. The present study required 3-6 mg of an inhibitor compound, and the whole experiment takes about 8-10 h to be completed by an individual in a laboratory having basic facilities. The current protocol is suitable for testing any compound to identify intolerable toxic or off-target effects of the compound in the early phase of drug discovery and to detect subtle toxic effects that may be missed in the cell culture or other animal models. The method reduces procedural delays and costs of drug development.

The critical part of the evaluation of toxicity is testing different concentrations of one or multiple chemical compounds in a single experiment. In the beginning, select the compounds for evaluation of toxicity, the number of concentrations to test for each compound, and accordingly, make a chart (Figure 3). We used a unique color for each compound to organize the samples (Figure 3). The use of solvent resistant marker and label...

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Rapid Evaluation of Toxicity of Chemical Compounds Using Zebrafish Embryos

Zebrafish embryos are used for evaluating the toxicity of chemical compounds. They develop externally and are sensitive to chemicals, allowing detection of subtle phenotypic changes. The experiment only requires a small amount of compound, which is directly added to the plate containing embryos, making the testing system efficient and cost-effective.

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has ...

The toxicity testing procedures will be demonstrated by Dr.Ashok Aspatwar, a senior researcher from my laboratory. Our protocol identifies off-target effects and detects subtle toxic effects of chemicals in the early phase of discovery that may be missed in cell culture or other animal models. The main advantages of our technique are:it is rapid and efficient, requires very small amount of chemical, and the basic facilities in the laboratory.Toxicity screening will help us in deciding the concentration for studying its efficiency against mycobacterium that causes tuberculosis and for further preclinical calculation. This method uses insights into off-target effects of chemicals, and hence this method can be used in any area of research where the toxicity of chemical is of primary importance. This method is very simple and can be carried out by anybody without prior experience.Individuals with no experience should plan the experiment carefully and use just one compound. This method provides toxic effects of the chemicals in the form of phenotypic changes in the developing zebrafish embryos and hence safety of the compounds tested. To begin, place two to five adult male zebrafish and three to five adult female zebrafish into mating tanks overnight.In the morning, turn on the light as breeding is induced by the automatic dark and subsequent light cycle. To avoid handling stress to the animals, allow the animals to rest for two weeks before using the same individuals for breeding. The next day before noon, collect the embryos using a fine mesh strainer, and transfer them onto a petri dish containing E3 embryo medium.Place the petri dish under a stereo microscope to examine each batch of embryos. Identify the opaque appearance and remove them as unfertilized or dead embryos. Keep the embryos at 28.5 degrees Celsius in an incubator overnight.The next morning, examine the embryos under a stereo microscope and remove any unhealthy or dead embryos. Carefully use a pasteur pipette to transfer one embryo into each well of a 24 well plate. Make sure each well contains enough E3 medium to cover the embryos.Take out the vials containing inhibitor compounds stored at 4 degrees Celsius in a refrigerator. Use an analytical balance to weigh appropriate amount of the compound, and prepare 250 microliters of 100 millimolar stock solution for each compound in E3 medium or another appropriate solvent. Then, use E3 medium to make serial dilutions of the stock solutions based on toxicity levels in 15 milliliter centrifuge tubes.From the wells that each contains one DPF embryo, use a pasteur pipette and 1 milliliter pipette to remove the E3 water one row at a time. Immediately distribute 1 milliliter of each diluent of the stock solutions into the wells of the 24 well plate starting from lower and moving to higher concentration. For the control groups, add E3 water or another relevant solvent.Label 24 well plates with the name and concentration of the compound and keep the plates at 28.5 degrees Celsius in an incubator. Take care that the dilutants of the chemicals in the wells is not evaporated in the incubator by sealing the sides of 24 well plate. Twenty four hours after exposure of the chemical compounds, use a pasteur pipette to transfer the larvae exposed to each concentration of the compound in a small petri dish containing 3%high molecular weight methyl cellulose.With a metal probe, lay it sideways. Place the petri dish under a stereo microscope attached to a camera. Take the images, and save the images in a separate folder each day until the end of the experiment.Enter all the observations in a table each day either in an online table or on a printed sheet. For neurotoxic compound exposure, the four to five DPF larvae may show abnormal swim pattern. In that case, make a record of such changes by capturing a short 30 seconds to one minute video of the larvae.After five days of exposure to the chemical compounds, note the concentration at which half of the embryos die as the half maximal lethal concentration 50 of each chemical. Construct a curve for mortality of embryos for all the concentrations using a suitable program. In this protocol, the critical part of the evaluation of toxicity is testing different concentrations of one or multiple chemical compounds in a single experiment.For the compounds that induce any phenotypic defects in the larvae, the defects were recorded every 24 hours over the period of one to five days post exposure to the chemical. The embryos treated with beta CA inhibitors at the concentrations of 250 micromolar and 125 micromolar exhibit various phenotypic defects compared with the control. For example, unhatched embryos even at Day 3, curved body structure, unutilized yolk sac and pericardial edema, and absence of otolith sacs in the larvae at five days after treatment.In another study, the embryos treated with CA inhibitors showed the absence of otolith sacs and swim bladder. The experiments identified a representative compound carbonic anhydrase 9, which induced minimal or no phenotypic changes during the embryonic development at 500 micromolar. The compound showed a high LC50 concentration.However, in a comparison with a control, the same compound at 300 micromolar was found to be neurotoxic and induced ataxia in the larvae after five days of exposure. Good quality of embryos are important for toxicity screening of chemicals. For obtaining good quality embryos, use young pair of adult zebrafish for breeding.The compounds that emerge as safe can be further characterized by performing relevant in vitro and in vivo experiments. In the field of antituberculosis drug development, this technique helped to set up further experiments for in vivo inhibition of mycobacterium marinum in zebrafish embryos. The protocol involves use of chemicals that may be toxic to humans.Persons involved in the experiments should take proper care when handling the chemicals.

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10.4K vistas.  Tampere University. Los procedimientos de prueba de toxicidad serán demostrados por el Dr. Ashok Aspatwar, un investigador senior de mi laboratorio. Nuestro protocolo identifica los efectos fuera del objetivo y detecta los efectos tóxicos sutiles de los productos químicos en la fase temprana del descubrimiento que pueden perderse en el cultivo celular u otros modelos animales. Las principales ventajas de nuestra técnica son: es rápida y eficiente, requiere una cantidad muy pequeña de productos químicos, y...