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Obtaining vegetable dyes as a didactic
resource in Biology laboratories
Obtención de Colorantes Vegetales como Recurso
Didáctico en los Laboratorios de Biología
Raúl Pozo-Zapata
Universidad Central del Ecuador, Quito, Ecuador
Facultad de Filosofía Letras y Ciencias de la Educación, Carrera de Pedagogía de las
Ciencias Experimentales Química y Biología
rpozo@uce.edu.ec
https://orcid.org/0000-0002-2808-9946
Adriana Barahona-Ibarra
Universidad Central del Ecuador, Quito, Ecuador
Facultad de Filosofía Letras y Ciencias de la Educación, Carrera de Pedagogía de las
Ciencias Experimentales Química y Biología
abarahonai@uce.edu.ec
https://orcid.org/0000-0003-2196-1954
Jonathan Tigasig-Urcuango
Universidad Central del Ecuador, Quito, Ecuador
Facultad de Filosofía Letras y Ciencias de la Educación, Carrera de Pedagogía de las
Ciencias Experimentales Química y Biología
jjtigasig@uce.edu.ec
https://orcid.org/0009-0009-2466-9669
Marxuri Vivar-Toapanta
Universidad Central del Ecuador, Quito, Ecuador
Facultad de Filosofía Letras y Ciencias de la Educación, Carrera de Pedagogía de las
Ciencias Experimentales Química y Biología
mvvivar@uce.edu.ec
https://orcid.org/0009-0003-6492-9273
(Received on: 19/10/203; Accepted on: 30/11/2023; Final version received on: 08/01/2024)
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Suggested citation: Pozo-Zapata, R., Barahona-Ibarra, A., Tigasig-Urcuango, J. y Vivar-
Toapanta, M. (2023). Obtaining vegetable dyes as a didactic resource in biology
laboratories. Revista Cátedra, 7(1), 109-125.
Abstract
Natural dyes are millenary discoveries that have been used since ancient Greece to color
numerous objects such as sculptures but have lost their usefulness and have been replaced
by artificial dyes that can be harmful to health. The purpose of this study is to obtain a
natural vegetable dye from the hibiscus flower using vinegar as a solvent, to be used as a
didactic resource in the experimental practices of the biology laboratory. The hypothesis of
the study indicates that a useful colorant can be obtained from the calyxes of the hibiscus
flower, demonstrating its function in the experimental observations in the practices of
mitosis and meiosis. The method used was descriptive and observational since
experimental activities were carried out applying the reagent and dye obtained. The dyes
and reagents required for such practices are of high cost and those obtained as a result of
this research were of low cost, the acetic orcein in the market has a value of $31.3 for 60 ml,
while in the research carried out the cost of the pigment was $0.76 for 60 ml and the reagent
that replaced the hydrochloric acid that in the market costs $6 for 500 ml, the reagent
obtained had a value of $2.36 for 500 ml; the products obtained had a similar performance
to those acquired in the industry. The results contribute with resources that will allow for
better.
Keywords
Learning, dyes, experimental practices, didactic resources, vinegar.
Resumen
Los colorantes naturales son descubrimientos milenarios que se utilizaban desde la Grecia
antigua para dar color a numerosos objetos como las esculturas, pero que han perdido su
utilidad siendo sustituidos por los colorantes artificiales que pueden resultar dañinos para
la salud. El propósito de este estudio es obtener un colorante vegetal natural a partir de la
flor de jamaica empleando como solvente el vinagre, para utilizarlo como recurso didáctico
en las prácticas experimentales del laboratorio de Biología. La hipótesis del estudio indica
que se puede obtener un colorante útil de los cálices de la flor de jamaica demostrando su
funcionamiento en las observaciones experimentales en las prácticas de mitosis y meiosis.
El método empleado fue descriptivo y de observación, ya que se realizaron actividades
experimentales aplicando el reactivo y el colorante obtenidos. Los colorantes y reactivos
que se requieren para dichas prácticas son de alto costo y, los que se obtuvieron como
resultado de esta investigación fueron de bajo costo, la orceína acética en el mercado tiene
el valor de $31,3 los 60 ml, mientras que en la investigación realizada el costo del pigmento
fue $0.76 los 60 ml y el reactivo que reemplazó al ácido clorhídrico que en el mercado cuesta
$6 los 500 ml, el reactivo obtenido tuvo un valor de $2.36 los 500 ml; los productos
obtenidos tuvieron un funcionamiento similar a los que se adquieren en la industria. Los
resultados aportan con recursos que permitirán mejorar la calidad de la enseñanza–
aprendizaje en el laboratorio de Biología.
Palabras clave
Aprendizaje, colorantes, prácticas experimentales, recursos didácticos, vinagre.
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1. Introduction
Obtaining dyes from vegetables is one of the activities that were performed for many
centuries as evidenced by the objects found in cultures since Greece. According to Yusuf,
Shabbir, and Faqeer (2017).
nature has always dominated the synthetic or artificial, since the
beginning of this world since nature was the only option for humans to
acquire elements, materials of natural origin present characteristics that
are advantageous over synthetics, giving them priority (p. 124).
"New technologies are allowing the obtaining of pigments from natural dyes, making them
efficient, causing synthetic pigments to be rejected" (Cuesta, 2018, p. 1). The high cost of
dyes in the industry where, the value of acetic orcein which is a pigment to give color to
chromatin reaches $31.3 the 60 ml in the chemical industry. The scientific experience allows
the student to give answers to questions, strengthening their meaningful, lasting and
practical learning, enriching their scientific knowledge, in the certainty of being able to live
in this world without destroying it and keeping it in good condition for future generations.
"Constructivism involves the student to be the constructor and protagonist of his own
learning, who brings his knowledge that serves as a support to strengthen the new
knowledge" (Miranda-Núñez, 2022, p. 81).
Experimental activities have been considered as a methodological and didactic instrument
that supports the academic-scientific training process of students. Teachers who teach
subjects in the area of natural sciences frequently incorporate laboratory practices based
on a pedagogical model, adapting them to the peculiarities of the environment, to the usable
inputs, and to the training needs, since, in addition to supporting the theoretical classes,
they awaken and develop the curiosity of the students, motivating them to solve problems
(Zorrilla et al., 2022).
The purpose of the research is the extraction of the dye from the calyxes of the hibiscus
flower using vinegar as a solvent to substitute acetic orcein, which is commonly used in
laboratory practices for the observation of the phases of mitosis and meiosis (chromatin);
likewise, lemon and vinegar will substitute hydrochloric acid in the softening of the plant
tissues of the samples used in these practices. This dye can be used in Biology laboratories
as a didactic resource for the realization of experimental practices, considering that the
experiences obtained in the laboratory are fundamental in the teaching-learning process.
This will allow the participation and development of students as active beings who
construct their own knowledge, obtaining a scientific experience, since they will develop
investigative thinking for the understanding of natural phenomena. This knowledge and
information can have a new application in the field of formal education and could contribute
to the creation of alternative didactic tools to improve the educational process of biological
sciences. Hence, it has become mandatory to look for alternatives to obtain dyes by
experimental methods such as the one presented in this research, where through the
calyxes of hibiscus flowers, the dye was obtained at a cost of $0.76 for 60 ml of the dye.
In this sense, it is necessary to stimulate the biological conditions that allow human beings
to construct knowledge. Science teaching is carried out based on pedagogical practices in
an interaction that focuses on teaching and learning the contents of the scientific discipline.
This space requires a constructivist and contextualized educational practice. The
knowledge based on the production of vegetable dyes constitute constructs in the biological
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sciences, the teacher must deepen in the inquiry which will allow the student to reflect and
conceptualize using knowledge and knowledge derived from nature.
Regarding the structure of the article, section 1 contains the introduction in which the
reader is familiarized with the context of the work. Section 2 presents the theoretical
reference where the documented information on the research topic is gathered. Section 3
details the methods and instruments used to develop this research. In section 4, the results
obtained as a result of the study carried out in a theoretical and practical manner are
detailed. Section 5 shows the discussion in which the results are extrapolated, interpreted
and summarized. In section 6, conclusions are drawn according to the results obtained.
2. Theoretical reference
It is possible that the taste for color in human beings is the result of the change of the seasons
of the year, and has been shaped by the use of pigments obtained from plants and flowers
and even blood and ashes. The accelerated development according to Dupey, and the
flourishing of craftsmanship supported mainly by the establishment of Mexican territories,
provoked and stimulated the leap from basic dyes (made with earth) to a wider and wider
range of dyes. For this reason there was a variety of resources with which colorations
applicable in pottery, fabrics, ideograms, rituals, food, and even in the bodies of kings,
priests and warriors were obtained (Dupey, 2016). In this regard, Villaño et al. (2016)
mention that "the origin of dyes and their classification are due to their biological properties
and based on the biological characteristics that dyes possess, the features of their physical
properties to their chemical structures are evident" (p. 1).
Thus, according to Valenzuela and Pérez, a variety of colors can be obtained using
vegetables, fruits and spices that have diverse applications, since they can be used to dye
fabrics, paint wood, color canvases, color homemade soaps, among others. Plant pigments
include a wide variety of ranges of components and colors, among the most important we
can mention flavonoids, anthocyanins, carotenoids, betalains, chlorophylls among others
(Valenzuela and Perez, 2016). "Since the appearance of synthetic dyes in the industry,
natural pigments have been relegated, since the synthesized ones present greater stability,
wide range of colors and lower cost" (Rolón, 2018, p. 11).
Amchova et al. (2015) state that "in the industry, a series of tests are carried out for the use
of different colorants in order to detect the different forms of toxicity" (p. 2). "Natural type
colorants are obtained from foods such as fruits and vegetables with intense color and
through maceration or boiling processes the colorant can be extracted" (Ulloa, 2017, p. 3).
The following table shows the types of colorants with characteristics and examples.
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Natural
Artificial
Vegetables
Animals: hematoxylin
Synthetics: obtained by
fractional distillation of the
footprint: Gentian violet
Tornasol, saffron.
Direct or substantive:
methylene blue and
hematoxylin.
Indirect: ferric
hematoxylin.
Indirect reversible: indirect
hematoxylin.
Dyes that require a
chemical reaction or
development: periodic
Schiff's acid.
Dispersed: sudans
Metachromatics: toluidine
blue.
Reagents: allow superior
durability and
homogeneity.
Hybrids: higher resistance
to degradation by radiation
at temperature.
By chemical affinity: blue
generated by
epifluorescence.
Acids: sulfonium and carboxyl groups
Basic: contain amino groups with organic
groups to form a salt.
Table 1. Dye classes Source: (Garrido, 2021).
Thus, "There is little knowledge of the properties of natural colorants" (Ulloa, 2017, p. 2).
According to Galarza (2013)
the production of synthetic pigments is no longer as convenient for
producers and buyers, as costs are high, and they are perceived by the
public as hazardous to health and the environment, and products
containing them are consumed in increasingly smaller quantities (p. 7).
In the words of Jácome et al. (2023) currently, there is "a great interest in natural additives,
especially colorants, because these natural compounds have no associated side effects and
most of them are functional ingredients, acting as health promoters" (pp.1477-1478). Due
to research that has been advanced in reference to the toxicity of synthetic pigments "the
interest in natural colorants is growing daily as a result of the continuous eliminations of
artificial colorants" (Marcano, 2018, p. 8). Nowadays, people pay more attention to the
ingredients in their food and the resources they use on a daily basis.
Hibiscus flowers also called Guinea sorrel, Obelisk, Rosamorada or Karkadé belongs to the
Malvaceae family and its scientific name is Hibiscus sabdariffa (Table 2), it is native to Africa
where it is considered a very important plant both in its medicinal and nutritional
properties, it can reach up to 3 meters in height and its reproduction is carried out by self-
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fertilization. The hibiscus "is a source of bioactive compounds such as polyphenols,
flavonoids, ascorbic acid, among others; this composition gives it antioxidant activity"
(Sumaya et al., 2014, p. 2).
Realm
Plantae
Sub-realm
Tracheobionta
Division
Magnoliophyta
Type
Magnoliopsida
Sub-type
Dieeniidae
Hierarchy
Malvales
Family
Malvaceae
Subfamily
Malvoideae
Genre
Hibiscus
Species
Hibiscus sabdariffa L.
Common name
Jamaica
Table 2. Taxonomic characterization of hibiscus Source: (Moposa, 2019).
)
According to Sumaya (2014) "the composition of hibiscus exerts pharmacological effects
that produce therapeutic functions in the body that are beneficial to health" (p. 3). According
to Pantoja (2022) "hibiscus has different uses, as a medicinal plant to lower cholesterol,
triglycerides, decreases body weight, stimulates the functioning of the liver and kidneys and
contributes in the absorption of certain minerals." (p. 27). Sumaya (2014) states that
the cultivation of hibiscus is widespread in Mexico, Central and South
America and Southeast Asia, its cultivation and production is expensive
because it is often damaged by excessive rainfall, drought or pests, and
its harvest requires a high amount of labor to avoid contamination and
loss of safety (p. 2).).
It is also known to possess phytochemical compounds such as flavonoids, phenolics, b-
carotene, polysaccharides and ascorbic acid. Cruz-Moreno et al. point out that the persistent
red color in its calyxes is what gives flavor and color to infusions and prepared beverages,
this is due to the content of anthocyanins and the acid flavor to the content of organic acids
such as malic, citric, hibiscus and tartaric acid (Cruz-Moreno et al., 2020). On the other hand,
flowers contain several natural antioxidant compounds that also act against various viruses
and bacteria. One of them "is hisbiscus acid and its derivatives, chemical compounds with
antimicrobial properties were identified in them" (Portillo-Torres et al., 2019, p. 2). In the
calyxes are found: anthocyanins 1.5 percent, organic acids 15-30 percent, mucilaginous
polysaccharides 50 percent, flavonoids, saponins, phytosterols, pectin and fiber. The
organic acids and anthocyanins have been shown to have antimicrobial and antioxidant
activity. All these components have good bioavailability and have shown therapeutic
potential. Other phytochemical components that calyxes possess "are composed of
elements rich in anthocyanins, phenolic acids, flavonoids and organic acids." (Izquierdo-
Vega, et al., 2020, p. 3).
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Jamaica, according to Urbina is an annual herbaceous plant, Malvaceae family that often
reaches 1 to 2 meters in height, the stem, leaf petiole and calyxes have a dark or light red
color with a tendency to purple or lilac; the flowers are usually born solitary in the axils of
the leaves with yellowish petals, and red calyx that usually take one to two days to fall then
appear the apices and the fruit or capsule of 5 compartments. The mature plant enveloped
by the fleshy calyx is ovoid in shape and contains numerous reniform seeds, pubescent with
reddish thread, which take 3 to 4 weeks to develop (Urbina, 2009). Figure 1 summarizes the
process of the Jamaican flower.
Figure 1. Processing of Jamaica
The process for the commercialization of the hibiscus flower has nine stages (Figure 1). "At
the time of ripening of the first calyxes and capsules, the plant is cut from the root, then the
calyxes are separated and promptly taken to dehydration" (Rosado, 2020, p. 40). According
to Galarza, there are several techniques for the separation of substances such as extraction
that allows the organic product to be separated from a reaction mixture or to be isolated
from its natural sources, observing the formation of two phases so that the process can be
carried out: in the first case a solid and a liquid phase, while in the second case there are
two immiscible liquid phases (Galarza, 2013).
There is a diversity of plants that "present special characteristics in their structure that
generate pigments such as the red Geranium (Pelargonium hortorum) that has a pale green
center contrasting with the margin and flowers arranged in umbels of red, violet pink or
white colors" (Estrada, 2021, p. 6). "As for the Red Dahlia (Dahlia pinnata), the expansion of
a number of types, colors and sizes offers a solid possibility to the increase of its production"
(Jimenez, 2015, p.). "Beetroot (beta vulgaris) is grown mainly for its juice and nutritional
value and its roots are blood red with thin skin" (Lopez et al., 2019, p. 368). "Red bell pepper
(Capsicum annuum) its odor and color make it attractive for consumption, come from its
biochemical components: flavonoids, phenols, epicatechin, rutin, β-carotene, capsanthin,
lutein, resveratrol and acids: gallic, chlorogenic and ascorbic" (Martinez et al., 2016, p. 38).
Knowing the properties of plants helps the development of experimental practices. Hence,
Jamaica flower
9
5
6
7
8
1
2
3
4
Marketing
Transport
Packaging
Calyx selection
Drying
Cleaning
Collection
Harvest
Storage
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according to López and Tamayo, they are fundamental in the teaching and learning process
of science, since, by relating theory with practice, the development of skills and abilities is
promoted, the promotion of reasoning abilities, critical and creative thinking providing the
student with the construction of knowledge and the strengthening of science with society
and culture (López and Tamayo, 2012). Directing the educational process of Natural
Sciences in function of satisfying the expressed demands "involves thinking and reasoning,
as he observes and experiences the different situations, which allow him to act and establish
his own theoretical understanding of the surrounding reality and everything related to it"
(Paladines-Condoy et al., 2018, p. 59).
Knowledge management is increasing so it is important to address issues related to
innovation and updating of the educational system. Precisely, education must "establish
methodological strategies for the development of the class, with the enormous purpose of
obtaining positive results for the improvement of teaching in its scientific development"
(Calero, 2019, p. 10). Biology laboratory practices should be "encouraged in students by
applying simple steps and feasible and easily available domestic resources to strengthen
creativity" (Susantini et al., 2017, p. 216). In the Natural Sciences teacher should "prevail
the need to be trained to be the student's guide to be a more independent being in the search
and assimilation of scientific knowledge through experimentation" (Ramirez, 2023, p. 6).
According to Castellanos (2017) "teachers seek different skills oriented to teaching not only
theory, but also towards the convergence of theory and practice, so that there is true
meaningful learning" (p. 235). In the same sense, Gil-Álvarez et al. (2017) point out that "the
critical-social paradigm, considers the dialectical unity of the theoretical and the practical,
as an inseparable whole" (p. 74). It has become visible that stimulation and imagination play
an important role in learning and understanding science (Gómez, Ortega, & Lafaid, 2017;
González & Palomeque, 2017).
For Reyes (2020) the laboratory is "a great opportunity where the student verifies
theoretical concepts that constitute the first contact with the reality that the future
professional will face" (p. 63). The use of experimental practices from the constructivist
framework "strengthens knowledge and achieves the development of scientific
competencies by promoting student participation, so that they are the ones who propose
and execute practices that address conceptual, procedural and attitudinal dimensions"
(Espinoza-Ríos et al., 2015).
For this reason, it is necessary to stimulate the biological conditions that allow human
beings to construct knowledge. Science teaching is carried out based on pedagogical
practices in an interaction that focuses on the teaching and learning of contents specific to
the scientific discipline. This requires a constructivist and contextualized educational
practice. The knowledge based on the production of vegetable dyes constitutes constructs
in the Biological Sciences; the teacher must deepen in the inquiry, which will allow the
student to reflect and conceptualize using knowledge and knowledge derived from nature.
Coronado and Arteta indicate that students are expected to strengthen some skills, for
example, seek or formulate reasons to phenomena or problems, create logical and
propositional arguments of what is perceived, explain equal phenomena using conceptual
characters appropriate to different degrees of complexity, establish cause-effect
relationships, combine ideas in the construction of texts, employ mathematical ideas and
techniques (Coronado and Arteta, 2015, p. 9). For this, didactic resources that are
fundamental in the development of laboratory practices in Biological Sciences are required,
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such as dyes, since they have been able to make organic matter visible and to identify the
cellular structure that allows life to exist.
3. Methods and instruments
En la investigación se aplicó el método descriptivo y de observación, ya que se realizaron
prácticas experimentales, aplicando el reactivo y colorante obtenidos, mismos que
permitieron la visualización de las diferentes fases que conforman la meiosis y mitosis.
Dichas prácticas fueron realizadas adaptando los protocolos de Andrade et al. 2005 en los
que se observa células sexuales y somáticas vegetales.
3.1 Material and Methodology
The material selected to replace acetic orcein which in the market reaches a price of $31.3
per 60 ml, was hibiscus flower which produces a very strong dye used in the manufacture
of textiles (Arrascue, 2018), and its cost is $0.84 per 30g and vinegar was used as solvent
which has a cost of $0.86 per 500 ml. Therefore, the total cost of dye obtained in the research
(acetic soda
1
) is $0.76 per 60 ml. On the other hand, the reagent (hydrochloric acid) with
which the softening of the vegetable tissues is carried out has a cost in the chemical industry
of $6 for 500 ml, being replaced by lemon juice which has a value of $2.75 for 700 g and
vinegar at 5%, thus the definitive value of the reagent that replaced the hydrochloric acid
(Auran acetic acid
2
) is $2.36 for 500 ml.
3.2 Obtaining the dye
3.2.1 Phase 1 test:
As shown in Table 3, prior to the final standardization of the dye, different plant tissues
possessing a red color were tested, thus 20 ml of 5% vinegar was diluted with 6 g of red
geranium Elargonium hortorum, 20 ml of 5% vinegar with 2. 3 g of red dahlia Dahlia
pinnata, 50 ml of 5% vinegar with 40 g of beet Beta vulgaris and 20 g of red bell pepper
Capsicum annuum with 20 ml of 5% vinegar and did not obtain a pigment that allows to
clearly color the chromosomes in the nucleus.
3.2.2 Phase 2 preparation of plant extract
The calyxes of hibiscus flowers available in the city's markets were acquired and those in
good condition were selected. Then, to extract the dye, 6 g of calyxes were subjected to two
tests, one with 70% alcohol and the other with 5% vinegar, which were used as solvents,
leaving them to stand at room temperature for 48 hours in an amber-colored flask. Then,
the dye was filtered and it was observed that the vinegar extracted the pigment from the
calyxes with a higher concentration.
3.2.3 Sample Standardization Phase 3
Different amounts of 5% vinegar were tested with different grams of Jamaican flower
calyxes (Table 4), showing that 6 g of calyxes and 20 ml of 5% vinegar resulted in an
optimum violet color for coloring the chromosomes in mitosis and meiosis. This substance
1
Sabda acetic acid is the name given to the dye obtained as a result of this project that replaces acetic
orcein in chromatin observation.
2
Auran acetic is the name given to the reagent obtained as a result of this research that replaces
hydrochloric acid in the softening of plant tissues.
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was left to stand at room temperature for 48 hours in an amber-colored flask, after which
the dye was filtered to remove organic residues.
3.3 Obtaining the reagent
3.3.1 Quantity and time modification Phase 1
1 ml of lemon juice was extracted and mixed with 1.5 ml of 5% vinegar, this reagent was
added to the root apices of Allium cepa and the anthers of Lilium candidum, placing these
elements on different watch glasses for 10 minutes, no flaming was used in this phase and
there was no softening of plant tissue (see Table 5).
3.3.2 Phase 2 quantity and time modification 2
1.5 ml of lemon juice was extracted and mixed with 1.5 ml of 5% vinegar, this reagent was
placed on the root apices of Allium cepa placed on a watch glass for 18 minutes. The same
process was carried out with the anthers of Lilium candidum. No flaming was used and there
was no softening of plant tissues (see Table 5).
3.3.3 Pre-standardization Phase 3
1.5 ml of lemon juice was extracted and mixed with 1 ml of 5% vinegar, this mixture was
placed on the root apices of Allium cepa placed on a watch glass, 2 intervals of flaming for 1
minute and 2 intervals of rest of 1 minute were performed, similar process was performed
with the anthers of Lilium candidum, only the external tissue was softened (see table 5).
3.3.4 Phase 4 reagent standardization
In 1 ml of lemon juice and 1 ml of 5% vinegar, this reagent was placed on the root apices of
Allium cepa located on a watch glass where 4 intervals of flaming for 1 minute and 4
intervals of rest of 1 minute were carried out, with the anthers of Lilium candidum the same
steps were followed. There was total softening of the root tissue and anthers (see Table 5).
4. Results
In graphs 2, 3, 4 and 5 it is appreciated that the obtained colorant (Sabda acetica) colors in
a similar way to acetic orcein, the obtaining of this pigment was made through the
dissolution of 6 g of calyxes of the hibiscus flower in 20 ml of vinegar at 5% (see table 4)
and to replace the hydrochloric acid that allows the softening of the vegetable tissues 1 ml
of lemon juice was used with 1 ml of vinegar at 5% (Auran acetica) for a total of 8 minutes.
By combining the flaming (4 intervals of 1 minute) with the resting time (4 intervals of 1
minute), which should be interspersed with 1 minute of flaming and 1 minute of resting to
complete the 8 minutes established for softening (see Table 5). This process allowed
coloring and softening the plant tissues in a similar way to the procedure developed with
acetic orcein and hydrochloric acid in the observation of the phases of mitosis and meiosis
(graphs 2, 3, 4 and 5).
Species
Grams of the species
5% vinegar
Red geranium
Elargonium hortorum
6 grams
20 ml
Red Dahlia
Dahlia pinnata
2.5 grams
20 ml
Beets
Beta vulgaris
40 grams
50 ml
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Table 3. Obtaining the material
Table 3 shows the experimental procedure describing how the optimum biological material
was chosen for the elaboration of the dye, which was obtained by relating the weight of the
different flowers of the various species with different ml of 5% vinegar.
Jamaica
5% Vinager
Color
3 grams
35 ml
Pigmented red
4 grams
35 ml
Munsell Red
4 grams
30 ml
Pantone Red
5 grams
25 ml
Crimson Red
6 grams
20 ml
Violet red
Table 4. Dye concentration standardization process.
Table 4 shows the experimental procedure describing the standardization of the colorant,
which was obtained by relating the weight of hibiscus to the ml of 5% vinegar.
Test of
experimentation
Lemon
5%
vinegar
Flamed
Resting
time
Result
1
1 ml
1.5 ml
0
minutes
10
minutes
No flaming was used.
No tissue softening.
2
1.5ml
1.5 ml
0
minutes
18
minutes
No flaming was used.
No softening of the
fabric.
3
1.5 ml
1ml
2
intervals
of 1
minute
2 intervals
of 1
minute
Joined flaming
intervals with rest
intervals.
Softening of external
tissue only.
4
1 ml
1ml
4
intervals
of 1
minute
4 intervals
of 1
minute
Joined flaming
intervals with rest
intervals.
Total softening of
plant tissue.
Table 5. Softening process of plant tissue for the observation of meiosis and mitosis.
Red bell pepper
Capsicum annuum
20 grams
20 ml
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En el cuadro 5 se aprecia el procedimiento experimental. Allí se describe la estandarización
de la substancia que permitió el ablandamiento de las raíces de cebolla Allium cepa y anteras
de lirio Lilium candidum para observar las fases mitosis y meiosis respectivamente.
Graph 2 clearly shows the mitosis carried out with onion roots (Allium cepa) using the
organic dye hibiscus (Sabda acetica) and the softening of the plant tissue with the mixture
of lemon juice and vinegar at 5% (Auran acetica), which allowed the visualization of the
phases. Figure 3 shows the mitosis using the dye acetic orcein and hydrochloric acid to
soften the plant tissue, showing that the dye produced with calyxes of hibiscus flowers
pigments in the same way as with acetic orcein and the mixture of lemon juice with vinegar
softens the plant tissues as well as hydrochloric acid.
Figure 4 clearly shows the meiosis carried out with onion lily (Lilium candidum) anthers
using the organic dye of hibiscus (Sabda acetica) and the softening of the plant tissue with
the mixture of lemon juice and vinegar at 5% (Auran acetica), which allowed the
Figure 2. Application of the dye Sabda acetica
for the observation of the phases of mitosis in
root tissue of Allium cepa.
Figure 3. Application of acetic orcein dye for the
observation of mitosis phases in root tissue of
Allium cepa.
Figure 4. Application of Sabda acetic dye
for the observation of meiosis phases in
anthers of Lilium candidum.
Figure 5. Application of Sabda acetic dye for
the observation of meiosis phases in anthers
of Lilium candidum.
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visualization of the phases. Figure 5 shows the meiosis using the dye acetic orcein and
hydrochloric acid to soften the plant tissue, demonstrating that the dye produced with
calyxes of hibiscus flowers pigments in the same way as with acetic orcein and the mixture
of lemon juice with vinegar softens the plant tissues as well as hydrochloric acid.
5. Discussion
The dyes found in the chemical industry used for laboratory practices are of high cost, which
prevents the execution of experimental practices in educational institutions both at middle
and higher levels. Therefore, it is necessary to look for new alternatives for the elaboration
of low-cost dyes that are affordable, such as the one developed in this research (Sabda
acetica), so that the practical component can be carried out in subjects such as Biology in
educational institutions that do not have the necessary economic resources.
Similarly, for the procedure of softening plant tissues, hydrochloric acid is commonly used
for experimental practices; however, it was proposed to obtain a substitute that fulfills the
same function of this reagent from the mixture of lemon with vinegar (Auran acetic acid),
demonstrating that its application has the same function as hydrochloric acid. It should be
emphasized that the methods used in the experimental part established the basis for the
collection of data and later analysis of the information obtained. At the time of the
experimentation, several tests were carried out until reaching an effective standardization,
once the softening and staining process was established, it was possible to observe the
division of plant cells.
6. Conclusions
Plant dyes are indispensable resources that have diverse applications in the industry and as
didactic resources in experimental practices in the teaching-learning process of biology;
therefore, it is important to strengthen research on obtaining this type of dyes. The study
employed a technique that significantly reduces the economic resources that are invested
to obtain the inputs that are used to carry out the laboratory practices in plant and animal
tissues in which chromatin (chromosomes in mitosis, meiosis, polytene chromosomes and
Barr bodies) can be clearly observed.
Generally, for the softening process of plant tissues, reactive hydrochloric acid is required,
which is expensive and presents a risk in its handling during experimentation. However,
mixing 1 ml of lemon juice plus 1 ml of 5% vinegar for 8 minutes and flaming this compound
for 4 minutes minimizes the danger and cost of its use. Acetic orcein is a high-cost dye used
in experimental practices to stain chromatin, which makes it necessary to look for
alternatives to obtain dyes that fit the budget of educational institutions. In the research
carried out after developing several tests, the dye was standardized by diluting 6 g of calyxes
of hibiscus flowers with 5% vinegar, which allowed a similar or better visualization of
chromatin in plant tissues in comparison with acetic orcein.
The resources used to replace acetic orcein, which in the market reaches a price of $31.3
per 60 ml, were the calyxes of the hibiscus flower that produce a very strong dye and its cost
is $0.84 per 30g and vinegar was used as solvent, which has a cost of $0.86 per 500 ml, the
total cost of the dye obtained in the research (acetic soda) is $0.76 per 60 ml. On the other
hand, the reagent (hydrochloric acid) used to soften the vegetable tissues has a reference
value in the chemical industry of $6 per 500 ml, being substituted by lemon juice which has
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a value of $2.75 per 700 g and vinegar at 5%. The definitive value of the reagent that
replaced hydrochloric acid (Auran acetic acid) is $2.36 per 500 ml.
Acknowledgment
We would like to express our gratitude to the First International Congress of Experimental
Sciences organized by the Pedagogy Career of Experimental Sciences, Chemistry and
Biology of the Faculty of Philosophy, Letters and Education Sciences of the Central
University of Ecuador, which took place from July 31 to August 4, 2023. This congress
allowed to share experiences, dialogue, and reflect on educational topics that strengthen the
teaching-learning process.
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Authors
RAÚL POZO-ZAPATA obtained his degree in Education Sciences. High school teacher
specializing in Chemistry and Biology. Doctor in Biology. Master in Educational
Management. Doctorate in the Doctoral Program in Educational Research.
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Currently teaching Molecular Biology and Genetics at the Universidad Central del Ecuador,
Faculty of Philosophy, Letters and Education Sciences, Pedagogy, Letters and Education
Sciences.
ADRIANA BARAHONA-IBARRA obtained her Bachelor's degree in Education Sciences,
specializing in Biological Chemistry. Master in University Teaching and Educational
Administration. Doctorate in the Doctoral Program in Educational Research.
Currently, she is a research professor of Chemistry and director of the Pedagogy of
Experimental Sciences, Chemistry and Biology at the Universidad Central del Ecuador,
Faculty of Philosophy, Letters and Educational Sciences.
JONATHAN TIGASIG-URCUANGO Currently a sixth semester student of the Pedagogy of
Experimental Sciences, Chemistry and Biology at the Universidad Central del Ecuador,
Faculty of Philosophy, Letters and Education Sciences, student of outstanding academic
performance and interest in research projects.
MARXURI VIVAR-TOAPANTA Currently a sixth semester student of the Pedagogy of
Experimental Sciences, Chemistry and Biology at the Universidad Central del Ecuador,
Faculty of Philosophy, Letters and Educational Sciences Student of great talent for research
and academia.