Education and Information Technologies

The use of image analysis in the improvement of knowledge and skills on the example
of teaching the biology of lower plants
--Manuscript Draft--

Manuscript Number: EAIT-D-24-00456

Full Title: The use of image analysis in the improvement of knowledge and skills on the example
of teaching the biology of lower plants

Article Type: Manuscript

Keywords: active learning; information technologies; non-vascular plants; science education;

teamwork

Abstract: This study monitored the impact of image analysis-focused teaching on gymnasia
students’ science process skills (SPS) and knowledge (using software Fiji) within the
subject of the so-called lower plants (algae and fungi). A total of 53 gymnasia students
(ISCED3a) from three independent classes participated in the study. The theoretical
teaching was followed by a practical one, during which the students, using the
mentioned software, measured the area or signal intensity on photos from scientific
papers (algae Ulva or Coccomyxa and the fungus Glomerella) and then formed
conclusions from the data and answered knowledge questions. Before the education
(in the pre-test), the students had problems with formulating assumptions and
hypotheses, data interpretation or argumentation, but they had no problem e.g. with
the creation of a graph. Post-test results showed that experimental teaching increased
the students’ science process skills/SPS (+41.5%) as well as their knowledge (+97.5%)
in comparison with pre-test score. Among the classes, students of 8-year gymnasium
program achieved greater improvement in SPS (+62.6%) than those of 4-year program
(+ up to 33.4%) and absolute post-test score was also higher. Results also showed
that experimental teaching had a more positive effect on the knowledge and SPS of
girls (overall +51.5%) than boys (overall +45.2%) and absolute score differed too. The
knowledge test and worksheets are attached for free use. The worksheets represent a
good basis for the use of IT tools even for topics less interesting to students, such as
botany and especially lower plants.

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1 Abstract
1
2 2 This study monitored the impact of image analysis-focused teaching on gymnasia students’
3
4
5 3 science process skills (SPS) and knowledge (using software Fiji) within the subject of the so-
6
7 4 called lower plants (algae and fungi). A total of 53 gymnasia students (ISCED3a) from three
8
9
10 5 independent classes participated in the study. The theoretical teaching was followed by a
11
12 6 practical one, during which the students, using the mentioned software, measured the area or
13
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7 signal intensity on photos from scientific papers (algae Ulva or Coccomyxa and the fungus
15
16
17 8 Glomerella) and then formed conclusions from the data and answered knowledge questions.
18
19 9 Before the education (in the pre-test), the students had problems with formulating assumptions
20
21
22 10 and hypotheses, data interpretation or argumentation, but they had no problem e.g. with the
23
24 11 creation of a graph. Post-test results showed that experimental teaching increased the students’
25
26
27 12 science process skills/SPS (+41.5%) as well as their knowledge (+97.5%) in comparison with
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29 13 pre-test score. Among the classes, students of 8-year gymnasium program achieved greater
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14 improvement in SPS (+62.6%) than those of 4-year program (+ up to 33.4%) and absolute post-
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34 15 test score was also higher. Results also showed that experimental teaching had a more positive
35
36 16 effect on the knowledge and SPS of girls (overall +51.5%) than boys (overall +45.2%) and
37
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39 17 absolute score differed too. The knowledge test and worksheets are attached for free use. The
40
41 18 worksheets represent a good basis for the use of IT tools even for topics less interesting to
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44 19 students, such as botany and especially lower plants.
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46 20
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49 21 Keywords: active learning; information technologies; non-vascular plants; science education;
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51 22 teamwork.
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 23 1. Introduction
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2 24 In the last 100 years, there has been a large increase in scientific information about plants, but,
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5 25 in the same period, there has also been a decline in the level of students’ knowledge of botany
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7 26 (Link-Pérez and Schussler 2013). Also, to overcome the challenges of the 21st century in the
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10 27 science and technology sector, students need to be equipped with skills to ensure their
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12 28 competitiveness in the globalization era (Turiman et al. 2012). There is an international
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29 consensus that science education should support not only knowledge but also students’ SPS
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17 30 (science process skills) development (García-Carmona et al. 2023). One way to improve
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19 31 students’ knowledge and SPS is the use of information technologies (IT), including image
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22 32 analysis, in education (Vebrianto and Osman 2011, Helendra et al. 2018, Haglund and
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24 33 Schönborn 2019, Petersen 2021, Wong and Subramaniam 2022).
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27 34 One of the consequences of technological development is the common use of image
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29 35 analysis in various biological fields (Schindelin et al. 2015). Microscopic techniques as well as
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36 modern technologies allow biologists to obtain a large amount of data in the form of images
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34 37 that can be analyzed by IT. For example, a 10% decrease in cell size or a 2-fold decrease in the
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36 38 intensity of a fluorescently labeled protein in an image is undetectable to the human eye, but
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39 39 image analysis using Fiji/ImageJ software can detect such negligible but biologically significant
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41 40 changes (Baviskar et al. 2011). This software is freely available, user-friendly, and easy to
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44 41 install. Although image analysis is widely used in research, its implementation in the teaching
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46 42 process is rather rare (Petersen 2021). There are several suggestions for activities using image
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49 43 analysis, e.g. neurite development (Pemberton et al. 2018), marine biodiversity (Gerringer et
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51 44 al. 2023), protein structure (Gokalp et al. 2018), RNA interference (Palmisano et al. 2020) or
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45 zebrafish (Danio rerio) nerve development (Petersen 2021). The mentioned activities are
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56 46 professionally demanding and applicable only in university education. For example, university
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58 47 students reported that education using image analysis improved their skills in the interpretation
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 48 of results, they had a better understanding of the research process and learned to work
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2 49 independently (Gerringer et al. 2023). In the Scopus database, we did not find works that were
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5 50 devoted to the use of image analysis in secondary education. To our knowledge, no paper
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7 51 studied the impact of the educational implementation of image analysis on students’ knowledge
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10 52 or science process skills in the area of plant biology. Indeed, a recent review focused on learning
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12 53 activities with plants and technology reported that only 1 out of the 9 analyzed studies involved
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54 a non-vascular plant (Leo-Ramírez et al. 2023).
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17 55 The present study aims to create practical activities with the use of image analysis that
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19 56 can be used at lower levels of education and to verify the impact of the worksheets on the
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22 57 student’s knowledge and science process skills. The worksheets are focused on so-called lower
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24 58 plants, as teachers also use e.g. algae in the classroom a little (Kováčik and Vydra, 2023). The
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27 59 more complex questions in the knowledge test and in the subsequent teaching are intended to
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29 60 highlight the need for more comprehensive teaching in secondary schools (and we personally
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61 observe deficiencies in this area also in university students).
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34 62
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63 2. Methods
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39 64 2.1. Participants of the study
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41 65 Students of three independent classes from Gymnasium in the town Lipany participated in the
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44 66 study: two 2nd-grade classes in a 4-year program (2. A. and 2. B.) and one 6th-grade class in an
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46 67 8-year program (Sexta), ensuring that all students were 16-17 years old (see discussion for the
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49 68 comments on 4-year and 8-year program). A total of 53 students participated in the study
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51 69 (56.6% were girls and 43.4% boys). There was a total of 18 students in 2. A., 17 students in 2.
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70 B. and 18 students in Sexta. Gymnasium is a type of high school (ISCED3) that provides general
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56 71 upper secondary education and prepares students for further university education.
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58 72
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 73 2.2. Experimental education
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2 74 During 3 weeks of teaching focused on biology of so-called lower plants (algae and fungi), we
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5 75 performed six lessons in each class (each lesson lasted 45 minutes). Four lessons were devoted
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7 76 to algae and two lessons to fungi. For each topic, half of the lessons were taught theoretically
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10 77 (2 hours about algae and 1 about fungi) and half of the lessons consisted of activities using
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12 78 image analysis and worksheets (2 hours about algae and 1 about fungi). Theoretical lessons
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79 were taught with presentation focused on the body/thallus structure, ecology, significance and
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17 80 slightly systematics of algae and fungi. As part of the practical activities, students analyzed
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19 81 photos from randomly selected scientific works using the FIJI (ImageJ) program (for details
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22 82 about the program, see Schindelin et al. 2012). Students analyzed photos of the habitus of the
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24 83 cultivated alga Ulva fenestrata (Steinhagen et al. 2022), photos from fluorescence microscope
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27 84 of the alga Coccomyxa subellipsoidea (Kováčik et al. 2017) and photos showing the effect of
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29 85 salicylic acid (SA) on symptoms caused by the fungal pathogen Glomerella cingulata (Zhang
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86 et al. 2016). Students worked in four-member groups, but each student analyzed at least one
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34 87 photo in the given software. Worksheets focused on image analysis and respective tasks and
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36 88 questions are included the supplementary materials. We created 3 worksheets:
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39 89
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41 90 • Algae as food and a source of nutrients
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44 91 • Algae, oxidative stress and antioxidant
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46 92 • Fungal disease and Aspirin-related compound
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49 93
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51 94 The main aim of this experimental education was to monitor the impact of IT combined with
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95 real scientific data on students’ knowledge (understanding the importance of given organisms,
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56 96 the basic principles of oxidative stress and plant defense, and the interaction between
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58 97 organisms) and on the development of science process skills (check variables, formulate
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 98 assumptions, interpret data, form conclusions, formulate relationships between variables, and
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2 99 construct graphs).
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5 100
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7 101 2.3. Research tool
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10 102 We used the pre-test and post-test to measure changes in students’ knowledge and science
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12 103 process skills in response to experimental teaching. The test contained graphs (Kováčik et al.
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104 2016), table (Coşkun et al. 2023), and microscopic photos (Kováčik et al. 2019) from scientific
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17 105 papers. We monitored “knowledge” with 14 tasks and “science process skills” with 11 tasks.
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19 106 The test contained only open questions and for each correct answer, the student received one
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22 107 point (maximum score of 25 points). We evaluated the individual areas, i. e. the knowledge and
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24 108 science process skills as well as the overall score. These results were used for subsequent
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27 109 statistical analysis. For easier interpretation, the results were converted into percentages. The
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29 110 reliability of the test was sufficient (0.75). Pre-test and post-test were carried out seven days
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111 before/after the experimental teaching.
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34 112
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36 113 2.4. Statistical analyses
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39 114 Normality of distribution was verified with the Shapiro-Wilk test. Considering that our data
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41 115 were not normally distributed (Shapiro-Wilk p > 0.05), we used the Mann-Whitney U test to
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44 116 compare the mean values of the pre-test and post-test. Correlations were measured by Person’s
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46 117 correlation analysis. Statistical analyses were performed in XLSTAT software (Addinsof, Paris,
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49 118 France) and graphs were constructed using GraphPad Prism version 10.1.1 for Windows
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51 119 (GraphPad Software, Boston, Massachusetts USA).
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 120 3. Results and discussion
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2 121 3.1. Results of experimental teaching in individual classes
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5 122 In the pre-test, students of individual classes achieved various results and the average overall
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7 123 success rate ranged from 23.00% to 32.47% with students of the 8-year program (class marked
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10 124 as Sexta) reaching the highest score (Fig. 1). The lowest score in the area of SPS was achieved
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12 125 by students in class 2. A. and in the area measuring students’ knowledge in class 2. B. (Fig. 1).
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126 The Ministry of Education itself claimed: “The current state of 8-year gymnasia has gradually
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17 127 reached a lower than expected quality” (MESRS-SR, 2014) and limits the number of admitted
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19 128 students on this program (MESRS-SR, 2018). Despite the growing criticism of the 8-year study
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22 129 program at gymnasia, we found that students in these classes have better knowledge and science
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24 130 process skills/SPS within the biology area we monitored (Fig. 1). Besides, 15-years old students
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27 131 of 8-year gymnasia achieved higher score in scientific literacy in the international study PISA
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29 132 2022 than 15-year students at primary schools and secondary vocational schools, but not than
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133 students of a 4-year program at gymnasia (NÚCEM, 2023). The question remains whether these
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34 134 results are due to more effective work with students of 8-year gymnasia, or better abilities of
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36 135 the admitted students already at the beginning of this program (in Slovakia, 8-year gymnasia
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39 136 select the number of students allowed by the state based on the success of the entrance test from
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41 137 the entire population of students in the given year). However, it seems that elite schools (and
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44 138 probably also 8-year gymnasia) have their importance, e. g. it has been proven in Mexico that
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46 139 admission to such type of schools is likely to increase the earnings of the admitted student in
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49 140 the future (Estrada and Gignoux, 2017). Also based on personal communication with several
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51 141 teachers, we can say that 8-year gymnasia place more emphasis on knowledge, homework and
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142 more scientific work with students and teachers are generally satisfied with these students.
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56 143 The lowest score in the post-test and pre-test was achieved in the SPS area by students
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58 144 of class 2.A. and the smallest difference between both tests were found here (only 8.22%),
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 145 which caused that the experimental teaching in this class did not have a significant effect on the
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2 146 SPS area (Fig. 1). On the contrary, the highest impact of the experimental teaching on the SPS
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5 147 area was found in the class Sexta (8-year program), representing increase in absolute score over
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7 148 24%. At the same time, the most expressive change in knowledge between the pre-test and post-
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10 149 test was found in class 2. B. with over 42% increase in score which may be related to the lowest
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12 150 pre-test knowledge score in this class (Fig. 1). Experimental teaching using image analysis had
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151 a positive effect on the overall test results in all three classes, leading to improvement by 12.57
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17 152 – 29.72% (Fig. 1).
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19 153
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22 154 3.2. The effect of gender on the results of experimental teaching
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24 155 In the pre-test, both genders achieved similar scores in the SPS area, but boys obtained a higher
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27 156 score (by 8.60%) in the knowledge area (Fig. 2). On the contrary, girls achieved a higher score
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29 157 in the post-test of SPS area (by 8.29%) while both genders achieved similar scores in the
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158 knowledge area (Fig. 2). In contrast to girls, non-significant effect of experimental teaching on
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34 159 SPS was found among boys. The greater improvement among girls is surprising, as boys prefer
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36 160 to use information technologies in biology classes (Kubiatko et al. 2009), therefore greater
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39 161 motivation and improvement in individual areas can be expected. On the other hand, female
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41 162 students may have better SPS than male students, as they are often more enthusiastic and more
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44 163 curious during practical activities. A higher level of critical thinking and partial SPS of girls
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46 164 was identified in Indonesia through descriptive research using observation sheet (measurement
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49 165 of SPS) and a test (measurement of critical thinking, Darmaji et al. 2022). In addition,
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51 166 questionnaire research on primary to high school students found that girls had significantly
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167 higher scores in all the components of science motivation (e.g. intrinsic motivation, career
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56 168 motivation or motivation in class) than boys (Daher et al. 2021). Using the same questionnaire
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58 169 slightly modified for secondary agriculture students from Mexico, the relationship between
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 170 gender and motivation was found in two of the five areas of motivation, namely motivation in
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2 171 class and self-efficiency (Chumbley et al. 2015), indicating that gender is important factor
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5 172 influencing motivation. Considering the differences between the genders, it is important to
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7 173 diversify science teaching methods to fit both male and female science students (Daher et al.
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10 174 2021), e.g. the use of IT in teaching. It is therefore a positive finding that experimental teaching
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12 175 using image analysis had an increasing effect on the overall test scores of students of both
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176 genders (Fig. 2). Based on these results, we recommend its wider application, at least at the
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17 177 high school/gymnasium level of education, with the aim of improving science process
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19 178 skills/SPS and knowledge, which are a good prerequisite for subsequent studies at the
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22 179 university.
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24 180
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27 181 3.3. Individual tasks and the overall effect of experimental education
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29 182 Science is not only about encyclopedic information, but it is a way of systematically
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183 understanding the environment (Turiman et al. 2012). That is the reason why we focused not
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34 184 only on the development of knowledge but also on the improvement of science process skills
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36 185 (SPS). In the pre-test, students had problems with task 1. a. (SPS), which was based on the
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39 186 ability to control variables (Tab. S1). In line with our data, curricular research revealed that
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41 187 students aged 13 – 18 years had problems with selecting the appropriate experimental setup and
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44 188 handling more than two variables (Tairab et al. 2015). Even more surprising was the finding
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46 189 that even experimental teaching did not improve students’ ability to control variables (Tab. S1).
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49 190 Another task (2. a.) that was problematic for students was focused on the ability to formulate
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51 191 an assumption or hypothesis (Tab. S1), so these areas require more attention in future
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192 experiments. However, this ability increased at least partially after the implementation of the
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56 193 experimental teaching and students achieved a higher score in the post-test (Tab. S1). In order
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58 194 to develop this ability, students must become active in class, and this is a task not only for the
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 195 teacher, but also for the student himself (e.g. to understand the benefit of this activity for his
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2 196 skills and future studies or work). Within genetics, it was found that teaching focused on science
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5 197 process skills and Mendel’s discoveries had a positive impact on the students’ ability to
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7 198 formulate hypotheses (Kim et al. 2011).
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10 199 In the pre-test, students also had a problem with data interpretation (5. b.) and
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12 200 argumentation (5. c., Tab. S1): lower score achieved in these tasks may have been due to the
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201 use of fluorescence microscopy images that the students had probably not seen before. When
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17 202 constructing arguments, students often selectively choose information that is consistent with
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19 203 their own beliefs and ignore information that contradicts their opinion (Zeidler, 1997). On the
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22 204 contrary, students had fewer problems with construction of the graph (task 6. a., Tab. S1),
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24 205 although they relatively often forgot to mark the individual axes/variables. They also had fewer
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27 206 problems when interpreting data from the graphs (tasks 3. a. – c.) or tables (tasks 4. a. and b.,
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29 207 Tab. S1).
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208 In only two tasks of SPS area, students did not achieve a higher score in the post-test
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34 209 than in pre-test (Tab. S1), resulting in a positive effect of experimental teaching on students’
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36 210 SPS (Fig. 3). In one of few similar studies, it was found that the use of information technology
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39 211 in “science” education also had a positive impact on science process skills compared to
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41 212 conventional teaching methods (score 81.36 versus 74.17, Vebrianto and Osman 2011).
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44 213 In the knowledge area, most of the pre-test questions were problematic for students and
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46 214 they had scores above 30% in only four questions (Tab. S1). Students were able to give
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49 215 examples of fruits and plants that contain a lot of vitamin C (task 2. b.) and were aware of the
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51 216 importance of this molecule in human food (task 2. c.) with the most common answer as
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217 “boosting immunity”. They also knew quite well that cadmium and nickel are metals (task 3.
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56 218 d.) and could define lichens (task 5. f.). Although more than one-third of the students were able
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58 219 to define lichens in the pre-test, students did not know their importance for the environment and
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 220 humans (task 5. g.). A positive finding was that experimental teaching improved students’
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2 221 knowledge in almost all questions of this area (Tab. S1), resulting in an increase in the total
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5 222 knowledge score (Fig. 3). Consistent with our data, research investigating the effect of ICT-
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7 223 based evolutionary university textbooks found a positive impact of these textbooks on students’
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10 224 conceptual knowledge (Helendra et al. 2018). Also, a 6 week-long experimental teaching using
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12 225 mobile phones designed for data collection during field trips improved the students’ knowledge
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226 about plants (parts of the flower, pollinations and pollinators, fertilization and interrelationship
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17 227 between animals and plants) more intensively compared to the group that did not use
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19 228 smartphones (improvement in score between pre-test and post-test: 22.68 vs. 13.62, Zacharia
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22 229 et al. 2016). The use of IT in today’s digital age represents a motivational and educational
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24 230 prerequisite for increasing the effectiveness of teaching and based on the positive results
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27 231 presented in this study, we recommend wider testing and possibly incorporation into the Slovak
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29 232 curriculum. In line with this assumption, curricular research in the United Arab Emirates in the
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233 field of “science” shows that the need to better prepare students for future scientific inquiry
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34 234 requires new curricula and teaching approaches that respond and focus not only on basic
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36 235 scientific content but also on the acquisition of advanced transferable skills (Tairab et al. 2016).
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39 236 The implementation of IT in teaching is also positively evaluated by students, e.g. after using
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41 237 mixed reality focused on learning about plants students (aged 11 – 12 years) mention that such
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44 238 teaching was useful and interesting (97.7%) and that enjoyed this education (90.9% of students,
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46 239 Liu et al. 2007).
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49 240 Since a positive effect of experimental teaching was found not only in knowledge but
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51 241 also in SPS area, it is not surprising that there was an overall increase in post-test score (Fig.
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242 3). This overall increase was evoked especially by the more intensive improvement of students
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56 243 who had greater deficiencies in the pre-test (Fig. 4). This more intensive improvement of
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58 244 students with lower scores in the pre-test is probably due to working in groups on individual
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 245 practical activities. It is group work that has several benefits in teaching, including higher
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2 246 motivation in the classroom, better teaching results, or the development of critical thinking
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5 247 skills (Laal and Ghodsi, 2012). However, teamwork is not implemented enough in teaching,
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7 248 e.g. a questionnaire survey of Finnish teachers educating students aged 7 to 16 found that only
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10 249 42.7% of respondents use group work weekly (Saloviita et al. 2018). We can expect that this
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12 250 situation is even worse in other countries (and e.g. Slovakia is under the OECD average in PISA
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251 tests, NÚCEM, 2023).
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17 252 The worksheets we have prepared (see supplementary materials) stimulate group work,
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19 253 discussion between students and the need to argue their opinion/result within the work team:
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22 254 this can represent a good basis for the use of IT tools even for topics less interesting to students,
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24 255 such as botany and especially lower plants. Indeed, a recent review focused on learning
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27 256 activities with plants and technology reported that only 1 out of the 9 analyzed studies involved
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29 257 a non-vascular plant (Leo-Ramírez et al. 2023).
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34 259 Conclusions
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36 260 By applying short-term activities combining a less interesting area of biology (plants and non-
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39 261 vascular taxa) with the use of IT, we found that experimental learning had a clearly positive
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41 262 effect not only on the overall score (science process skills +41.5% and knowledge +97.5%) but
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44 263 we also found differences between classes (8-year gymnasium program +62.6% vs. 4-year
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46 264 program +33.4%) and genders (girls +51.5% vs. boys +45.2%). Although the absolute score
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49 265 exceeded 60% in only one area, in view of the wider focus of the topics and the qualitative-
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51 266 quantitative parameters of the individual test questions, we can state that the absolute score
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267 typically exceeding 50% is gratifying considering the age of the students (second year of high
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56 268 school). Linking biological topics with chemical and environmental themes represents a
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58 269 challenge for future research with the aim of a complex and more scientific education linking
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 270 topics, as is also the aim of the “science” curriculum in Slovakia. We hope that the worksheets
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2 271 presented in this work can also be a stimulus for this change towards the linking of knowledge.
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5 272
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7 273 Disclosure statement: The authors declare that there are no conflicts of interest.
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10 274 Availability of supporting data: Supporting data are presented in the supplementary materials.
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12 275 Pre-tests and post-tests of students, in Slovak language, are available from the corresponding
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276 author upon reasonable request.
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17 277 Funding: The present research did not receive any funding.
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19 278
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22 279 References
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24 280 Baviskar, S.N. (2011). A quick & automated method for measuring cell area using ImageJ.
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26 281 American Biology Teacher, 73(9), 554-556.
27 282 Chumbley, S.B., Haynes, J., Stofer, K. (2015). A measure of students’ motivation to learn
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29 283 science through agricultural stem emphasis. Journal of Agricultural Education, 56, 107-
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31 284 122.
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33 285 Coşkun, F., Alptekin, Y., Demir, S. (2023). Effects of arbuscular mycorrhizal fungi and
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35 286 salicylic acid on plant growth and the activity of antioxidative enzymes against wilt
36
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287 disease caused by Verticillium dahliae in pepper. European Journal of Plant Pathology,
38 288 165(1), 163-177.
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314 Kim, S.O., Kim, B.S., Seo, H.A., Kim, Y.M., Park, J.S. (2011). Improvement of students’
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47 326 acid protects Coccomyxa subellipsoidea against metal toxicity through modulation of
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49 327 ROS/NO balance and metal uptake. Journal of Hazardous Materials, 339, 200-207.
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60 333 with plants and technology: a systematic literature review. Applied Sciences, 13(6), 3377.
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2 335 district teach about plants. Plant Science Bulletin, 59(3), 99-110.
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27 349 of the international study PISA 2022 and the first results for Slovakia). available at:
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381 Sciences, 15, 346-350.
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33 385 data collection in supporting elementary school students’ conceptual understanding about
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35 386 plants. International Journal of Science Education, 38(4), pp. 596-620.
36 387 Zeidler, D.L. (1997). The central role of fallacious thinking in science education. Science
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38 388 Education, 81(4), 483-496.
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40 389 Zhang, Y., Shi, X., Li, B., Zhang, Q., Liang, W., Wang, C. (2016). Salicylic acid confers
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42 390 enhanced resistance to Glomerella leaf spot in apple. Plant Physiology and Biochemistry,
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44 391 106, 64-72.
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 1
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90 2. A. 90 2. B.
3 80 80
4 *
70 * 70 ***
5 ns ***
6 60
* 60
score (%)

score (%)
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score (%)

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36 SPS knowledge overall
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38 393
39 394 Figure 1. Comparison of pre-test and post-test results in three independent classes. The
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41 395 knowledge test monitored either science process skills (SPS) or knowledge (and sum of both
42 396 parts is shown as columns “overall”). Data are in percentage ± SDs. Values above columns with
43 397 *, ** and *** indicate significant differences at 0.05, 0.01 and 0.001 levels of the Mann-
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45 398 Whitney U test between the pre-test and post-test. Values with ns are not significantly different
46 399 according to the Mann-Whitney U test (P > 0.05). Classes 2. A. and 2. B. are students of 4-year
47 400 gymnasium program (meaning only gymnasium education, ISCED3) while class Sexta are
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401 students of 8-year program (meaning lower secondary followed by gymnasium education, i. e.
50 402 ISCED2 and 3).
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 1 boys girls
2 90 90 pre-test
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4 80 ns 80 ** post-test
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6 ** ** ***
60 60
score (%)

score (%)
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8 50 50
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10 40 40
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17 SPS knowledge overall SPS knowledge overall
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21 405 Figure 2. Comparison of pre-test and post-test results within boys and girls. The knowledge
22 406 test monitored either science process skills (SPS) or knowledge (and sum of both parts is shown
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24 407 as columns “overall”). Data are in percentage ± SDs. ** and *** indicate significant differences
25 408 at 0.01 and 0.001 levels of the Mann-Whitney U test between the pre-test and post-test. Values
26 409 with ns are not significantly different according to the Mann-Whitney U test (P > 0.05).
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410
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 1 90
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3 80 *** pre-test
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6 ***
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score (%)
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SPS knowledge overall
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22 412 Figure 3. Comparison of pre-test and post-test results in all classes. The knowledge test
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24 413 monitored either science process skills (SPS) or knowledge (and sum of both parts is shown as
25 414 columns “overall”). Data are in percentage ± SDs. *** indicate significant differences at 0.001
26 415 level of the Mann-Whitney U test between the pre-test and post-test.
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 1 100
2 r = 0.477**
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4 deficit in pre-test (%) 90
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22 improvement (%)
23 416
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417 Figure 4. Graph of Person’s correlation analysis between the deficit in pre-test (%) and
26 418 improvement after the experimental education (%). We defined “deficit” as the difference
27 419 between 100% – the pre-test result (%) and “improvement” as the difference between the post-
28 420 test and pre-test results. Only a minimum of students had a negative improvement (that is, a
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30 421 worse post-test result than the pre-test), and the positive correlation of most students shows that
31 422 the worse the pre-test deficit, the greater the improvement. ** indicates significant differences
32 423 at 0.01 level.
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blind supplementary materials Click here to view linked References

Supplementary materials for the work:

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5 The use of image analysis in the improvement of knowledge and skills on the
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8 example of teaching the biology of lower plants
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 Table S1. Comparison of pre-test and post-test results as a sum of all students. The test
1 monitored science process skills (grey rows) and knowledge (white rows).
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6 question no. pre-test post-test
7 1. a. 20.0% 11.1%
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9 1. b. 25.6% 41.1%
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11 1. c. 7.8% 23.3%
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13 1. d. 6.7% 17.8%
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15 2. a. 18.9% 25.6%
16 2.b. 92.2% 91.1%
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18 2. c. 44.4% 60.0%
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20 3. a. 92.2% 75.6%
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22 3. b. 36.7% 51.1%
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3. c. 28.9% 45.5%
25 3. d. 30.0% 46.6%
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27 3. e. 22.2% 35.6%
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29 4. a. 53.3% 70.0%
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31 4. b. 34.4% 51.1%
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4. c. 15.6% 52.2%
34 5. a. 26.7% 65.6%
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36 5. b. 17.8% 33.3%
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38 5. c. 14.4% 20.0%
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40 5. d. 13.3% 35.6%
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5. e. 0.0% 33.3%
43 5. f. 37.8% 58.9%
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45 5. g. 14.4% 52.2%
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47 6. a. 46.7% 68.9%
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49 6. b. 2.2% 15.6%
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 Pre-test and post-test
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2 Gender: male/female
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Identification code:
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5
6 1. Thomas tried to determine the effect of Ni on the content of ROS (reactive oxygen species) in
7 the algae Scenedesmus quadricauda. He proposed the following experiment:
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Different nutrition media have various mineral compositions. After 2 days, the content of ROS in
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24 the experimental group was higher than in the control group, and therefore Thomas formulated
25 the following conclusion: „Nickel induces a higher production of ROS and has a negative effect on
26 Scenedesmus quadricauda.“
27 a. Could Thomas validly formulate this conclusion based on his experiment? Justify your answer.
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30 b. What is oxidative stress?
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35 c. Do you think that ROS occur only in algae and plants or also in other organisms? Justify your answer.
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38 d. Why hydrogen peroxide molecule occurs in cells?
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2. Ascorbic acid (vitamin C) is a metabolite synthesized mainly by plants and is important in
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44 regulating the constant level of ROS. The ROS are formed in larger quantities in plants under
45 unfavourable conditions (e. g. higher cadmium/Cd concentrations). Plants therefore need to
46 reduce the ROS content in their body. Simone wants to carry out an experiment in which she will
47 investigate the effect of lycorine (inhibitor of vitamin C synthesis, note: inhibit means to block) on
48 the content of ROS in the alga Scenedesmus quadricauda exposed to Cd.
49 a. Help Simone make a hypothesis about the amount of ROS in the algae exposed to lycorine + cadmium
50 compared to cadmium alone:
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56 b. Can you give examples of plants/fruits that contain a lot of vitamin C?
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59 c. Why is it important to get enough vitamin C through food?
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 3. Dominika found a scientific article with graphs showing the content of Cd and Ni in old and
1 young cultures of alga Scenedesmus quadricauda exposed to 1 and 10 μM Cd or Ni.
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17 a. Did the algae contain more Cd or Ni?
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21 b. Did the age of the algal culture affect the uptake of Cd and Ni, and if so, how?
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25 c. Does metal absorption by algae depend on the concentration that was applied?
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28 d. Describe what you know about cadmium and nickel.
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31 e. What is the effect of Cd or Ni on the organisms?
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36 4. Sebastian found a scientific article with table presenting the effect of the fungal pathogen
37 Verticillium dahliae (Vd), salicylic acid (SA) and mycorrhizal fungus (AMF) on shoot length (SL)
38 of Capsicum annum seedlings. Help Sebastian formulate the conclusions from the table.
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57 a. What was the effect of Verticillium dahliae on shoot length?
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 b. What was the effect of salicylic acid and mycorrhizal fungi on the shoot length of Capsicum annum
1 infected with the pathogen Verticillium dahliae?
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4 c. Do you know what effect acetylsalicylic acid has on the human body?
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8 5. Daniela found a scientific article with microscopic photos of a lichen showing the impact of
9 cadmium (Cd) and nitric oxide inhibitor (cPTIO) on nitric oxide content (column A) and on ROS
10 content (column B). Nitric oxide (NO) is a molecule that usually has a positive effect on organisms
11 and protects them from ROS. The ROS are generated as a normal product of cellular metabolism,
12 but various factors can lead to their excessive production. These microscopic photographs (always
13 a similar part of the lichen) were observed under a fluorescence microscope. With the help of
14 fluorescence microscopy, it is possible to identify where and in what concentration (more intense
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red or blue color = higher quantity of the given substance) specific substances (in this case, nitric
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17 oxide and ROS) are found in the tissue. Help Daniela formulate conclusions about the relationship
18 between Cd, NO and ROS.
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46 a. What is the effect of Cd on ROS and NO content?
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49 b. What is the effect of cPTIO on ROS and NO content during co-application of Cd?
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52 c. Does nitric oxide have a positive effect on Cd-induced changes? Justify your answer.
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57 d. Do you know any effects of nitric oxide on the human body?
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 e. Nitric oxide is a substance that usually has a positive impact on organisms. Do you know how this
1 or other oxides of nitrogen affect the environment?
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5 f. What are lichens?
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10 g. What is the importance of lichens in nature and for people?
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13 6. Mark carried out an experiment and wrote the results in a table. Help him to construct a graph
14 showing the dependence of the phenolic content on the harvest day of the alga Ulva lactuca. Create
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a graph based on the data in the table (use a ruler):
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18 harvest day phenolic content
19 1. 80 mg of phenols per gram of dry matter
20 5. 50 mg of phenols per gram of dry matter
21 7. 30 mg of phenols per gram of dry matter
22 10. 20 mg of phenols per gram of dry matter
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25 a. Graph drawing:
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46 b. Phenols are a group of plant substances that can make up to 45% of the organic matter in plants. Do
47 you know the effect of phenols on humans?
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53 c. If you know, give examples of foods that contain a lot of phenols.
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 Worksheet no. 1: Algae as food and a source of nutrients
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Introduction: One of the biggest problems of the 21st century is the provision of sufficient
4 resources for the constantly growing world population. A partial solution to this problem is the
5 cultivation of seaweed. Ulva lactuca, also known as sea lettuce, is an edible green alga. This
6 alga is used locally in soups and salads, other species of the genus are used in different parts of
7 the world. Just as farmers try to maximize the yield and quality of the plants they grow, seaweed
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9 farmers also try to maximize the yield and quality of the algae. A Swedish team of biologists
10 also dealt with it, trying to find out the optimal day for harvesting Ulva fenestrata during the
11 year. One of their conclusions are these photos:
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37 Research question: What is the effect of time on the size/area of the alga?
38 Photos can be objectively compared using the image analysis method. It is a method of image
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40 processing when assessing the properties and quality of an object, in which subjective judgment
41 by a human is replaced. We will use the Fiji program to analyse the image. Note that each photo
42 has own scale. Why is a scale necessary for every photo?
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Instructions for measuring the size of alga:
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46 1. Open the Fiji program on your computer.
47 2. In groups of four students, divide the pictures, which will be analysed by individual
48 group members. Each member selects one picture.
49 Subsequently, open the file that you will analyse in the Fiji program (Fig. 2).
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23 3. Set the scale of the photo:
24 • Click on the line icon (Straight) and mark the scale, which represents 5 cm (Fig.
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3, red arrows).
27 • After marking the scale length, click on Analyze → Set Scale, set the scale
28 parameters according to Fig. 3 (don’t overwrite the row Distance in pixels).
29 Then click on the OK button (yellow arrow).
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 4. Threshold the photo: click on Image → Adjust → Color Threshold, set the threshold
1 according to Figure 4 and close the given window.
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27 Figure 4
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30 5. In the program, you need to set what you want to measure: click on Analyze → Set
31 Measurements. Set the window that appears according to Fig. 5 (Area) and click OK.
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55 Figure 5
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 6. Select the object whose area you want to measure: click on Analyze → Tools → ROI
1 Manager.
2 7. A window will appear. Click on the wand icon (Wand tool, Fig. 6, red arrow) and click
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4 on the object you want to measure. Then click on Add in the “ROI Manager” window
5 (Fig. 6, yellow arrow).
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Figure 6
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32 8. By clicking on Measure in the “ROI Manager” window, you will see a table showing
33 the size/area data. Write your result.
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36 From the results of all members of your group, construct a graph that will show the
37 dependence of the size of alga on the harvesting time. Is it possible to conclude when is the best
38 time to harvest algae from a growth point of view?
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Graph:
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53 Your conclusion: ..........................................................................................................................
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56 Based on Figure 1, formulate an assumption about the change in chlorophyll content of the
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algae during various days of harvest: ...........................................................................................
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 Growth/size is not the only parameter of quality, and the chemical composition is also
1 important. Scientists also determined other parameters (the content of proteins, fatty acids,
2 chlorophylls, and phenols) using analytical methods.
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6 Study these graphs and answer the questions below (DW = dry weight):
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37 Figure 7
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39 a) Is it possible to conclude from the graphs when is the best time to harvest algae in terms
40 of metabolite content?
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45 b) Is it true that the largest algae size also has the highest metabolite content?
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50 c) Based on the metabolite content graphs on this page and the algal size graph you created
51 on the previous page, suggest the best time to harvest algae to achieve maximum
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growth/area and highest metabolite content simultaneously.
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 Worksheet no. 2: Algae, oxidative stress and antioxidant
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Introduction: Coccomyxa is a genus of unicellular green algae (so-called microalgae) with a
4 small ellipsoid to spherical shape. About 40 species are known (free-living and symbiotic
5 species in lichens). Some of the species have potential use for biofuel production. Biofuel is a
6 substance derived from organic material that can be used as an energy source. Coccomyxa is
7 often used as a model organism since its entire genome is known.
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9 Plants, including green algae, are exposed to changing environmental conditions
10 throughout their life. Unlike animals, they cannot migrate to another place and must cope with
11 unfavourable conditions. In such conditions, reactive oxygen species (ROS) are excessively
12 formed in all aerobic cells, which damage the basic components – proteins, lipids, or DNA. The
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ROS formation is significantly stimulated by an elevated concentration of metals. Nitric oxide
15 (NO) is a gas that was originally discovered in animal cells. It has many functions in plants,
16 from regulating basic growth processes to alleviating the effects of various stresses.
17 The results of research that investigated the formation of ROS and nitric oxide in algae
18 exposed to mercury (Hg) and the possibilities of alleviating the negative effect of this metal are
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20 shown in Fig. 1 as microscopic photos. The researchers also investigated the effect of vitamin
21 C (ascorbate), a molecule potentially mitigating the negative effects of the metal. Using a
22 fluorescence microscope, it is possible to monitor the intensity of ROS (green signal) and nitric
23 oxide (blue signal) formation (more intense green or blue color = higher quantity of the given
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substance).
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48 Figure 1
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51 Research questions:
52 • Does the Hg induce ROS formation in algae?
53 • Does the content of nitric oxide change after addition of Hg?
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55 • What is the effect of vitamin C on these changes?
56 Photos can be objectively compared using the image analysis method. It is a method of image
57 processing when assessing the properties and quality of an object, in which subjective judgment
58 by a human is replaced. We will use the Fiji program to analyse the image.
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 Instructions for measuring signal intensity:
1 1. In groups of four students, divide the pictures, which will be analysed by individual
2 group members. Each member chooses one column (ROS without vitamin C, ROS with
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4 vitamin C, nitric oxide without vitamin C, or nitric oxide with vitamin C) with two
5 photos for each team member (control and Hg).
6 2. Open the Fiji program on your computer.
7 3. Subsequently, open the file that you will analyse in the Fiji program (Fig. 2).
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24 Figure 2
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4. In the program, you need to set what you want to measure: click on Analyze → Set
28 Measurements. Set the window that appears according to Fig. 3 and click OK.
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47 Figure 3
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49 5. Now we will measure the signal intensity of individual microscopic photos. Using the
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Rectangle under the File, mark the first photo (control) you want to analyse and click
52 Analyze → Measure. A table with the measured data will be displayed. Area represents
53 the size of the area you marked and Mean represents the relative intensity of the signal.
54 6. Repeat the fifth step for the second image and save the table with results or copy it to
55 an Excel file.
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57 7. From the results of all members of your group, construct two graphs showing the
58 impact of metal Hg and vitamin C on the formation of ROS and nitric oxide.
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 Graph:
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29 Questions:
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32 a) What was the effect of mercury on the formation of ROS and nitric oxide in the alga?
33 Is the relationship between them positive or negative?
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39 b) What was the effect of vitamin C on the formation of ROS and nitric oxide induced by
40 the presence of mercury?
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46 c) Did higher nitric oxide concentrations after vitamin C application contribute to better
47 protection against ROS? Justify your answer.
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d) What is the effect of nitric oxide on the human body? If you do not know the answer, use
55 the internet to help you.
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 e) Can you write the chemical formula of nitric oxide? Do you know other oxides of
1 nitrogen? One of them is a very dangerous greenhouse gas, 1 molecule of which released
2 into the atmosphere is equivalent to 265 molecules of released carbon dioxide. What
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4 molecule is it? If you do not know the answer, use the internet to help you.
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8 The graph below shows the content of Hg in algae exposed to mercury without or with the
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10 addition of vitamin C. Abbreviation DW = dry weight.
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f) Based on the graph, explain how vitamin C affected the mercury content in algae.
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36 g) The graph above shows that the algae contain 37 mg of Hg in 1 g of dry weight (black
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38 column). Express the Hg content as % of dry weight:
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h) Is it possible to assume that, for example, dandelions produce ROS in the presence of
44 metals in a similar way to the algae? If yes, why do you think so?
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49 i) Give examples of plants that contain a lot of vitamin C. What is its importance for
50 humans?
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55 j) Is mercury useful or harmful for humans and the environment? Give any example you
56 know.
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 Worksheet no. 3: Fungal disease and Aspirin-related compound
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Introduction: Plant diseases cause great economic losses to farmers worldwide. The Food and
4 Agriculture Organization (FAO) estimates that plant pests and diseases are responsible for
5 approximately 25% of crop losses. Plant diseases are caused mainly by viruses, bacteria and
6 fungi. One of the fungal pests is Glomerella cingulata (belongs to the Ascomycota), which
7 damages many varieties of apple trees, e.g. “Gala”, is sensitive to this fungus. On the leaves,
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9 the disease is first manifested by several small black spots which quickly increase in size,
10 causing premature leaf fall.
11 The results of the research that investigated the influence of Glomerella cingulata on
12 apple leaves are shown in Fig. 1. In this research, the authors applied sprays containing different
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concentrations of salicylic acid (SA) to the leaves and determined whether this substance
15 slowed down the development of the fungal disease. SA is formed naturally in plants.
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Figure 1
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40 Research question: What is the effect of salicylic acid on the spread of the disease caused by
41 the fungus Glomerella cingulata?
42 Photos can be objectively compared using the image analysis method. It is a method of image
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processing when assessing the properties and quality of an object, in which subjective judgment
45 by a human is replaced. We will use the Fiji program to analyse the image.
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47 Procedure for measuring leaf area and disease area:
48 1. Open the Fiji program on your computer.
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50 2. In groups of four students, divide the pictures, which will be analysed by individual
51 group members. Each member chooses one picture.
52 3. Open the file to be analysed in Fiji. Click File → Open and open the file.
53 4. Set the scale of the photo:
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55 • Click on the line icon (Straight) and mark the scale which represents 1 cm (Fig.
56 2, red arrows).
57 • After marking the scale length, click on Analyze → Set Scale, set the scale
58 parameters according to Fig. 2 (don’t overwrite the row Distance in pixels).
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19 Figure 2 Figure 3
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23 5. To compare the effect of salicylic acid, you need to know the area of individual leaves.
24 Measure the area of the leaf using the already known procedure. Threshold the photo:
25 click on Image → Adjust → Color Threshold, set the threshold according to Fig. 3
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and close the given window.
28 6. In the program, you need to set what you want to measure: click on Analyze → Set
29 Measurements. Set the window that appears according to Fig. 4 and click OK.
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53 Figure 4
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56 7. Select the object whose area you want to measure:
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 • A window will appear. Click on the wand icon (Wand tool, Fig. 5, red arrow)
1 and click on the object you want to measure. Then click on Add in the “ROI
2 Manager” window (Fig. 5, yellow arrow).
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Figure 5
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28 8. By clicking on Measure in the “ROI Manager” window, you will see a table that shows
29 the leaf area data. Write the result.
30 9. Close the given photo and open the same file. Click File → Open and open the file.
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32 10. Find out the area of the leaf that is damaged by the disease. Threshold the photo: click
33 on Image → Adjust → Color Threshold and set the threshold according to Fig. 6
34 (don’t forget to click Dark background and close the given window).
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 11. Now edit the photo to black and white: Click Process → Binary → Make Binary.
1 12. Now color the area you don’t want to measure. Double-click the left mouse button on
2 the brush (Paintbrush tool, red arrow, Fig. 7) and set the window according to Fig. 7
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4 (yellow arrow), click OK, and color black the parts of the photo whose area you do not
5 want to measure.
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28 Figure 7
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31 13. Measure the area of the disease: Click on Analyze → Analyze Particles, a window will
32 appear that you set according to Fig. 8 (red arrow) and click OK. You will see the results
33 of the leaf disease area in a table. Write this result.
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 14. Using the results obtained from the image analysis, calculate how much % of the leaf is
1 infected by the given fungal disease depending on the applied concentration of SA.
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12 15. From the results of all members of your group, create a graph showing the effect of
13 salicylic acid on the spread of disease on apple leaves.
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17 Graph:
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33 Questions:
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36 a) Give examples of other fungal diseases on plants that commonly occur.
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42 b) What was the effect of salicylic acid on the disease caused by Glomerella cingulata?
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48 c) The researchers used apple trees of the same size in the experiment. They also used only one
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50 apple variety. Why are these factors important to the experiment?
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 d) The picture shows 2 molecules. One molecule is salicylic acid, and the other is acetylsalicylic
1 acid. What is the difference between these two molecules? Highlight it:
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16 We can say that acetylsalicylic acid is a derivative of salicylic acid. In chemistry, a derivative
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is a compound that is formed from a similar compound by a chemical reaction.
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21 e) Because of what effect do people use preparations containing acetylsalicylic acid?
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f) Do you know the names of medications that contain acetylsalicylic acid?
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36 Salicylic acid is a substance synthesized by plants. Plants are interesting for humans due to
37 many health-positive compounds. Many drugs/medications and their derivatives commonly
38 used in practice are of plant origin.
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42 g) Which medicinal plants do you use most often?
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graphical abstract