Teaching biological sciences can be made more stimulating for students through the use of experimentation. This manuscript presents two different yet complementary protocols that can be utilized in the classroom to encourage students to formulate and test hypotheses related to high-calorie diets, starvation, and aging.
Caenorhabditis elegans (C. elegans) is a transparent, non-parasitic nematode with a simple biology, which makes it a great tool for biological sciences teaching through the staining of the cells or their molecular content. Lugol dye (iodine-potassium iodide solution) has been widely used in biochemistry to stain glycogen stores. In this context, it is possible to observe differences between fed and starved animals, besides the effects of different conditions, such as different diets and oxygen levels. Erioglaucine is a blue dye that indicates the loss of the intestinal barrier. When the intestinal barrier is intact, the blue dye stains inside the lumen; however, when this integrity is disrupted, the dye leaks into the body cavity. Using a stereomicroscope or a microscope, teachers can demonstrate physiological and biochemical alterations, or they can instigate students to ask a scientific question and hypothesize and test their hypothesis using these assays. The present protocol describes two staining techniques in C. elegans that can be easily carried out by students.
Biological sciences teaching in high school is a continuous challenge. Notably, the access and use of technology have brought important advances in the teaching-learning process, however, tools such as artificial intelligence chatbots make rationalizing and seeking evidence more difficult due to easy (and sometimes incorrect) responses obtained1. Because of that, the use of a scientific method with practical experimentation in an inquiry-based approach in the classroom is an important strategy to develop or stimulate critical thinking, creativity, and technical skills in the students2.
In this context, the free living nematode Caenorhabditis elegans has been successfully used in experimentation for teaching purposes3 because of its particular advantages: It is not a parasite and the Escherichia coli used for feeding is biosafety level-1, therefore reducing near to zero the biological hazard; it has an elegant and quantifiable locomotion movement, which is interesting for students to observe; and it is transparent, which allows organ observation, but also staining with pigments that can indicate the presence of biomolecules or the occurrence of physiological alterations4. Therefore, it is possible to hypothesize and test in the classroom simple postulations related to biochemistry and physiological changes such as aging.
Glycogen is a storage carbohydrate, formed by a long and branched chain of glucose molecules formed by glucosyl residues with (1→4)-α glycosidic linear linkages and (1→6)-α glycosidic bonds at branch points and is particularly important for muscle contraction, cell differentiation and glycemia maintenance5. Glycogen is synthesized after feeding due to insulin activation of the enzyme glycogen-synthase. During exercise or fasting, epinephrine or glucagon, respectively, activate glycogen phosphorylase and, therefore, break down the polysaccharide to provide glucose-6-phosphate to the muscle cells or release free glucose to circumvent hypoglycemia6,7. Alterations in glycogen levels impacts cell differentiation, signaling, redox regulation, and stemness under various physiological and pathophysiological conditions, including cancer8. In C. elegans, glycogen is mainly found in esophageal muscle, hypodermis, intestine, neurons and mainly in body wall muscles9. The glycogen content can be measured by using Lugol's Iodine solution, since iodine binds into the helical coils forming an iodine-glycogen complex, giving a visible sharp blue-black or brown-black color, which has been successfully used to demonstrate glycogen content in C. elegans10. It has been demonstrated that glycogen accumulation caused by high glucose feeding can reduce worm's lifespan, therefore accelerating the aging process11,12. In addition, metabolic disturbances, other hormones, and exposure to xenobiotics can alter glycogen metabolism as well13,14. Therefore, experimentation on glycogen content in C. elegans is quite interesting, since diverse factors may disturb its metabolism and can stimulate an in-class discussion on basic biochemistry associated with transversal themes such as exercise, diets, diseases, and aging.
Aging is a time-dependent functional decline caused by cellular damage. This damage can be associated with oxidative stress, telomere attrition, loss of proteostasis, inflammation and even by accumulation of insoluble polyglucosan bodies15, just to name a few. One of the hallmarks of aging is the reduction of intestinal integrity, associated with several chronic conditions that occur during the life of an organism16. Maintenance of intestinal homeostasis depends on the integrity of the intestinal epithelium, which is supported by junctional proteins forming a physical barrier and connecting adjacent epithelial cells. When there is damage to this epithelium, leakage of luminal content into the interstitium occurs17. Based on this mechanism, the smurf test has been used to verify intestinal integrity in several animal models, since this blue dye Erioglaucine disodium salt does not cross the intestinal membrane, remaining in the lumen18. When worms are infected with a pathogen, contaminated with some toxicants or age, altering the interstitial integrity, the dye crosses the barrier and spreads all over the worm, which becomes all blue. This assay allows discussion on the physiology of aging and experimenting on factors that can accelerate or delay this process by exposing worms to different conditions. The protocols here will describe in detail these two, dye-based methods that can be easily done in class to instigate and stimulate students to formulate and test hypotheses related to biochemistry and physiology.
The first part of protocol shows its applicability to analyze qualitatively and quantitatively the glycogen content in C. elegans model10. The purpose of second part of the protocol is to assess the integrity of the C. elegans intestine. This technique allows for the monitoring of C. elegans aging by evaluating the integrity of the intestinal membranes. Furthermore, it allows evaluating whether a substance accelerates or delays aging and whether any substances have toxic potential on the intestinal barrier19.
The C. elegans strain used for the present study was Bristol N2 wild type. However, the procedure can be replicated using strains that exhibit comparable growth rates, or the method must be adjusted based on need of equipment replacement, considering they have the same or similar function, or depending on the strain used, as certain strains have specific maintenance and/or sensitivity requirements; this information can be obtained from the Caenorhabditis Genetics Center (CGC) or WormBase website. These changes should not impact the reproducibility of the method.
NOTE: Escherichia coli OP50 (E. coli OP50) bacteria and Bristol N2 wild type strains can be obtained from the CGC, University of Minnesota, USA or from donation from a C. elegans laboratory. For researchers' safety, it is imperative to use Personal Protective Equipment. Although the concentrations of reagents like hypochlorite and sodium hydroxide are low, it is essential to wear the recommended PPE, as highlighted in the manuscript, to minimize any potential risks associated with these chemicals.
1. Glycogen content
Figure 1: Overall glycogen content assay schematic in C. elegans. A schematic of the experimental performed here to carry out the glycogen content assay. Please click here to view a larger version of this figure.
2. Evaluation of intestinal permeability
Figure 2: Overall intestinal permeability assay schematic in C. elegans. (A) C. elegans preparation. (B) Staining with Erioglaucine disodium salt. Please click here to view a larger version of this figure.
The glycogen content assay provides a robust and rapid method for screening various testing conditions, such as comparative studies of different strains that may influence glycogen synthesis or degradation. In this study, L4 worms were subjected to three distinct test conditions: fasting, feeding, and glucose-enriched groups. The assay was performed three times, with each condition replicated twice in each assay; a representative image is displayed in Figure 3. Following each assay, worms stained with Lugol's Iodine solution were observed using a stereomicroscope, and a qualitative comparison was made among the groups regarding the staining of the worms. In terms of visual observation, worms in the fasting group (starvation) displayed a less intense stain compared to the worms fed with E. coli OP50 (regular), as well as those fed with E. coli OP50 and D-glucose (high glucose). Additionally, the worms fed with E. coli OP50 and D-glucose (high glucose) exhibited the most intense staining.
Figure 3: Representative images from glycogen content assay. Glycogen levels in C. elegans were evaluated through iodine staining. Microscopic images depict representative C. elegans worms subjected to (A) starvation, (B) regular, or (C) high glucose conditions for 48 h. The worms were stained by immersing them in a solution of Lugol's iodine/M9 buffer (1%) for 5 min, following the procedure detailed in step 1.4. Please click here to view a larger version of this figure.
The results of intestinal permeability show the difference in staining of young worms (L4) with intact intestine and old worms (7th day of adulthood) with damaged intestinal membranes due to aging (Figure 4). The intestine of C. elegans is a tissue of central importance to health, as it plays critical roles in digestion and metabolism and serves as a signaling center for stress response and aging regulation21. In young worms, the intestine of C. elegans is constituted of a strong barrier that prevents the leakage of intestinal contents. However, as these worms age, the intestinal barrier function decreases and becomes more sensitive to disruption due to progressive degradation of cells and microvilli and accumulation of bacteria in the aging intestine21,22. When young C. elegans ingest the dye Erioglaucine disodium salt, the intestine of the nematode is stained (Figure 4A). On the other hand, it is possible to observe that aged worms presented an extravasation of the dye, permeating the entire body of the worm, which indicates damage to the intestinal membrane (Figure 4B).
Figure 4: Representative image of C. elegans staining with Erioglaucine disodium salt. The microscopic image shows C. elegans with intact intestine (stage L4) and worms with ruptured intestine (7th day of adulthood). The worms were stained by immersion in a 25% Erioglaucine disodium salt solution for 3 h, following step 2.2.4. Please click here to view a larger version of this figure.
Supplementary Figure 1: ImageJ screenshot for selecting C. elegans contour. Please click here to download this File.
Supplementary Figure 2: ImageJ screenshot for calculating mean. Please click here to download this File.
Supplementary Table 1: Media, buffer, and culture recipes. Composition of different media, buffers, and cultures used in the current protocol. Please click here to download this File.
Supplementary File 1: Detailed description of the ImageJ Software for quantifying glycogen content in worms. Please click here to download this File.
In summary, this protocol provides a qualitative evaluation of glycogen content in individual C. elegans worms using Lugol staining: a straightforward, robust, and swift assay. Lugol staining is a label-free and non-invasive approach that facilitates the acquisition of molecular data at subcellular resolutions, allowing for the monitoring of glycogen content fluctuations within single worms10. Furthermore, the assay offers the advantage of cost-effectiveness due to its minimal equipment requirements. Only a microscope or stereomicroscope is necessary to capture the images, which can be easily obtained using a cell phone camera as long as the same light/brightness/exposure settings for all pictures. Additionally, certain procedural adjustments can enhance the effectiveness of this protocol. More complex laboratory equipment, such as a flow hood and centrifuge, can be substituted with alcohol lamps and gravity sedimentation. These alternatives make the method even more budget-friendly and accessible to a wide range of researchers. In addition to researchers, educators can also employ this assay for educational purposes in schools to illustrate changes associated with glycogen storage and its biosynthesis.
Although conventional enzymatic assays can quantify glycogen levels, Lugol staining shows promise as a sensitive, accurate, cost-effective, and high-throughput method for assessing glycogen stores in C. elegans. In our study, the data revealed noticeable differences in staining among the three conditions, suggesting a potential correlation between the feeding conditions and the intensity of staining. In other words, it indicates variations in glycogen content among the different feeding groups, as staining intensity is directly proportional to glycogen content. With these initial observations and evidence, students can inquire and investigate whether various factors, such as different foods, beverages, or chemical reagents, can also influence glycogen content in the nematodes.
One drawback of this approach arises from its reliance on manual procedures and subjective interpretation. The subjective nature of the staining assessment, where scores are assigned based on visual observation, can lead to variability among different analysts, potentially introducing fluctuations in result interpretation.
This report also demonstrates the evaluation of the intestinal integrity of C. elegans using the dye Erioglaucine disodium salt. It is possible to evaluate the loss of intestinal barrier function in C. elegans by feeding the worms with a blue dye that is not absorbable through the cuticle. This test makes it possible to check for leakage of dye out of the intestine, a test known as the smurf test because the animals turn blue like the cartoon Smurfs23. This methodology was previously applied to Drosophila melanogaster and Danio rerio using a similar method for intestinal evaluation24,25. In C. elegans we used the same principle of the technique with the Erioglaucine disodium salt, being considered a simple and reproducible technique to verify the intestinal integrity of C. elegans19,26.
This protocol can be used to evaluate the induction or delay of aging of nematodes and also to verify the toxic potential of substances in the intestine of C. elegans in an easy and fast way, without the need to use complex equipment, which makes the method economically viable19,27. In addition to its use in scientific research, teachers may also employ this assay for educational purposes in educational institutions to investigate changes in intestinal permeability of C. elegans. With a simple test, it is possible to investigate hypotheses about aging and the effect of chemical reagents on the intestinal barrier of nematodes.
The assay can be adapted to use other C. elegans strains and larval stages according to the purpose of the study, as long as the replacements serve the same or similar function to maintain method reproducibility. For a successful analysis, it is important that the aged worms are stained for 3 h, as mentioned in the protocol. Subsequently, the washes to remove excess dye are a critical step in the method and must be done carefully so that the worms are not removed along with the supernatant. In the case of evaluating substances, one of the limitations found in the protocol is the difficulty in finding an easily accessible positive control that lyses the intestinal membrane of nematodes.
The authors have nothing to disclose.
D.S.A acknowledges funding from Conselho Nacional de Pesquisa e Desenvolvimento (CNPq/Brazil), grant number #301808/2018-0, #313117/2019-5, Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS/Brazil), grant number, 21/2551-0001963-8, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Finance Code 001 for N.S.J and A.C.S)
1.5 mL microtubes | Local suppliers | – | |
37-degree incubator | KS 4000i | 97014-816 | |
50 mL conical tube | Local suppliers | – | |
6 cm Petri plates | Local suppliers | – | |
Agar bacteriological | Dinâmica Química Contemporânea Ltda. | 9002-18-0 | |
C. elegans Bristol N2 (wild type) | Caenorhabditis elegans Genetic Center (CGC, Minnesota, USA) | – | |
CaCL2 | Dinâmica Química Contemporânea Ltda. | 10035-04-8 | |
Cholesterol | Sigma-Aldrich Brasil Ltda | 57-88-5 | |
D-(+)-Glucose anhydrous | Neon | 50-99-7 | |
Distilled H2O | Local suppliers | – | |
Erioglaucine disodium salt | Sigma-Aldrich Brasil Ltda | 3844-45-9 | |
Escherichia coli OP50 | Caenorhabditis elegans Genetic Center (CGC, Minnesota, USA) | – | |
Flow hood | Mylabor | ||
Incubator | Panasonic Healthcare company of North America, MIR-254-PA. | – | |
KH2PO4 | Dinâmica Química Contemporânea Ltda. | 7778-77-0 | |
Levamisole hydrochloride | RIPERCOL L 150F | – | |
Lugol solution | Sigma-Aldrich Brasil Ltda | L6146 | |
MgSO4 | Synth | S1063-01-AH | |
Microcentrifuge | Centrifuge 5425R Eppendorf SE, Germany | ||
Na2HPO4 | Dinâmica Química Contemporânea Ltda. | 7558-79-4 | |
NaCl | Dinâmica Química Contemporânea Ltda. | 7647-14-5 | |
Nystatin | Sigma-Aldrich Brasil Ltda | N6261 | |
Peptone bacteriological | êxodo científica | 91079-38-8 | |
Stereomicroscope | Leica S8 Apo Stereomicroscope (São Paulo, Brazil) | ||
Streptomycin Sulfate | Estreptomax | – |
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