The overall goal of our study is the development and spread of novel experiments, with readily available materials and simple procedures, in order to improve the working rate of the observations and experiments in elementary and secondary school classes. In the basic biology of high school, students study the connection between the constancy of the internal environment and various blood components. In this study, we developed an effective laboratory study to understand erythrocyte and homeostasis using blood from bivalves ark shell, Scapharca broughtonii. The ark shell is one of a few invertebrates containing erythrocytes. The color of the blood in the ark shell is red, since its hemocytes contain the respiratory pigment erythrocruorin and the heme group as its oxygen carrier, similar to hemoglobin. The ark shell contains one nucleus and granules in its erythrocytes, unlike human erythrocytes, which do not contain a nucleus. We purchased living ark shells with their shells intact from fresh markets or mail-order markets and obtained the blood-containing erythrocytes from the organisms by opening the shells. We constructed a suitable laboratory study method using the erythrocytes from the ark shell, such as applying the blood to a glass slide, staining nuclear, the concentration of hypo- and hyper-osmotic solution. All students and teachers who participated in this laboratory study achieved sufficient data (stained and non-stained erythrocytes with nuclei and an understanding of the influence of osmotic solutions under the microscope). These observations could be seen within one class period (45–50 min). This laboratory study generated interest for students and teachers not only related to the erythrocytes under homeostasis but also for diversity through the presence of erythrocytic nuclei, unlike that of humans. Thus, we succeeded in developing an observation experiment using ark shell blood erythrocyte.

Teaching materials were developed for performing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The aim of the practical was the detection of tissue-specific proteins in a large volume of differentiated animal cells. The procedures were practiced in class. Electrophoresis samples were prepared by adding a trace amount of material to the sample treatment solution. As a result of the experiment, I demonstrated that myosin heavy chain, actin from muscle tissue, and hemoglobin from blood or bone marrow could be detected in pigs, chickens, and horse mackerel. Molecular groups of crystalline proteins from the lens could be detected in tissues of pigs and horse mackerel. In pigs, by separating blood into serum and cells, I confirmed that serum albumin is present in blood serum and hemoglobin is present in blood cells. Pigs are considered a suitable experimental subject, and students performed experiments using pig tissues in class. Preparation of gels used for SDS-PAGE is very easy for teachers because precast gel is readily available from stores. When this experiment was conducted, students were encouraged to investigate the amount and molecular weight of each protein by themselves as much as possible. From experimental results, students easily estimated the types of proteins in the major bands on electrophoretograms. Moreover, by combining SDS-PAGE with microscopic observation of muscle and blood, students were able to better understand the locations of proteins detected in the tissues.

In biological experiments in schools, students need to acquire a large amount of numerical data in science class in order to perform graphing and statistical analysis. Microplate reader, a kind of the spectrophotometers used in various research such as drug discovery and physiology, can measure light absorbance of sample solutions in microplate at once. The most common microplate format used in academic research laboratories or clinical diagnostic laboratories is 96 wells with a typical reaction volume between 100 ~ 200 μL per well. As an educational experiment to realize physiology and ecology, a method for determining the ammonia concentration by colorimetry has already been established. However, since the density is determined based on the color chart, it is difficult to perform a numerical analysis. Therefore, we developed ammonia assay using the microplate leader by indophenol method. In particular, the volume of reagents and samples was unified at 200 μL to make it easier for students to handle. We found that, light absorbance in 650 nm, an absorptivity wavelength of indophenol blue, reached the peak when sodium hydroxide and effective chlorine of sodium hypochlorite were 125 mM and 0.0065% at the final concentration. We next performed a student experiment based on this result, all the students could make standard curve and determine 1 ~ 3 ppm ammonia in water kept goldfish. Since the microplate reader can measure the light absorbance of 96 samples in approximately one minute, it will be an effective instrument to realize inquisitive biological experiments using numerical values.

There are a lot of laboratory activities conducting polymerase chain reaction (PCR). In most of them, electrophoresis gel images of PCR products themselves or the products treated with restriction enzymes lead to a certain discrimination or recognition. Because the protocol is thoroughly predetermined by the instructor in these activities, experiencing students are warranted to get the expected results as long as they conduct the protocol properly. If students need to investigate and predict what restriction enzymes they should use to discriminate samples in such activities, it will give them an opportunity of scientific thinking. In this study, we developed the PCR-RFLP (restriction fragment length polymorphism) activity, in which students need to decide appropriate restriction enzymes to discriminate samples through the comparison of their DNA sequences. We found that rbcL regions are suitable for students to predict RFLP by sight when using some combinations of vegetables such as sprouts. We conducted the developed activity for the students belonging to the teacher training course in the university, and confirmed that the difficulty is in a proper level. Some students commented that they engaged actively and it helped their understanding of the restriction enzyme.