Undergraduate students exploring microorganisms in fermented milk products using basic microscopes and smartphone cameras

ABSTRACT

Students of an undergraduate class were trained to explore microorganisms in milk products. Fermented curd/yogurt prepared at home was compared with commercial milk products for the presence of bacteria and yeast. Students visualized Gram-stained samples with a basic microscope and captured images by adjusting their smartphone on the eyepiece. They estimated the dimensions of the organisms using the images, after factoring in the smartphone’s magnification and the microscope’s field of view (FOV). Students could appreciate the health benefits of fermented milk products prepared at home by monitoring the prevalence of Gram-positive bacilli. Undergraduate teachers can readily adopt this pedagogy approach to give hands-on training to students in large labs, even within economically constrained setups.

INTRODUCTION

Students reach a critical phase in academics while transitioning from early education in schools to higher education in colleges and universities. They should not only be able to solve tasks given by their teachers but also identify new questions and acquire the ability to solve them (1). Thus, students need proper training through laboratory courses, in addition to textbook-based conventional education (2). Hands-on training is essential for developing practical skills, with laboratory courses playing a crucial role in developing students’ interest in a particular subject (3).

The task of imparting practical skills in Biology laboratories can be challenging due to the complex and time-consuming nature of the tasks (4). While new and unexpected outcomes enhance learning, training large classes in discovery mode can be demanding. The uncertainty and variability associated with the outcomes involving living organisms add to the challenges. In addition, teachers may sometimes be constrained to try new approaches because of tight schedules and limited equipment (5). Students are therefore advised to perform activities by following established protocols and collecting data in standard ways. Despite the challenges associated with conducting laboratory courses, training in discovery mode enhances students’ problem-solving skills (6, 7). Once students get to answer queries through experiments, they get motivated and feel accomplished from their learning (8, 9). New training methods also give educators and teachers a sense of achievement and motivation to evolve and innovate (10–12).

We encouraged students to ask real-life questions using readily available equipment in an undergraduate biology laboratory. The students were advised to utilize their skills acquired in earlier sessions of the course to observe the prevalence of microorganisms in fermented milk products. They performed Gram staining of prokaryotic and eukaryotic cells, took pictures using basic microscopes and smartphone cameras, and reverse calculated their dimensions from the magnified images. They also compared the health benefits of the milk products by recording the presence of beneficial Gram-positive bacteria (Lactobacilli and Lactococci) and yeast. The approach trained students with microorganisms in real-life examples and enhanced their analytical capabilities.

PROCEDURE

Organization of the course and management of large classes

Classes of ~275 undergraduate students, admitted after their intermediate education in the academic years 2022, 2023, and 2024, were trained with various equipment and basic techniques. Subsequently, they observed different microorganisms and distinguished bacteria with respect to their Gram nature. The class was divided into three sections with ~90 students. A workbench with essential equipment, such as a micro-pipette set and a basic microscope with 10×, 40×, and 100× objectives without a camera, was assigned to teams of five students. Handouts of detailed protocols were provided. Instructors who are faculty introduced theoretical concepts and coordinated the sessions. A teaching assistant (TA) (graduate students as well as lab technicians) provided guidance during the activities and data analysis to two teams each. The course typically covers 11–12 sessions, with one weekly session of 3 h for a group of students.

Genesis of the approaches

Gram staining has always been a part of this laboratory course. Culturing Gram-positive Lactobacilli requires anaerobic conditions, which were not readily available in the laboratory. The exercise with Gram-negative Escherichia coli was less effective in the absence of Gram-positive organisms. Therefore, we explored sources of Gram-positive bacteria and adopted the staining exercise by using homemade curd and fermented milk products. Furthermore, imaging of microorganisms was not possible with basic microscopes without cameras, and advanced microscopes were not available for hands-on training of large classes. Therefore, smartphone cameras were considered for imaging. Estimation of cell sizes through back calculations from magnified images was added to the activities, since the total magnification could be deduced by combining the magnifications of the microscope and smartphone cameras.

Cultures of microorganisms and milk products

Handling of microorganisms under aseptic conditions was a part of the course. Only non-pathogenic standard laboratory strains were used in the exercise. Students were advised to wear laboratory coats and gloves. The bacteria E. coli, the budding yeast Saccharomyces cerevisiae, and the fission yeast Schizosaccharomyces pombe were cultured by TAs following standard protocols. Cells were stained with diluted safranin dye to obtain better contrast for focusing under 100× objectives. Starter cultures for curds (yogurt) can be sourced from households and markets. Homemade curd was prepared by mixing pre-boiled lukewarm milk (~40°C) with a spoonful of curd from an older batch. Commercial yogurt and probiotics were procured from the market. Microorganisms used in commercial yogurt were not known. Commercial probiotics are milk products containing Lactobacillus casei strain Shirota.

Gram staining of microorganisms in milk products

The Gram staining method, devised by Hans Christian Joachim Gram, was adopted for staining and differentiating the Gram-positive and Gram-negative bacteria (13). Yeast cells appeared Gram-positive. A thin smear of milk products was spread on glass slides. Products of thicker consistencies were diluted in phosphate buffer saline (PBS). After air drying, the smear was heat fixed by gently passing the slide 5–6 times over a burner. The smear was covered with a drop of Gram’s crystal violet stain for 1 min. Next, the smear was covered with a drop of Gram’s iodine for 1 min. The smear was covered with a few drops of 95% ethanol to fix the stain in the cells’ interior and remove the excess stain. The air-dried smear was covered with safranin for 1 min. After every step mentioned above, debris and excess stains were gently washed away by rinsing the slides in water. Students examined the slides under low magnification, followed by oil immersion objectives (100×). They took pictures of focused slides by carefully adjusting a smartphone camera on the eyepiece and used the images for studying microorganism diversity, dimensions, and Gram nature.

Size estimation of microorganisms

The following two approaches were used for cell size estimation and comparison. For calculations, the images were magnified on a computer. Students calculated the size of a cell relative to the image’s diameter on the computer screen. Images of graph/ruled paper were advised to verify the relative measurements.

1. Field of view (FOV): The FOV is often indicated on the eyepiece and objectives of a microscope. Since the eyepiece had a 20-mm FOV, it was expected to show an image of 20-mm diameter. With a 10× objective, students could see a field of 2-mm diameter. Thus, the FOV with the 10× objective was 20/10 = 2 mm. Similarly, with the 100× objective, the FOV was ~20/100 = 0.2 mm. If an image taken by the 100× objective had 100-mm diameter on a computer screen, and the cells’ average size in that field was 5 mm, the actual size of the cell would be 5/100th of the FOV, i.e. 5/100 × 0.2 mm = 0.01 mm or 10 µm.

2. Combined magnification of the microscope and smartphone: In an image taken with a 10× eyepiece and 100× objective, a cell would be magnified by 1,000×. If an image captured with no magnification (zoom) in the mobile phone is magnified 10 times on a computer screen, the total magnification would be 10,000×. The cell’s size would be equivalent to its size measured on the computer divided by the total magnification.

Data collation

Students recorded their observations and calculations of each session in their lab journals. TAs and instructors collated the results of the class through a Google Form questionnaire asking for the nature and dimensions of the organisms in different samples and plotted the data using GraphPad Prism 8.0.2 to show the prevalence of different microorganisms. The data were shared with the students for them to see their values within the cohort. The values were also compared with the known sizes of the microorganisms.

Activities and outcomes

Microscopic images of microorganisms captured with smartphone cameras

Students had limited prior laboratory exposure. They were first trained with basic equipment, including micro-pipets, pH meter, centrifuges, weighing balance, microscope, and spectrophotometer. In subsequent laboratory sessions, they were advised to ask real-life questions using their skills. Instructors briefed the students about the activities to be performed in the 3-h laboratory session. The laboratory setup ensured the availability of equipment for hands-on training for every student within the schedule. Three laboratory sessions were dedicated to the activities discussed here. Students were trained with microscopes and microorganisms in the first session, Gram staining of microorganisms and milk products in the second, and further inquiries with samples of students’ choice, data analysis, and discussions on the health benefits of the microorganisms in milk products in the third.

Teams of five students were given pure cultures of E. coli, S. cerevisiae, and S. pombe for microscopy. Students were advised to prepare and image slides of at least one sample, and the team was instructed to observe the preparations of their members and analyze the data together. Finding focus planes with 100× objectives was often challenging for many. Staining with safranin eased the task. The exercise also familiarized the students with different microorganisms. They performed Gram staining of these organisms and captured images with their smartphones. The task of adjusting the smartphone for imaging required some practice; only specific orientations of the smartphone on the eyepiece could capture the slides (Fig. 1).

Fig 1.

Gram-stained samples of microorganisms visualized with a basic microscope and imaged using a smartphone camera. A student visualizing a Gram-stained slide using a basic microscope. A representative image taken with a smartphone camera of Gram-negative bacteria with a 100× magnification objective. Scale bar, 10 µm.

Sizes of various microorganisms were estimated using two methods: the FOV of the objective and the total magnification of the images. The class was encouraged to verify the FOV and magnifications by imaging a graph/ruled paper on the microscope stage. Students transferred the images to a computer and measured the dimensions of magnified cells in relation to the FOV. In the second method, total magnifications from the eyepiece, objective, smartphone, and computer were estimated. The size of a cell was back-calculated from the measured dimension divided by the total magnification. Students measured the sizes of E. coli, S. pombe, and S. cerevisiae using the magnification and FOV methods and found similar values (Fig. 2A and B). The values reported by the students (shown as median on the box plots) were close to the expected sizes of the microorganisms. The variation in the measured cell sizes might have resulted from differences in the cells’ growth phase, varied nutrient availability, or manual errors.

Fig 2.

Variations in cell size estimations done using magnification and FOV methods by students across different academic years. Sizes of E. coli, S. cerevisiae, and S. pombe measured by the classes. Box and whisker plots compare variations in the measurements obtained by the approaches of the microscope’s FOV (blue boxes) and the total magnifications of the microscope and smartphone cameras (pink boxes). Students were advised to count all cells in a FOV and calculate the average size of ten cells. The Y-axis represents the percentage of students reporting the prevalence of each type of microorganism in the given sample. Each box represents the interquartile range (IQR), with the horizontal line indicating the median cell size. Whiskers extend to the minimum and maximum values. (A) Cell size was measured using the magnification method by the class of 2022 and the FOV method by the class of 2023. (B) The class of 2024 used both methods. The numbers on the top of the boxes represent the median values. The known size ranges of E. coli, S. cerevisiae, and S. pombe are 1–3, 3–4, and 5–10 µm, respectively.

Microorganisms in homemade curd, commercial yogurt, and probiotics

After training with microscopic visualization of cells, measurement of cell dimensions, and Gram nature, students were asked to apply their skills to study microorganisms with potential health benefits (14). We provided homemade curd, commercial yogurt, and commercial probiotics and advised the students to observe the organism contents in the samples by Gram staining in one or more samples. Homemade curd fermented from milk was expected to contain Gram-positive rod-shaped Lactobacilli and round Lactococci, and oval-shaped yeasts, which also stain Gram-positive. A small number of students could not see any organisms in the samples. The team members were advised to analyze the data together (Fig. 3). Interestingly, students could readily observe Gram-positive bacilli and cocci in homemade curd, in addition to yeast and Gram-negative bacilli (Fig. 3H and I). By contrast, commercial yogurt was rich in Gram-positive cocci, and Gram-positive bacilli were not seen (Fig. 3F, G and I). Although commercial probiotics were expected to contain a dense population of Gram-positive Lactobacillus casei Shirota strain, the bacilli were seen by a small number of students only in a few batches of probiotics for reasons unknown (Fig. 3D, E and I).

Fig 3.

Representative images of the probiotics and microorganisms’ abundance in different samples. Images of different cells and probiotic samples with different types of microbes and their abundance. Images were captured with smartphone cameras without zoom. (A-C) E. coli, S. pombe, and S. cerevisiae stained with safranin. (D and E) Gram-stained probiotics showing cocci-shaped bacteria. (F and G) Gram-stained commercial curds (packaged sweet curd and unflavored curd) show rod-shaped bacilli. (H) Gram-stained homemade curd shows rod-shaped bacteria, cocci-shaped bacteria, and budding yeast. (I) Stacked bars represent the percentage of students reporting each microorganism in the samples. Scale bar, 10 µm. A few students could not see any organism in their samples (marked as “none”).

CONCLUSIONS

Our approaches gave hands-on training to a large undergraduate class and encouraged the students to ask real-life questions. Each student of the team tested at least one sample and analyzed the data within their team. Comparisons across teams and the rest of the class let students see their skills within the larger cohort. Most students captured good-quality images. Estimated cell sizes were close to the actual dimensions of microorganisms. In some instances, however, the values showed significant variations from the rest of the class, reflecting the heterogeneity of skills in large classes.

Students noticed striking differences in microorganism contents in homemade and commercial curds. Homemade curd had a heterogeneous population of Gram-positive and Gram-negative bacilli, cocci, and yeast, whereas the latter was homogeneous and rich in Gram-positive cocci. It likely reflected the difference in starter cultures used in preparations at home and in the industry. A large number of students could not find Gram-positive Lactobacilli in multiple batches of probiotics, in contrast to homemade curd. The observation suggested batch-to-batch variations of probiotics, lower organisms count, or a Gram-variable form of the bacteria, which could not be readily stained (15). These outcomes induced curiosity among students and encouraged intense discussions on the health benefits of various milk products (16).

Instructors, TAs, and laboratory assistants were integral to conducting the laboratory experiments. They invoked curiosity among the students through questions. Students learned problem-solving skills and team spirit. Some students went beyond the activity and came up with interesting ideas. For instance, some tested curd from their dining places for the presence of Gram-positive bacteria, while others tested commercial probiotics they were consuming to understand their health benefits. Interested students can continue their investigations of milk products in future laboratory courses by biochemical and molecular approaches described previously (17–19).

In conclusion, the students acquired problem-solving skills through real-life questions without adding financial constraints to the laboratory. They performed the tests, collected data, compared the results with other team members and the class, drew inferences, and kept the records. It also instilled teamwork and healthy competition. For many students, the exercise and its outcomes were the first real-life investigations with implications for health. The training was taken enthusiastically, as was obvious from their anonymous feedback. The course also gave a sense of accomplishment to the TAs and instructors.

ETHICS APPROVAL

Activities presented here are the outcomes of the instructors' interventions in a pre-approved laboratory course. They did not require ethical approval.