A practical low-cost course module for teaching bacteriophage isolation to undergraduate students

ABSTRACT

Bacteriophages are viruses that infect bacteria and have gained attention as promising tools against antimicrobial-resistant pathogens. Despite their relevance, phage biology remains underrepresented in undergraduate curricula. This study describes a low-cost course designed to introduce students to fundamental virology concepts through hands-on phage isolation, characterization, and genome analysis. Participants collected environmental water samples, performed phage enrichment, and detected viral plaques using the double-layer agar technique. The course also incorporated web-based bioinformatics activities using BLASTN and PhageScope, enabling students to explore genome size, GC content, gene annotation, and taxonomy without the need for coding knowledge or powerful computers. These activities strengthened the integration between laboratory and computational virology. Students had access to key concepts such as viral diversity, lytic cycles, biosafety, host specificity, and plaque quantification. This accessible educational model promotes active learning, democratizes access to virology training, and helps prepare future professionals for research in bacteriophage biology and phage therapy.

INTRODUCTION

Bacteriophages, or phages, are viruses that can infect only bacterial cells. After infection of host cells, these viruses can replicate and either induce cell lysis (lytic phages) or genome integration (lysogenic phages) (1). These viruses were described in the early 20th century, and due to their ability to control bacterial growth, they are recognized as a potential therapeutic tool against bacterial infections (2, 3). However, with the discovery of antibiotics and the wide use of these molecules by most countries worldwide, the use of phages as a viable option to treat bacterial infections was restricted to the former Soviet Union and Eastern European countries (4).

Nowadays, with the increase in antimicrobial resistance in several clinically important bacterial species, such as bacteria belonging to the ESKAPE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) (5, 6), and the failure to discover new antibiotics (7), phage therapy has gained attention, and it could be considered an important tool to combat antibiotic resistance (8). In general, the undergraduate health and bioscience programs still offer limited instruction on phages and their clinical applications, restricting students’ access to this emerging knowledge. Most of the undergraduate courses focus on bacteria, fungi, and viruses of medical or veterinary importance, with few or even no citations about the potential use of bacteriophages in clinical therapeutics. This gap can be attributed to both the lack of updated teaching materials and the lack of inclusion of this topic in official undergraduate courses.

To address this limitation, several educational initiatives have incorporated bacteriophage discovery into undergraduate research experiences (9–11). The semester-based Science Education Alliance–Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program is a widely implemented two-semester course that integrates environmental phage isolation, genome sequencing, and bioinformatic analysis within an authentic research framework (10, 11). SEA-PHAGES has engaged thousands of students across diverse institutions and has demonstrated strong educational outcomes, including increased engagement, scientific identity development, and student co-authorship in peer-reviewed publications (10, 11). More broadly, course-based undergraduate research experiences (CUREs) have been consistently associated with enhanced student motivation, persistence in STEM fields, and development of higher-order thinking skills (12, 13). These findings support the pedagogical value of integrating authentic research into the undergraduate curriculum.

However, while highly impactful, programs such as SEA-PHAGES typically require a full academic semester or year, access to sequencing infrastructure, and sustained institutional investment. Such requirements may limit adoption in institutions with restricted time allocation or limited laboratory resources. In contrast, the model described here was designed as a short-duration (5-day), low-cost module that can be implemented using commonly available laboratory materials without requiring genome sequencing infrastructure. By combining environmental phage isolation, plaque visualization, host range assessment, and introductory web-based genome analysis, this course offers a scalable and accessible alternative for institutions seeking to introduce phage biology within constrained curricular structures.

In this context, we describe a low-cost, biosafety-conscious course designed to disseminate fundamental concepts of bacteriophage biology and phage therapy while promoting active learning and research-based training. By integrating essential theoretical and practical components within a condensed format, this model addresses the limited curricular exposure to phage biology and expands equitable access to authentic research experiences in microbiology education. The course provides undergraduate students with complete hands-on experience from environmental sampling to phage isolation and introductory genome analysis, using commonly available laboratory materials. By integrating essential theoretical and practical components within a condensed format, this model helps address the limited exposure to phage biology in undergraduate education, while promoting broader and more equitable access to research-oriented learning opportunities. By democratizing access to this knowledge, the course can represent an important step toward scientific inclusion, stimulating new research interests and strengthening the critical training of future health professionals.

PROCEDURE

Materials and methods

Course structure and participants

The course was designed as a standalone training program rather than a module within a longer academic discipline, allowing flexible implementation across different curricula. It was offered once, in August 2025, and primarily targeted undergraduate students enrolled in Biological Sciences, Biomedicine, Pharmacy, and Biotechnology programs, with limited participation from graduate students. Most participants had little or no prior experience with bacteriophage research, making the course suitable for early-stage scientific training. The program was structured as an intensive short-term course combining theoretical lectures, hands-on laboratory activities, guided discussions, and collaborative data analysis (Table 1). On the first day, participants were introduced to basic concepts on the subject and received training on sample collection procedures (1:00–2:00 p.m.). For this purpose, sterile universal collection containers and gloves were provided, along with a detailed explanation of the environments suitable for sample collection, such as bathroom drains, sinks, toilets, grease traps, rivers, and streams (day 1). In addition, detailed biosafety information was presented, including an explanation of the risks associated with handling these samples and the need to avoid direct contact with samples and the necessity to use personal protective equipment, such as protective gloves. It is important to note that sample collection was performed before the laboratory activities by the students.

TABLE 1.

Timeline of activities in the bacteriophage course

Day	Main activities	Key procedures	Expected outcome
Day 1	Introduction and sample collection	Instructions about sample collection and biosafety Collection of environmental samples	Samples ready for processing
Day 2	Laboratory introduction and sample preparation	Biosafety training Introduction to the equipment Introduction to bioinformatic analysis (genome assembly, genome assembly, and available tools) Sample centrifugation and filtration Preparation of enrichment cultures	Enrichment cultures prepared and incubated (overnight) Bioinformatic analysis
Day 3	Phage detection (initial screening)	Processing enrichment cultures (chloroform treatment) Preparation of double agar plates Spotting enrichment cultures Spotting filtered samples	Plates incubated for phage detection (lysis plaques expected next day) Bioinformatic analysis
Day 4	Analysis and experimental expansion	Detection of lysis zones Preparation of phage stocks Preparation of new plates for phage titration and host range assays	New plates incubated (titration and host range results expected next day) Bioinformatic analysis
Day 5	Final analysis and presentation of the results	Phage titration (PFU/mL) Host range evaluation Genomics discussion Group presentations	Interpretation of results and course conclusion

The experimental activities were delivered over 4 consecutive days, in the afternoon (1:00–5:00 p.m.), totaling 16 contact hours (4 h per day). Activities were organized sequentially to simulate an authentic research workflow, including environmental sampling, phage isolation, purification, amplification, characterization, and preliminary genomic analysis. Each daily session integrated a conceptual background with immediate experimental application. Students worked in small groups to promote collaborative problem-solving and scientific discussion, and each group was responsible for documenting experimental procedures and recording observations in laboratory notebooks. The preliminary results were presented at the end of the course (fifth day). Assessment was formative and based on participation, laboratory performance, data interpretation, and a final oral presentation summarizing the phage discovery process.

All laboratory activities were conducted under Biosafety Level 1 (BSL-1) conditions, in accordance with internationally recognized biosafety guidelines for undergraduate microbiology teaching laboratories (BMBL) (14). Only non-clinical environmental samples were processed, and all host bacterial strains used were either standard laboratory strains or reference strains widely employed in educational settings. Students received formal instruction on risk assessment, safe handling of environmental samples, surface decontamination using 70% ethanol and sodium hypochlorite, and proper biological waste disposal through autoclaving. The inclusion of chloroform treatment during enrichment processing further ensured inactivation of residual bacterial cells prior to plaque assays. These measures align with recommendations for safe implementation of course-based undergraduate research experiences in microbiology (15). Instructors implementing this module may consider the use of pre-screened environmental samples, particularly when working with early-stage undergraduate students or in institutions with more restrictive biosafety policies. This approach can help balance the pedagogical benefits of environmental sampling with institutional safety considerations.

Bacterial strains and growth conditions

Five bacterial strains were used as host organisms: Escherichia coli DH5α, E. coli BL21, E. coli ATCC 25922, Proteus mirabilis ATCC 25933, and Pseudomonas aeruginosa PA14. All strains were cultivated in Luria–Bertani (LB) broth or LB agar at 37°C. For double-layer agar assays, bottom agar contained 1.5% agar, whereas the soft agar overlay consisted of LB with 0.7% agar. It is important to note that some of the strains used in this course (P. mirabilis and P. aeruginosa) require Biosafety Level 2 facilities. Therefore, for instructors that might not have access to BSL-2 facilities, these organisms may be replaced by BSL-1 equivalents, such as non-pathogenic, well-characterized, safe hosts. This substitution preserves the educational objectives of phage isolation, host range assay, and characterization while ensuring compliance with biosafety regulations.

Sample collection and enrichment of phages

On the first day of the course, all participants (n = 20) received a sterile 15 mL tube and disposable gloves to collect environmental house water samples. The students chose to collect water from a bathroom sink, a grease trap, a toilet, and a bathroom drain. Tutors gave instructions to the students regarding sample collection procedures and biosafety. Specifically, students were instructed to wear gloves during sample collection to avoid direct contact with samples and ensure that collection tubes remained closed except during sampling. Samples were collected in well-ventilated household environments, avoiding high-risk locations such as hospital effluents or industrial waste sources. After collection, tubes were securely sealed, externally disinfected when necessary, and transported in secondary containment to prevent leakage.

Students were also instructed to perform proper hand hygiene immediately after sample collection and to dispose of gloves appropriately. All procedures were conducted under Biosafety level 1 (BSL-1) guidelines. The next day (day 2), students were divided into five groups (n = 4) and received a manual of all experimental protocols used in the course (supplementary material). Students shared information about the collected samples (type of sample and visual aspect). All samples were centrifuged at 1,000 × g for 10 min at room temperature to remove debris. The supernatant of samples from the same group was collected and used to create a unique pooled sample for that group. The pooled samples were filtered through 0.22 µm syringe filters to eliminate microbial cells, and the filtrates were stored at room temperature. For enrichment of phages, 4 mL of the pooled sample was added to a sterile 50 mL tube containing 0.5 mL of host bacterial culture grown overnight and 1 mL of 5× LB medium to a final volume of 5 mL. Tubes were incubated overnight at 37°C under agitation. To maximize the chance of isolating bacteriophage during the course, a trained microbiologist collected 10 mg of dog feces and spiked it in sterile water. This sample was considered the positive control for the course and processed by one of the trained microbiologists as described above for the environmental samples. The dog feces sample was obtained from a healthy domestic animal with no history of infectious disease and handled exclusively by trained personnel under BSL-1 precautions. As an alternative to the use of fecal material as a positive control, instructors may employ pre-isolated laboratory phage stocks or previously screened environmental samples known to contain bacteriophages infecting the selected host strains. This approach eliminates the need to handle fecal material while still ensuring reliable demonstration of plaque formation. The choice of control should be guided by institutional biosafety policies and the level of student experience.

Bioinformatic analysis

To introduce core concepts of phage genomics, each group received a FASTA file containing a complete genome deposited in the NCBI database (FLC2: NC_027125.1; FLiP: NC_047837.1; ISP: NC_047720.1; T4: NC_000866.4; and VAC3: PQ466445.1). An overview of genome size, genome organization, and the importance of annotation was provided using the PhageScope platform (16) (https://phagescope.deepomics.org/) and BLASTN (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Regarding the PhageScope platform, students uploaded the phage sequence (FASTA format) and used the Genome Annotation tool in the analysis menu. In the module list, they should mark all modules (Completeness Assessment, Phenotype Annotation, Structural Annotation, and Functional Annotation). Similarly, the phage nucleotide sequences were uploaded in FASTA format into the BLASTn interface. The “nr/nt” database was selected as the reference data set, and viruses (Taxid: 10239) were selected as the model organism. Default parameters were used and searches were performed using the “Highly similar sequences (megablast)” option to prioritize identification of closely related phages.

Students were instructed to analyze key output metrics, including percentage identity, query coverage, E-value, and alignment length, to infer sequence similarity and potential taxonomic relationships. Comparative analysis was conducted by examining the top hits and identifying conserved regions across related phage genomes. After guided instruction by the tutors, each group performed basic genomic analyses, including determination of GC content, genome size, number of predicted genes, functional classification of coding sequences, and taxonomic inference based on sequence similarity. The objective of this activity was to introduce students to fundamental principles of phage genome organization, annotation, and comparative analysis. Students were guided through structured analytical questions designed to connect genome features (e.g., GC content, gene density, and functional modules) with biological concepts discussed in the laboratory component. Integrating genome analysis into undergraduate research experiences has been shown to strengthen conceptual understanding and promote higher-order scientific reasoning (17). Moreover, incorporation of bioinformatics into phage-focused CUREs, such as SEA-PHAGES, has demonstrated that genome annotation activities enhance student engagement and interdisciplinary skill development (10). By combining wet-lab isolation with computational genome exploration, the course reinforces the interconnected nature of modern microbiological research.

Processing of enrichment cultures

The following day (day 3), 0.9 mL of enrichment culture was mixed with 0.1 mL chloroform, homogenized, and stored on ice for 15 min to lyse remaining bacterial cells. Phage detection was performed using the double agar overlay technique (18). Briefly, 0.5 mL of overnight exponential-phase host culture was mixed with 3 mL of soft agar (0.7% agar, pre-warmed at 55°C) and overlaid onto LB agar plates (1.5% agar). After solidification, 10 µL aliquots of enrichment supernatants or directly filtered samples were spotted onto the bacterial lawns. Each group used their own enrichment cultures after chloroform addition (containing only viable phages), as well as enrichment cultures from the other groups. Plates were incubated at 37°C, and lysis zones (plaques) were recorded after 18–24 h.

Phage stock preparation and titration

On day 4, students came to the lab in order to see if there were areas of lysis on their agar plates. If positive, they scraped the lysis area from the plates using 1 mL sterile tips and suspended it in 0.9 mL sterile LB broth. A volume of 0.1 mL chloroform was added to each suspension, mixed, and stored at 4°C for further use. Serial 10-fold dilutions (10⁻¹ to 10⁻⁵) of phage suspensions were prepared in LB broth. Ten microliters of each dilution, as well as undiluted stock, were spotted on double-layer agar plates. After incubation (day five), plaques were counted, and titers were expressed as plaque-forming units (PFU) per milliliter. To evaluate host specificity, 10 µL of phage suspensions were spotted onto lawns of all bacterial strains used in the study. Plates were incubated at 37°C, and plaque formation was assessed to determine the phage host range.

Activities and outcomes

Throughout the 5-day course, all groups completed each experimental step, from environmental sampling to phage enrichment and detection. Clear lysis zones were observed in plates (Fig. 1) after enrichment, indicating that students successfully isolated active bacteriophages using simple and low-cost materials. Each group pooled its environmental samples and performed enrichment using a designated bacterial host strain. After incubation, the enrichment supernatants were collected and screened for lytic activity by spot-test assay against all bacterial strains available in the course, not only the strain used for enrichment. Therefore, Fig. 1 is representative and indicates positive (left) and negative (right) results of the initial cross-screening of enrichment supernatants across multiple potential hosts. Table 2 shows that four groups (80%) were able to isolate bacteriophages. The host range assays revealed that two isolates were able to infect more than one bacterial strain, reinforcing the biological diversity of environmental phages (Table 2). The use of common laboratory strains of E. coli (e.g., DH5α) and the characteristics of environmental samples collected reinforce the idea that coliphages were one of the most abundant bacteriophages in the environment (19–21). It is important to note that among all samples, 10 (50%) were collected from drains and 4 (20%) were collected from toilet water. The high number of phages recovered from bathroom-related samples is consistent with the current knowledge about the human intestinal virome (22, 23). Human feces have high concentrations of bacteriophages (10⁹–10¹⁰ PFU/g), and more than 90% of the intestinal virome is composed of phages (24). In addition, toilet flushing produces aerosolized particles capable of dispersing viral particles efficiently across surfaces (25–27). These mechanisms may explain why bathroom environments represent hotspots for phage presence and why samples from drains and toilet water in our course yielded active coliphages. Collectively, these observations reinforce that phage-rich locations increase the likelihood of successful isolation by students using simple enrichment procedures.

Fig 1.

Detection of bacteriophages isolated by student groups using the spot-test assay after enrichment of pooled environmental samples from each student group. Each plate corresponds to the enrichment culture obtained by pooling environmental samples collected by a single group and incubating them with a specific host bacterial strain. After enrichment, supernatants were tested for lytic activity using the spot-test assay on lawns of the same bacterial host used during enrichment. The plates are divided into numbered sectors corresponding to independent enrichment samples from different student groups (1–5). Sector six represents the positive control enrichment sample. In the left plate (E. coli ATCC 25922), the circled dotted regions indicate clear lysis zones, confirming the presence of active bacteriophages capable of infecting the host bacterial strain used. The plate on the right (P. mirabilis ATCC 25933) shows no detectable lysis, indicating that no infectious phages were recovered from those samples under the conditions tested. Legend: F1: enrichment culture from group 1, F2: enrichment culture from group 2, F3: enrichment culture from group 3, F4: enrichment culture from group 4, F5: enrichment culture from group 5, and F6: enrichment culture from positive control.

TABLE 2.

Experimental outcomes of environmental sampling and phage isolation performed by five student groups

	Group 1	Group 2	Group 3	Group 4	Group 5	Positive control
Sample type	Water	Water	Water	Water	Water	Water spiked with dog feces
Sample collection site	Toilet Grease trap Bathroom sink River	Bathroom drain Laboratory drain Water from plant pot tray Toilet	Sink drain Bathroom drain Bathroom drain Public square fountain	Laundry drain Bathroom drain Water tank Bathroom drain	Bathroom drain Toilet Toilet Bathroom drain	Not applied
Host bacterial strain	E. coli DH5α	E. coli BL21	E. coli ATCC 25922	P. aeruginosa PA14	P. mirabilis ATCC 25933	E. coli DH5α
Presence of infective phages	Yes	Yes	Yes	Yes	No	Yes
Host range of isolated phages	E. coli DH5α	E. coli BL21 E. coli ATCC 25922	E. coli DH5α E. coli ATCC 25922	P. aeruginosa PA14	Not applied	Not tested

Additionally, it is important to note that among the different environmental samples, the strategy used to pool samples increases the rate of phage isolation and reduces the cost to course implementation. Use of pooled samples is a very common strategy to manage a high number of samples and increase the rate of isolating bacteriophages (28, 29). Besides the different outcomes in isolation, this experience helped students to connect theoretical microbiology concepts with real applications. The detection and quantification of viral plaques help students to visualize and understand the biological impact of viruses on bacterial populations and the rational use of phage therapy as an alternative to antimicrobial resistance (8).

Before the course, most students were unfamiliar with phage therapy, and at the end, all students were able to correctly define phage therapy and its benefits. Additionally, other important scientific concepts were also understood by students, such as biosafety, sample handling, aseptic techniques, viral isolation, and quantification by plaque-forming units (PFU). Although no formal quantitative assessment instrument was applied during this first implementation, student learning was inferred from active participation in laboratory activities, successful execution of protocols, correct interpretation of plaque assays, and final oral presentations. Future iterations of the course may incorporate validated pre- and post-course assessment tools to generate quantitative measures of learning gains, in accordance with recommendations for evaluating course-based undergraduate research experiences (12, 13).

Although sequencing was out of the scope of the course and would make it costly, the students were also exposed to basic phage bioinformatics by the use of friendly web-based bioinformatic tools to analyze bacteriophage genomes from the literature. The two main platforms used (BLAST N and Phagescope) and different genomes were used to improve some important aspects in bacteriophage field research. The students conducted research in these two online platforms to gain knowledge about the necessity of determining genome size and annotation. This helps students to understand how bioinformatics can connect wet-lab to computational data to characterize bacteriophage. A high level of student engagement was observed throughout all steps, and since the first day of the course, all students have expressed satisfaction with the results obtained. On the final day, all students showed their results in a seminar, where all laboratory and bioinformatic results were presented to other groups. The incorporation of a genome analysis component, using freely available online tools such as BLASTN and PhageScope, reinforced the interdisciplinary nature of virology by integrating computational skills into the experimental workflow. Beyond the experimental outcomes, our short course demonstrated how hands-on, low-cost phage isolation activities can substantially enhance students’ conceptual understanding of core virology principles.

The estimated consumable cost per student was approximately USD 5, primarily covering culture media, disposable tubes, pipette tips, and agar plates. No sequencing costs were incurred, as genome analysis relied on publicly available data sets and free online tools. This contrasts with comprehensive semester-long CURE programs such as SEA-PHAGES (10, 11, 13), which include genome sequencing and annotation components and therefore require greater financial and infrastructural investment. By prioritizing accessibility and reducing operational costs, our short-duration module increases feasibility for institutions with limited laboratory resources.

Current virology curriculum guidelines emphasize that undergraduate training should prioritize foundational concepts, such as virus–host interactions, replication strategies, viral diversity, and ecological roles (30). By isolating and characterizing bacteriophages from real environmental samples, students directly engaged with topics such as viral ecology, lytic cycles, and host specificity. This experiential approach mirrors the recommendations for active learning and course-based undergraduate research experiences, which are recognized as best practices for cultivating higher-order thinking and scientific reasoning in virology education (31).

This initial implementation has limitations. The short duration of the course did not allow for full genome sequencing of isolated phages, and educational outcomes were assessed qualitatively rather than through validated quantitative instruments. Additionally, the small cohort size limits the generalization of the educational impact. Nevertheless, as a proof-of-concept, the model demonstrates feasibility and scalability in resource-limited educational settings.

The first implementation of the course provided important insights for future iterations. High levels of student engagement underscored the effectiveness of integrating theoretical instruction with hands-on and introductory bioinformatic activities within a condensed format. Detailed protocol manuals were critical for supporting student autonomy and ensuring efficient use of laboratory time. Additionally, pooling environmental samples increased phage recovery while minimizing costs, reinforcing the scalability of the model for resource-limited institutions. Future offerings may benefit from dedicating additional time to data interpretation and structured scientific discussion to further strengthen analytical skills and critical thinking.

CONCLUSION

One of the most important characteristics of this course is the possibility to show the students the phage therapy concepts with minimal resources. The entire course was designed to be done with a low-cost investment, using reagents and materials commonly available in undergraduate teaching laboratories. The main experimental steps (e.g., sample collection, phage enrichment, and plaque visualization) required only standard consumables such as Luria-Bertani broth and agar, sterile saline, and basic plasticware. Using these materials and the environmental samples as phage sources, the total cost per student was estimated at approximately USD 5 per student. This value makes the course financially adequate for institutions with limited budgets and does not compromise educational quality. The use of real environmental samples and scientific methodology strengthened the connection between theory and practice, increasing the curiosity and critical thinking of students. Another point is the alignment of this course with broader educational goals of inclusivity and democratization of science. By reducing the financial barriers, this model course allows institutions with all types of budgets (high, medium, and low) to offer experimental learning opportunities. Moreover, the modular structure of the course facilitates adaptation by educators in diverse academic contexts, including colleges, schools, and universities in developing regions. In conclusion, this low-cost model of course can be used strategically as a powerful pedagogical tool to enhance microbiology education and promote equity in access to scientific experiences, and inspire the next generation of researchers in the field of bacteriophage biology.

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/jmbe.00065-26.

Supplemental material. jmbe.00065-26-s0001.pdf.

Manual of experimental protocols used in the course.

ASM does not own the copyrights to Supplemental Material that may be linked to, or accessed through, an article. The authors have granted ASM a non-exclusive, world-wide license to publish the Supplemental Material files. Please contact the corresponding author directly for reuse.