Microbial life in a winogradsky column: from lab course to diverse research experience.

INTRODUCTION

Students struggle to understand the transition between teaching labs and authentic research (7). This presents a difficulty in encouraging student research and inquiry-based learning. The goal of this course was to design a lab in which students developed individualized research, engaged in cooperative problem-solving, presented their findings in a meaningful manner, and gained experience in the mechanics of authentic research. An additional benefit of the lab was that it correlated lecture with lab, promoting student engagement in lecture and overall comprehension.

Winogradsky columns are easily constructed enclosed ecosystems that enable students to observe growth and changes in microbial populations, as well as the impacts that additives and contaminants yield within the columns. Such columns have previously been used to visually observe microbial communities, to demonstrate unique microbial metabolism (4), and as a portion of general microbiology labs (8). This lab course is novel in that the Winogradsky column was the central focus for the lab and provided a framework to achieve all general microbiology lab learning objectives. Additionally, this lab uniquely provided students with authentic research experiences throughout the entire course.

PROCEDURE

The course was designed such that students began constructing their Winogradsky columns on the first day of lab. Subsequent weeks were used to perturb the columns and allow sufficient time for the columns to incubate. During the incubation time, students learned basic biochemical and molecular assays (Appendices 1, 2), which they later applied to the columns for characterization purposes.

Winogradsky column construction

Following basic instruction regarding the historical usage of Winogradsky columns, students were given soil from a single site, distilled water, and 500-mL graduated cylinders. Students were instructed to choose one or more carbon sources from several possibilities (newspaper, un-bleached paper towels, cellulose, calcium carbonate) and a sulfur source (calcium or magnesium sulfate). Students were instructed to make two columns, filling them approximately with 1/3 soil, 1/3 water, and leaving the rest as head space. Columns were sealed with parafilm. One column was the control; to the experimental column, students were encouraged to add in a contaminant of interest, based on individual research. Such contaminants included, but were not limited to, phosphate, nitrate, sulfur, oil, nitroaromatics, and organophosphates. Contaminant concentrations and mode of addition varied per student, and were based upon data found in current scientific literature.

Winogradsky column assessment

Columns were assessed weekly via pH, observation, and documentation of column development. Pipettes were used to extract samples from targeted regions of the columns. Microbial isolates were obtained via repeated passage, using minimal media plates (5) supplemented, appropriately, with column chemical constituents. Isolates were then assayed biochemically (Appendix 1) and molecularly, using 16S-rRNA amplification and sequencing (Appendix 2) for characterization.

In addition to standard biochemical assays, several diffusion-based assessments were conducted to determine sensitivity to, and catabolism of, varied substrates. Isolates were tested for antibiotic sensitivity using the Kirby-Bauer Disk Diffusion protocol (Appendix 1). Isolates were also tested for carbon, nitrogen, and contaminant metabolism and resistance using auxanography (5). Briefly, sterile Whatman disks were placed on inoculated spread plates. Disks were loaded with appropriate concentrations of column-specific substrates. Plates were incubated and potential zones of growth and/or inhibition were measured.

Polymerase chain reaction (PCR) amplification of a partial 16S-rRNA sequence was conducted on colony preparations, using universal prokaryotic primers 341F and 926R (2). Amplicons were purified using Promega Wizard PCR Clean Up Systems (Madison, WI) and sent to Genewiz (South Plainfield, NJ) for sequencing. BLASTN analysis (1) was conducted on sequences following comparison in Gen-Bank (http://www.ncbi.nlm.nih.gov/GenBank/index/html) to determine the most likely identification of each isolate. As appropriate, identification of key metabolic (per student-chosen contaminants) genes was conducted following student-identified published protocols.

Student assessment

Students maintained authentic lab notebooks and engaged in weekly progress meetings, during which the entire lab participated in presenting results, troubleshooting, and brainstorming for further experimentation. Lab notebooks were checked periodically for protocols, observations, and data. Students were encouraged to use their lab notebooks to maintain ideas for further experimentation. Weekly lab meetings allowed students to compartmentalize key data, express it to lab mates, and provide feedback to lab mates regarding experimentation and hypotheses. Such meetings were facilitated by both the instructor and a graduate teaching assistant. Students were required to submit periodic progress reports and conduct group meetings with the instructor to discuss findings and logical progression for their research. Students wrote three lab reports throughout the semester. The first report detailed the setup and basic characterization of the columns. The second report presented data regarding carbon and nitrogen utilization, antibiotic sensitivity, 16S-rRNA characterization, and key genetic characterization of prokaryotic isolates from the columns. Students constructed a final report and presentation of their semester-long experimentation and subsequent results from their columns. Presentations were held as a seminar to encourage students to share their methods and findings and compile all results from the initial soil samples into a more composite understanding of microbial diversity.

Safety issues

Appropriate BSL2 procedures (3), including personal protective gear, signage, and disposal, were required for the above-described lab activities. Due to the unknown nature of potential isolates, all incubation of columns and plates was conducted at room temperature. Student-selected additives and contaminants were investigated and appropriate MSDS guidelines followed, and posted, during experimentation and disposal.

CONCLUSION

Winogradsky columns are ideal lab activities because they encourage customization and personalization of the lab experience. Activities with such columns can easily be adapted to all levels of students, including nonmajor, introductory level, and advanced students. Other components of the columns (chemistry, metabolism, genetics, ecology, etc.) can be easily assayed and effectively correlated with lecture topics.

This lab experience was unique in requiring students to engage in authentic research. Students gained experience and confidence with novel methods, as well as with designing and troubleshooting experiments. Such an opportunity enlivened students and encouraged their creativity. Additionally, the lab correlated well with Introductory Microbiology lecture throughout the semester (cell structure, metabolism, biogeochemical cycles, antibiosis, etc.), offering a way to reinforce lecture material throughout lab and to introduce students’ research into lecture. Furthermore, using the Winogradsky lab encouraged student learning of microbiology principles, as well as their learning of the underlying purpose and process of authentic research.

Appendix 1: List of biochemical and molecular assays utilized in the Winogradsky column lab, as well as purposes and protocol references

Appendix 2: 16S-rRNA primers and PCR conditions