A Multi-Unit Project for Building Scientific Confidence via Authentic Research in Identification of Environmental Bacterial Isolates.

INTRODUCTION

This is an open-ended, authentic research project for an upper-level undergraduate microbiology laboratory course. It is intended to push students who have little experience with real research, and build their confidence in their own problem-solving skills. These types of course-based research experiences are rapidly becoming a new and important model for undergraduate course design (1). The project was completed in groups of three to four students, to encourage teamwork and reduce materials and costs. The goal of the project was the identification of an unknown bacterial species isolated from a mixed-species biofilm from local tap water using a CDC biofilm reactor (3). These biofilm bacteria were part of a larger scholarly research project of the faculty teaching the course. Thus, students were engaged in an authentic research project that helped create real, publishable results. Three units, as described below, were completed as overlapping projects to allow time for data collection, troubleshooting, and repeated experiments.

PROCEDURE

Prior to student experiment

Environmental bacteria were collected and isolated from local tap-water. Water was used to inoculate a CDC-type bioreactor, which was fed dilute (1:100) R2A media (1 mL/min) at ∼25°C for eight days (3), prior to removal of PVC coupons and biofilm disruption by a sonicating water bath. After collection of environmental isolates on R2A at 30°C, the biofilm was inoculated with the human water-borne pathogen Legionella pneumophila. The biofilm population was able to sustain survival of this important pathogen, and identification of the bacteria from the biofilm is of interest in understanding the ecology of Legionella in biofilms present in human-made water systems. The unknown bacterial isolates for lab were ensured of being free of this BSL2 pathogen, since Legionella will not grow on R2A agar (3). However, these environmental isolates could be human pathogens and must be treated as BSL2 (see ASM Biosafety Guidelines; 2). Alternative methods for building simple biofilm growth chambers can be utilized (see Chapter 11 in the Biofilm Hypertextbook for examples, http://www.hypertextbookshop.com/biofilmbook/v004/r003/).

Unit one: metabolic, phenotypic analysis and Bergey’s manual

Students were provided with an R2A agar plate containing a pure isolate from the biofilm reactor (from a 50% glycerol cryostock at −80°C) and completed various staining tests (Gram, endospore, etc.) to classify their species within Bergey’s manual. Students also completed four metabolic tests from a list provided by the instructor (Appendix 1). All media, reagents, and design of appropriate negative and positive controls were the student’s responsibility. If any test had unsuccessful controls, students were required to repeat or attempt alternative tests. Here, students gained confidence in preparation and execution of microbiology experiments and collected data for tentative identification of their species.

Unit two: PCR amplification and sequencing of 16s rRNA gene for species-level identification

In each lab class, students were provided with one set of primers for PCR amplification of portions of the 16s rRNA gene (Appendix 2). Short products were used to increase the likelihood of successful amplification and minimize PCR repeats. DNA templates were obtained from a single colony of bacteria using a detergent-based lysis protocol (MICROLYSIS, Microzone, Haywards Heath, UK). The PCR reagents used were in premixed, freeze-dried beads (PURETAQ READY-TO-GO PCR BEADS, GE bioscience), to limit the likelihood of human error in assembly of PCR reactions. Qiagen spin columns were used to clean up PCR prior to sequencing, and samples were mailed to a regional facility (http://www.biotech.cornell.edu/brc/genomics) for sequencing. The amplification primers were successful when used as sequencing primers. Students were directed to edit and analyze their sequences following the steps in Table 1. All species with >97% similarities in 16s sequence were considered candidates for identification and were used to create phylogenetic trees of closest neighboring sequences.

TABLE 1.

Steps for 16s data analysis.

Goal	Resource
1) Change orientation of reverse data	http://www.bioinformatics.org/sms/rev_comp.html
2) Compare forward and reverse sequence (from Step 1) for differences	http://www.ebi.ac.uk/Tools/msa/clustalw2/
3) Consult raw chromatogram to resolve differences	http://www.geospiza.com/Products/finchtv.shtml
4) Compare consensus sequence to database	EzTaxon at http://www.ezbiocloud.net/
5) Assemble multiple alignment and phylogenic tree of highest hits on EzTaxon	http://www.ebi.ac.uk/Tools/msa/clustalw2/

Unit three: ability of a bacterial isolate to grow as biofilms

This unit required students to design all aspects of the study. The media/nutrient source, growth temperature, growth surface/container (24-well plates, glass tubes, or plastic tubes were provided), and length of the experiment for biofilm growth were student selected. Students were provided generic protocols for determining total biofilm content by crystal violet staining or by total CFU measurements (Appendix 3). Only one attempt was required, though most students had time to repeat unsuccessful attempts.

Assessment

Students completed weekly notebook entries outside of class, based on their notes from each week, which were due one week after each lab session. These described all experiments and results collected during one lab session, including all units worked on that day. The final project results were compiled into a single group presentation given in the final week of class.

Safety issues

Environmental isolates could include human pathogens; isolates should be treated as BSL2. For metabolic testing, chemical safety could be a concern; some reagents carry hazardous risks and require careful management of the hazardous waste generated.

CONCLUSION

This general experimental protocol has been used twice in my upper-level microbiology lab course. In each case, all of the groups obtained usable DNA sequencing data. Most groups had excellent results, with data from both forward and reverse primers for assembly of a consensus sequence, increasing the accuracy of the data. Many of the species identified have only been described recently (within the last five years), thus very little data regarding their metabolic phenotypes or biofilm growth capabilities is available, increasing the value of the student data. Once full-length 16s sequences are completed, these data are likely to be utilized in a publication by the instructor.

Educational outcomes self-reported by the students were highly favorable. Students reported more confidence in the design of scientific experiments and in the use of positive and negative controls (Fig. 1, Table 2). Many students reported they had no research experience prior to this course (50% in 2013 and 38% in 2014). In addition, each course was composed of over 80% seniors (88% and 84% in 2013 and 2014, respectively). Thus, it is clear that this project helped to provide at least one independent research experience to graduating Biology majors.

FIGURE 1.

Student responses to post-course survey regarding their own confidence and skill in experimental design after the first semester of the project in 2013. Similar results were obtained from the 2014 lab section.

TABLE 2.

Student responses to research project.

My favorite part of this project was
“Developing individual projects from our own ideas. Having the freedom to choose the path of the experiment. Seeing real results from the successful tests and sequencing.”
“…designing the experiments and getting a chance to see my mistakes through the write ups”
“…designing the biofilm experiments, because we didn’t have any strict guidelines to follow. We were able to test any conditions we wanted”
“The identification… really made the group feel accomplished”
“It helped me to be more confident with keeping notes in lab and understanding what’s really going on, rather than just following a given procedure”
“I loved performing our own choice of tests based on the results… It felt like a treasure hunt.”
“…the metabolic tests… the use of positive and negative controls was really helpful and educational”

Appendix 1: Metabolic testing assignment sheet

Appendix 2: 16s PCR and sequencing primers

Appendix 3: Two methods for quantification of biofilm growth