We designed a microbiology project that fully engaged undergraduate biology students, high school students, and their teachers in a summer research program as part of the Research Education Vitalizing Science University Program conducted at California State University Bakersfield. Modern molecular biological methods and microscopy were used to detect and identify microcrustacean species in ponds around Bakersfield, California, that harbor the amphibian pathogen Batrachochytrium dendrobatidis (Bd). The students learned about the amphibian decline in California and worldwide due to chytridiomycosis and how microcrustaceans as natural predators of Bd-zoospores can be used in mitigation strategies for amphibian conservation.

To spark an interest in biology for students in K–12 institutions and beyond, it is important to bring current research topics into the classroom. Freshwater environments such as ponds are ideal for making students aware of the diversity and complex interactions of its inhabitants. In the project described here, we focused on the detection of the fungal pathogen Batrachochytrium dendrobatidis (Bd), which is responsible for the decline of many amphibian populations worldwide. At the same time, we also investigated the diversity of its natural predators, filter-feeding microcrustaceans that prey on motile zoospores of the fungus. It is exciting to include modern technologies in lab experiments similar to those used in environmental and clinical biology research facilities throughout the world. The cost of supplies for these technologies is becoming more affordable, and students are excited to realize that these “complicated techniques” are, in fact, not that complicated at all. Students who are confident about what they can achieve in a science class, and who have been trained in modern molecular biological methods in addition to traditional techniques, will be better prepared for graduate school and possible careers in microbiology. Being included in a meaningful research project also enforces critical-thinking skills that are important for literature research, development and understanding of a project, evaluation of research data, and the presentation of results. Along with acquiring these skills, students also become educated citizens who gain an understanding of microbial interactions based on a topic of global concern: the ongoing amphibian decline due to chytridiomycosis.

Concept Explanation

Batrachochytrium dendrobatidis is a fungal pathogen that has been identified as the causative agent in the decline of many amphibian populations in California and worldwide (for an extensive discussion of amphibian decline due to chytridiomycosis and amphibian conservation strategies, see Woodhams et al., 2011).

A decline of diverse microcrustacean species, which can be natural predators of Bd (Buck et al., 2011; Woodhams et al., 2011), has been observed in some areas (e.g., Lake Tahoe and the Sierra Nevada Mountains) (Goldman et al., 1979; Nielsen et al., 2000). The identification of diapausing eggs from microcrustaceans can serve as an indicator for the diversity of microcrustaceans in the past (Limburg & Weider, 2002).

After obtaining water and sediment samples from different ponds, we determined the microcrustacean diversity in the water samples using microscopy, and then used molecular tools to detect Bd and to analyze the diversity of micro-eukaryotes in water and sediment samples. We also identified the amphibian species present at each pond.

Modern clinical and environmental research labs are nowadays using molecular techniques to investigate microbial diversity and community structure in various environments and to identify microbial pathogens. Molecular techniques have revolutionized biological sciences by allowing organism identification without cultivation. Unfortunately, as currently structured, many microbiology teaching labs do not expose undergraduate students to these current, and now somewhat standard, techniques. To address this, we developed and successfully conducted a lab that links traditional microbiological techniques such as microscopy, with electrophoresis techniques based on the polymerase chain reaction (PCR). Along with making students excited about microbiology and teaching important techniques that are standard in many laboratories, the aim of this lab exercise was to make students and their teachers aware of the connection between amphibians, an amphibian pathogen, and microcrustaceans as predators of single-celled prokaryotes and eukaryotes (including motile zoospores of the chytrid pathogen). This was accomplished by investigating water and sediment from ponds of different altitudes and environments near Bakersfield, California.

Materials Needed

The equipment and lab supplies indicated are the ones that were used in our laboratory and can be seen as suggestions.

Sampling

  • Sites investigated:

    1. Pond at the Environmental Studies Area on campus at California State University Bakersfield (CSUB)

    2. Pond at Hart Memorial Park, northeast of Bakersfield

    3. Lake Wollomes, near Delano, CA

    4. Tom Sawyer Lake in Tehachapi, CA (foothills of the Sierra Nevada)

    5. Pond and creek at Wind Wolves Preserve near Maricopa, CA (foothills of the Sierra Nevada)

  • Plankton net (150 µm mesh size; e.g., Bigelow Laboratories)

  • Critter keepers for water samples (e.g., critter totes, Petsmart)

  • Sterile plastic containers for sediment and water samples (e.g., 50-mL Falcon Tubes, Fisher Scientific)

Microscopy & Photography

  • Microscope slides and 1-mL plastic pipette tips (e.g., Fisher Scientific)

  • Bright-field light microscope (for max. 400× magnification; e.g., Olympus CH3ORF-100)

  • Dissecting scope with camera system and software (e.g., Leica EZ 4D)

  • Camera with ocular that can be attached to a microscope or dissecting scope (e.g., Nikon Coolpix 950)

Suggestions for Literature to Identify Microcrustaceans

  • Needham, J.G. & Needham, P.R. (1962). A Guide to the Study of Fresh-Water Biology. San Francisco, CA: McGraw Hill.

  • Hebert, P.D.N. (1995). The Daphnia of North America – An Illustrated Fauna, version 1. [CD-ROM.] Guelph, Ontario, Canada: University of Guelph.

  • http://www.cnas.missouristate.edu/zooplankton (references within)

Tools for Molecular Analysis

  • Centrifuge (e.g., Accu Spin Micro 17, Fisher Scientific)

  • Pipettes and sterile pipette tips for p200 and p10 micropipettes (e.g., from Fisher Scientific)

  • DNA extraction kits (e.g., Microbial DNA extraction Kit and Powersoil DNA extraction kit from MoBio Laboratories)

  • 1.5-mL Eppendorf tubes to store extracted DNA and 0.6-mL PCR tubes (e.g., Fisher Scientific)

  • PCR primers for micro-eukaryotes and Bd (see van Hannen et al., 1998; Annis et al., 2000; can be ordered online from, e.g., IDT DNA or Invitrogen)

  • PCR Mastermix (e.g., GoTaq Green Mastermix, Promega Laboratories)

  • PCR marker (e.g., 100-bp low-scale DNA ladder, Fisher Scientific)

  • Thermocycler (e.g., PTC200, MJ Research)

  • Agarose and gel electrophoresis tank and material (e.g., from Thermo Scientific)

  • Tris borate EDTA (TBE) and Tris acetate EDTA (TAE) running buffer (can be made or purchased from, e.g., Fisher Scientific)

  • Denaturing gradient gel electrophoresis (DGGE) system (e.g., CBS Scientific or BioRad Laboratories)

  • Formamide, urea, and polyacrylamide for making DGGE gels (e.g., BioRad Laboratories and Fisher Scientific)

  • Exo-sap-it to purify PCR products (Affymetrix Laboratories)

  • UV-gel box to visualize DNA fragments in AGE and DGGE gels (e.g., Fisher Scientific)

Schools are usually eligible for special education discounts by the vendors suggested above. High school teachers might also be able to borrow more expensive equipment, such as a thermocycler or UV-gelbox from a neighboring community college or university, if they want to perform this exercise on their own. Used laboratory equipment for reasonable prices is also available online from several providers (e.g., http://www.labx.com/, http://www.labequip.com, http://www.labrecyclers.com/, and http://www.sciquip.com, to mention only a few).

Experimental Details

To compare the microcrustacean diversity in different freshwater environments, five ponds were chosen and plankton samples were taken with a plankton net (500 mL of fresh water, and 25 g of sediment collected from each site). The freshwater samples were transferred into critter keeper boxes (15 × 8 × 11 cm) and transported to the lab. Once in the lab, the microcrustaceans that were visible to the naked eye were transferred with 1-mL plastic pipettes onto microscope slides or Petri dishes to be visualized with the help of a light microscope or dissecting scope and documented. Published literature was used to identify the microcrustacean species. We centrifuged 100 mL of the water samples (10,000 rpm for 10 minutes) to pellet all microorganisms present. DNA was extracted from water samples using the Microbial DNA Isolation Kit and from the sediment samples using the Powersoil DNA extraction kit, following the protocol provided by the manufacturer. An aliquot of the extracted DNA was visualized on a UV-table after agarose gel electrophoresis (AGE) using 2% agarose and 1X Tris Borate EDTA (TBE) buffer (10X TBE Electrophoresis Buffer: 108 g Tris Base, 55 g Boric Acid, 20 mL 0.5M EDTA [pH 7]), distilled water to 1000 mL), followed by ethidium bromide staining (0.001%) of the gel in 1XTBE buffer for 20 minutes. Once the successful extraction of DNA was confirmed, two PCRs were performed: the first using a primer pair specific to Bd (Annis et al., 2000), and a second one using a primer pair specific for eukaryotes (van Hannen et al., 1998). AGE of the amplificates followed by ethidium bromide staining was used to verify a successful amplification of the desired fragments. A PCR marker was used in the AGE to confirm the correct fragment size of the amplicons. The 18S rDNA PCR amplicons were then separated by DGGE choosing a gradient between 30% and 55% (urea and formamide) and 9% polyacrylamide and running conditions of 80 V for 18 h in 1XTris Acetate EDTA buffer (TAE) (10X TAE: 48.4 g Tris, 11.4 mL Acetic Acid, 3.7 g EDTA, distilled water to 1000 mL) at 60°C (for more details about the procedure, see Yan et al., 2007; Wu et al., 2009). After staining, the DGGE gels in 1XTAE buffer containing ethidium bromide (0.001%), the gels were documented on a UV screen with attached camera (Gel Doc system, BioRad Laboratories). Distinct bands were excised with a sterile scalpel and transferred into 0.6-mL PCR tubes that contained 50 µL of sterile water. The excised bands were stored in a refrigerator overnight to allow the DNA to migrate out of the polyacrylamide gel before reamplification of the DNA with the same primer pair and procedure used before. The resulting amplicons were purified using exo-sap-it to degrade remaining primers and enzymes and were sent for sequencing to a sequencing facility. Once the DNA sequences were analyzed, they were entered in a software program that algorithmically aligned the sequence to all sequences that have been deposited in the nucleotide database of GenBank (http://www.ncbi.nlm.nih.gov). Such an alignment generates a sequence-similarity hierarchy, in which the most closely conserved sequence is listed first, thereby revealing the possible identity of the unknown micro-eukaryote that was represented by a DGGE band. The identifications of micro-eukaryotes from DGGE bands were compared to the results obtained by microscopy. By combining microscopy with molecular tools, the students were able to also determine which microcrustaceans were dominant and which ones were less abundant in the water samples. See Figure 1 for an overview of techniques used in this project.

Figure 1.

Experimental flowchart. After students sampled freshwater and sediment samples and documented the amphibian species present, the samples were further investigated using the techniques described earlier.

Figure 1.

Experimental flowchart. After students sampled freshwater and sediment samples and documented the amphibian species present, the samples were further investigated using the techniques described earlier.

Student Instruction

To get our students excited about using molecular biology and microscopy, we developed a student-centered approach that involved them in all stages of the exercise. At the beginning of any research project there is a general inquiry or question(s) that can be formed, followed by investigations based on the scientific method. Students were encouraged to state hypotheses and hypothesis-based predictions that could be addressed, such as the following:

  1. 1. The amphibian population decline around Bakersfield is linked to chytridiomycosis.

  2. If this hypothesis is true, we predict that we will find Bd in the water samples.

  3. 2. Microcrustacean diversity declines in ponds that have fish present.

  4. If this hypothesis is true, we predict that we will find mostly members of fast-swimming Cyclopidae and not large, slow-swimming cladocerans in the water samples.

  5. 3. Micro-eukaryote diversity in the water samples will be different from micro-eukaryote diversity in sediment samples.

  6. If this hypothesis is true, we predict that we will obtain different DGGE banding patterns for water/sediment samples.

  7. 4. Micro-eukaryote diversity from ponds in the SJV will differ from those of higher elevations.

  8. If this hypothesis is true, we predict that we will obtain different DGGE banding patterns for water/sediment samples as well.

Along with providing hands-on experience with various techniques, we also promoted critical-thinking skills. We discussed possible pitfalls of the molecular methods and talked about seasonal variation of microcrustacean diversity. We discussed amphibian decline and the potential that microcrustaceans might have for reducing chytridiomycosis outbreaks in amphibians. We also talked about microcrustacean decline due to introduced shrimp and fish predators. Last but not least, the students were guided to make a poster and were instructed about how to present it to a large student/teacher audience.

Student Assessment

Students were assessed in two ways. The first was a conceptual assessment in the form of a discussion that included technical understanding of microscopy and molecular methods used. The second was a practical assessment of the experimental results: Were the students able to perform the techniques (e.g., prepare a gel for AGE and DGGE, make a buffer, load samples correctly in a gel)? Were they successful in identifying microcrustacean groups based on microscopy and literature? Did they successfully extract DNA and generated PCR products (for Bd and micro-eukaryotes)? Could they interpret DGGE banding patterns? If not, they were expected to provide potential explanation(s) for the failure to obtain a particular result, describing their reasoning in the discussion. A lab report could be used to assess the students’ knowledge and to improve their writing skills, but this was not part of our exercise, which culminated with the making of a poster for presentation to a student/teacher audience. Figures 2, 3, 4 and 5 display results obtained with molecular tools and microscopy.

Figure 2.

Agarose gel electrophoresis (2%) showing successful PCR results obtained with primers to amplify a fragment of the 18S rRNA gene (∼250 bp) from micro-eukaryotes (water and sediment samples).

Figure 2.

Agarose gel electrophoresis (2%) showing successful PCR results obtained with primers to amplify a fragment of the 18S rRNA gene (∼250 bp) from micro-eukaryotes (water and sediment samples).

Figure 3.

Agarose gel electrophoresis (2%) showing successful PCR results (∼350 bp) obtained with a primer pair specific to Bd (water and sediment samples).

Figure 3.

Agarose gel electrophoresis (2%) showing successful PCR results (∼350 bp) obtained with a primer pair specific to Bd (water and sediment samples).

Figure 4.

Denaturing gradient gel electrophoresis of PCR products obtained with micro-eukaryote-specific primers for all (A) water samples and (B) sediment samples. The strong bands visible in the water samples originated from different species in the family Cyclopidae.

Figure 4.

Denaturing gradient gel electrophoresis of PCR products obtained with micro-eukaryote-specific primers for all (A) water samples and (B) sediment samples. The strong bands visible in the water samples originated from different species in the family Cyclopidae.

Figure 5.

Microscopy of dominant microcrustacean species observed in the water column (35×, Leica EZ4D). (A, B, E–H) Different cladoceran species in the family Daphnidae. (C) An ostracod species (seed shrimp). (D) A female copepod (Cyclopidae) carrying eggs.

Figure 5.

Microscopy of dominant microcrustacean species observed in the water column (35×, Leica EZ4D). (A, B, E–H) Different cladoceran species in the family Daphnidae. (C) An ostracod species (seed shrimp). (D) A female copepod (Cyclopidae) carrying eggs.

Teacher Implementation/Suggestions

The exercise as described here was the result of a collaboration between high school students, undergraduate biology students, and their teachers. It would ideally be performed as a summer project over a 4-week period. The equipment necessary for this exercise might not be available in all high school laboratories but is standard at most universities. The National Science Foundation (NSF) is offering special grants that support research in the sciences for high school students, through programs such as the Math and Science Partnership (MSP) or the Transforming Undergraduate Education in Science, Technology, Engineering and Mathematics (TUES) program. Details about these opportunities can be found at the NSF website under “Undergraduate Education (DUE)” (http://www.nsf.gov/div/index.jsp?org=DUE). Funding might also be provided through locally based businesses, such as Chevron, which supported this lab exercise at CSUB.

In summary, this lab exercise is unique in focusing on the detection of an amphibian pathogen (Bd) of global impact and focusing at the same time on the diversity of its potential predators. Although the project described in this article is very specific in regard to the organisms being studied, it would be possible to apply this process to other microbiology projects. It might be necessary to alter the focus of this project in order to attract a university research partner. Specific funding sources might also change the focus of a future project, as might geography and the availability of local organisms to study. We believe that our project contributed in a positive way to higher education. The student participants were motivated, self-confident, and knowledgeable concerning microbiology, and they are now better prepared for future scientific goals that they want to achieve.

Acknowledgments

We acknowledge Chevron for generous sponsoring of this lab exercise in the Research Experience Vitalizing Science – University Program (REVSUP) at CSUB. Furthermore, we want to thank all high school students and undergraduate biology students who participated in this summer program: Valerie Sanchez, Marilla Jeffers, Yesenia Calderon, Jackeline Muñeton, Francisco Mosqueda, Humberto Melgoza, Nanse Mendoza, and Greg Gonzales.

References

References
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Goldman, C.R., Morgan, M.D., Threlkeld, S.T. & Angeli, N. (1979). A population dynamics analysis of the cladoceran disappearance from Lake Tahoe, California–Nevada. Limnology and Oceanography, 24, 289–297.
Kriger, K.M. & Hero, J.-M. (2007). Large-scale seasonal variation in the prevalence and severity of chytridiomycosis. Journal of Zoology, 271, 352–359.
Limburg, P.A. & Weider, L.J. (2002). ‘Ancient’ DNA in the resting egg bank of a microcrustacean can serve as a palaeolimnological database. Proceedings of theRoyal Society of London, Series B, 269, 281–287.
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