Located in Barnard Hall next to the Strand Union Building, the Center for Biofilm Engineering comprises more than 20,000 square feet, and includes offices and conference rooms for faculty, staff, and students; a computer lab; and 13 fully equipped research laboratories. The full-time CBE Technical Operations Manager oversees the research laboratories, provides one-on-one training for students, ensures safe laboratory practices, and maintains equipment. State-of-the-art instruments and equipment are available for use by all CBE faculty, staff, and students. General use areas include an analytical instrument lab, a microbiology lab with media preparation area and autoclaves, and a general molecular area with two thermocyclers, a gel running and imaging station, as well as an isolated radioactive isotope lab. Facilities of particular note are described below.
The microscopy facilities are coordinated by the Microscopy Facilities Manager who maintains the equipment and trains and assists research staff and students in capturing images of in situ biofilms via optical microscopy and fluorescent confocal microscopy. The microscopy facilities include three separate laboratories—the Optical Microscopy Lab, the Confocal Microscopy Lab, and the Microscope Resource Room and Digital Imaging Lab—which are detailed below.
- The Optical Microscopy Lab houses two Nikon Eclipse E-800 research microscopes which are used for transmitted light and epi-fluorescent imaging. Both microscopes are equipped with cooled CCD cameras from Photometrics (we have a CoolSnapfx, and a CoolSnapEZ) and use Universal Imaging Corporation’s MetaVue software (v 7.4.6) for digital image acquisition. We have a large collection of fluorescence filter cubes for the Nikons, including those optimized for the following fluorescent stains: FITC (gfp), TRITC (propidium iodide), DAPI, CTC, ELF-97, CY5, cfp, and we also have a B2E cube. Both Nikons are equipped with Nomarski/DIC. Other equipment in the Optical Microscopy Lab includes a Nikon SMZ-1500 barrel zoom stereomicroscope equipped with a color camera, a Leica CM1800 cryostat, a Zeiss Palm Laser Capture Dissection microscope and a dry ice maker
The Confocal Microscopy Lab contains two brand-new (2011) Leica SP5 Confocal Scanning Laser Microscopes (CSLMs).
One is an inverted confocal microscope with 405, 488, 561 and 633 nm laser excitation lines. It is equipped with a tandem scanner, so it can be switched from standard scanning mode to operate in Resonant Scanner mode, which enables scanning at exceptionally high frequencies for fluorescent imaging. This faster scanning is necessary for most live cell imaging (note: “live cell imaging” doesn’t generally refer to imaging bacterial cells, but rather mammalian cells and processes). This inverted SP5 also includes a heated stage with an environmental control chamber (i.e. it can be used to provide an enclosed CO2 atmosphere), and a motorized stage with Mark-and-Find and image tiling capabilities.
The second, new SP5 is an upright confocal microscope, also with 405, 488, 561 and 633 nm lasers, a motorized stage, Mark-and-Find, and tiling capabilities. This upright has a removable heated chamber that encloses the entire microscope, so that larger, incubated flow cell systems can be accommodated over long periods of time. This enables high-resolution time-lapse monitoring of biofilm development, treatment and detachment phenomena. Additionally, this microscope is equipped with Fluorescence Lifetime Imaging (FLIM) capability, which is also referred to as Single Molecule Detection.
The CSLM is capable of imaging biofilms on opaque surfaces, so a wide variety of materials can be used in the experimental flow cells. As biofilm formation proceeds in an experiment, representative areas of the colonized surface are scanned with the use of the automatic stage. Digital data is collected from sequential scans, and stored data can be viewed in the x, y, z coordinates to yield a 3-dimensional image of the biofilm architecture. Quantitative and qualitative information about biofilm architecture can be retrieved easily from examination of CSLM data, in both the x-y and x-z planes, and the existence or absence of structural features, such as microcolonies and water channels, can be determined.
- The Microscope Resource Room / Digital Imaging Lab is where CBE researchers examine and reconstruct the stacks of collected image data using our image analysis software. For quantitative analysis, such as intensity or particle-size measurements, we use Universal Imaging Corporation’s MetaMorph software. We use Bitplane’s Imaris software for qualitative analysis—for example, putting together a stack of 200 red and green flat images, to get a 3-dimensional image of a biofilm microcolony that can be rotated in space and examined from every angle. The lab consists of three dedicated computers, SCSI drives for storing large files, CD and DVD burners and readers, and a color printer. In addition to providing CBE students, staff, and researchers with an imaging workplace, the resource room gives us a place to hold group tutorials and WebEx group software training sessions.
Mass Spectrometry Facility
In 2005 an equipment grant was awarded for an Environmental and Biofilm Mass Spectrometry Facility through the Department of Defense University Research Instrumentation Program (DURIP). The grant funded the acquisition of an Agilent 1100 series high performance liquid chromatography system with autosampler and fraction collector, an Agilent SL ion trap mass spectrometer, and an Agilent 6890 gas chromatograph with electron capture detector, flame ionization detector, and 5973 inert mass spectrometer. Since then, an Agilent 7500ce inductively coupled plasma mass spectrometer with autosampler, liquid, and gas chromatographic capabilities has also been added. Mass spectrometers are very well suited for unknown compound identification and high sensitivity speciation measurements of organic and inorganic compounds; this equipment enhances the CBE’s research capabilities significantly. The Environmental and Biofilm Mass Spectrometry Facility is operated as a user facility and allows access for academic and non-academic researchers.
Specialized CBE Laboratories
The Ecology/Physiology Laboratory lead by Dr. Matthew Fields has general microbiology equipment, anaerobic gassing stations, Shimadzu UV-VIS spectrophotometer, biofilm reactors, protein and DNA electrophoresis, Qbit fluorometer, 2 PCR machines (96-well), incubators, laminar/fume hoods, microcentrifuges, table-top centrifuges, and a microcapillary gas chromatograph with dual TCDs. The lab also has a light-cycle controlled photo-incubator as well as photo-bioreactors for the cultivation of algae and diatoms, and the lab maintains one -20C freezer and a -70C freezer for sample storage.
This laboratory also houses a 454 GS-Jr. pyrosequencer. The GS-Jr. uses 454 technology at an intermediate scale (100,000 reads with up to 40-50 Mb of sequence). The GS Jr. offered by Roche is a high-throughput, multi-parallel sequencing instrument that is capable of delivering the information of approximately 100,000 different DNA molecules of approximate 450 nucleotides long within 48 hours. We have been using the Gs-Jr. for approximately 1 year, and have developed an in-house Python script that deals with filtering raw sequences based upon qulaity scores (Bowen DeLeon et al., in revision). We have also modified the protocols for the GS-Jr. in consultation with Roche to improve quality scores (Ramsay et al., manuscript in preparation).
Medical Biofilm Laboratory
The Medical Biofilm Laboratory (MBL) has earned a reputation for being a university lab that responds quickly to real world needs in the area of health care as it relates to biofilms. Dr. Garth James (PhD, microbiology), Randy Hiebert (MS, chemical engineering), and Dr. Elinor Pulcini (PhD, microbiology) have been the innovative leaders and managers of this respected, flexible, and adaptable lab group. The MBL team also includes three full- time research scientists, two technicians, one graduate student, and two undergraduate research assistants.
Currently, fifteen companies, including CBE Industrial Associates, sponsor MBL projects. The MBL is also collaborating with small businesses on two Phase I Small Business Innovation Research (SBIR) grants. In addition, MBL principal investigator Kelly Kirker recently received an R03 research grant from the National Institutes of Health (NIH) to investigate staphylococcal biofilm induction of apoptosis in human epithelial cells. Other MBL projects include evaluating treatments for oral biofilms, testing needle-free connectors, catheters, and other medical devices, as well as evaluating novel treatments for medically-related biofilms. The MBL is a prime example of integration at the CBE, bringing together applied biomedical science, industrial interaction, and student educational opportunities.
Standardized Biofilm Methods Laboratory
The Standardized Biofilm Methods Laboratory (SBML) was designed to meet research and industry needs for standard analytical methods to evaluate innovative biofilm control technologies. SBML staff and students develop, refine, and publish quantitative methods for growing, treating, sampling, and analyzing biofilm bacteria. The SBML members work with international standard setting organizations on the approval of biofilm methods by the standard setting community. Under a contract with the U.S. Environmental Protection Agency (EPA), the SBML conducts laboratory research to support the development and standardization of test methods for measuring the performance of antimicrobial products—including those for biofilm bacteria—and provide statistical services related to EPA's Office of Pesticide Programs Antimicrobial Testing Program. In addition, they conduct applied and fundamental research experiments and develop testing protocols. Methods include: design of reactor systems to simulate industrial/medical systems; growing biofilm and quantifying cell numbers and activity; testing the efficacy of chemical constituents against biofilms; and microscopy and image analysis of biofilms. SBML staff offer customized biofilm methods training workshops for CBE students, collaborators, and industry clients.
Microbial Ecology and Biogeochemistry Laboratory
Research in the Microbial Ecology and Biogeochemistry Laboratory lies at the intersection of microbial and ecosystem ecology and uses a combination of field and laboratory studies, as well as approaches ranging from the single-cell to the community level. Staff in this lab are interested in understanding how the environment controls the composition of microbial communities and how, in turn, those microbes regulate whole ecosystem processes such as nutrient and organic matter cycling. Ongoing research examines carbon flux through microbial communities, with the long-term goal of improving predictions of carbon fate (metabolism to CO2, sequestration into biomass, long-term storage in ice) in the context of a changing environment. Additionally, they are interested in physiological adaptations to life in icy environments. Regardless of the environment, the group employs microbiological, limnological, biochemical and molecular biology approaches to investigate fundamental processes carried out by microbes.
OTHER Montana State University facilities available for collaborative research
MSU Nuclear Magnetic Resonance (NMR) Facility
A state-of-the-art NMR facility is available on campus on a recharge basis for research projects. This facility is a 5-minute walk from the College of Engineering and CBE laboratories. All the instruments in the facility are Bruker Avance instruments. The facility houses 300, 500 and 600 MHz NMR instruments for high resolution spectroscopy analysis.
MSU Magnetic Resonance Microscopy (MRM) Facility
A state-of-the-art MRM facility is available on a recharge basis for research projects. This facility is located in the College of Engineering in the same building as the Center for Biofilm Engineering. Both instruments in the facility are Bruker Avance instruments. The facility houses 250 MHz standard/wide bore and a 300 MHz wide/super-wide bore instruments for imaging and fluid dynamics applications. The imaging systems are capable of generating NMR image and transport data with spatial resolution on the order of 10 μm in a sample space up to 6 cm diameter.
MSU ICAL Laboratory
The Image and Chemical Analysis Laboratory (ICAL) in the Physics Department at Montana State University is located on the 3rd floor of the EPS Building, adjacent to the Center for Biofilm Engineering. ICAL is a user oriented facility that supports basic and applied research and education in all science and engineering disciplines at MSU. The laboratory provides access to state of the art equipment, professional expertise, and individual training to government and academic institutions and the private sector. Laboratory instrumentation is dedicated to the characterization of materials through high resolution imaging and spectroscopy. ICAL promotes interdisciplinary collaboration between the research, educational and industrial fields.education, and industry, and to strengthen existing cooperation between the physical, biological, and engineering sciences by providing critically needed analytical facilities. These facilities are open to academic researchers.
A new critical point dryer―jointly purchased in 2007 by the CBE and the Image & Chemical Analysis Laboratory―has been set up in the ICAL lab for the processing of biological samples for electron microscopy. This equipment allows our researchers to remove water from soft samples without distorting the sample.
The ICAL currently contains eleven complementary microanalytical systems:
- Atomic Force Microscope (AFM)
- Field Emission Scanning Electron Microscope (FE SEM)
- Scanning Electron Microscope (SEM)
- Small-Spot X-ray Photoelectron Spectrometer (XPS)
- Time-of-Flight Secondary Ion Mass Spectrometer (ToF-SIMS)
- X-Ray Powder Diffraction Spectrometer (XRD)
- Scanning Auger Electron Microprobe (AUGER)
- Epifluorescence Optical Microscope
- Microplotting System
- Critical Point Drying
- Video Contact Angle System
For more information on each system, see the ICAL web site at: http://www.physics.montana.edu/ical/
CBE Computer Facilities
CBE staff and students have access to workstations connected to the MSU College of Engineering computer network. A student computer laboratory offers ten state-of-the-art PCs along with scanning and printing services. In addition, the COE maintains computational PCs and a computational cluster for data manipulation, mathematical modeling, and graphic image analysis.