Center for Biofilm Engineering

Facilities Overview
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. 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, and spectrophotometers for nucleic acid quantification, as well as an isolated radioactive isotope lab. See below for a comprehensive list of shared equipment available.

MICROSCOPE FACILITIES
The microscopy and chemical imaging 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, fluorescent and Raman confocal microscopy. The microscopy facilities include four separate laboratories—the Optical Microscopy Lab, the Confocal Microscopy Lab, the Chemical Imaging 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 Photometrics MYO cooled CCD cameras 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, and we have a 100x oil phase contrast objective and condenser especially for use with imaging spores.

Our microscope collection has expanded with the acquisition of a new Leica LMD6 Laser Microdissection System equipped with a color camera, fluorescence filter cubes (FITC, TRITC, DAPI), and a UV laser for sample dissection. Another recent addition is the GAN210 Optical Coherence Tomography (OCT) imaging system. OCT is a high resolution, non-contact, non-invasive, and non-fluorescent based technique that is well suited for imaging thick specimens. The OCT light source centered around 930 nm with a bandwidth >100 nm and has a scan rate of up to 36 kHz with an axial field of view of 2.9 mm / 2.2 mm. Depending on the scan objective the field of view (FOV) and resolution can be adjusted and vary between a larger FOV of 16x16 mm² at 12 µm resolution, and a FOV of 10x10 mm² with a higher resolution of 8 µm.

Additionally, within the Optical Microscopy Lab is a Leica M 205 FA computer-controlled stereomicroscope and a Leica DFC3000G fluorescence camera. This stereoscope can be used to image samples using fluorescence, brightfield with or without polarization or Rotterman contrast, and reflected white light. The software will also allow a z-stack of images to be collected and recombined using simple deconvolution. Other equipment in the Optical Microscopy Lab includes a Nikon SMZ-1500 barrel zoom stereomicroscope equipped with a color camera, a Leica cryostat, and a dry ice maker.

The Confocal Microscopy Lab contains two 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 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 Chemical Imaging Lab contains a Horiba Confocal Raman Microscope. Raman is a vibrational spectroscopic method that provides a fingerprint of the molecular, and to some extent the isotopic composition of a sample. The Horiba LabRam HR Evolution NIR is dedicated to studying the molecular composition of a sample. This is a fully integrated high resolution Raman microscope for confocal Raman analysis, optimized for the visible to IR range (400nm-2500nm) microscope. It includes a confocal Raman microscope with an automated xyz-stage with fast-mapping capabilities, transfer optics, stigmatic spectrometer equipped with two gratings (600 and 1800 l/mm gratings), multichannel air-cooled CCD detector, and computer package with the latest version of the LabSpec6 software and the KnowItAll Raman spectra library, Horiba edition. It is equipped with 532nm 100mW laser, HeNe 633nm laser, 785nm 90mW laser, and 10x, 50x, 100x, 20xLWD and 50xLWD objectives.

The Microscope Resource Room / Digital Imaging Lab is where CBE researchers examine and reconstruct the stacks of image data they have collected 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 computer-intensive data analysis like particle tracking and 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, a server 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 (GC) 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 have been added as well as an additional Agilent 1100 series high performance liquid chromatography system with autosampler and an Agilent 6890 GC with autosampler and flame ionization detector. The chromatographs and 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

Ecology/Physiology Laboratory
The Ecology/Physiology Laboratory headed by Dr. Matthew Fields has general microbiology equipment, anaerobic gassing stations in two lab spaces, Shimadzu UV-VIS spectrophotometer, Ultra-Centrifuge, Anaerobic Chamber, biofilm reactors, protein and DNA electrophoresis, Qubit fluorometer, two Eppendorf Mastercylcers, incubators, laminar/fume hoods, microcentrifuges, table-top centrifuges, and a microcapillary gas chromatograph with dual TCDs. The lab has two light-cycle controlled photo-incubators as well as photo-bioreactors for the cultivation of algae and diatoms, and maintains two -20°C freezers and three -70°C freezers for sample storage. Additionally, the lab has a large capacity refrigerated incubator (5-70°C) for temperature critical studies.

This laboratory houses an Illumina MiSeq Sequencing System. The MiSeq desktop sequencer allows the user to access more focused applications such as targeted gene sequencing, metagenomics, small genome sequencing, targeted gene expression, amplicon sequencing, and HLA typing. This system enables up to 15 Gb of output with 25 M sequencing reads and 2x300 bp read lengths by utilizing Sequencing by Synthesis (SBS) Technology. A fluorescently labeled reversible terminator is imaged as each dNTP is added, and then cleaved to allow incorporation of the next base. Since all four reversible terminator-bound dNTPs are present during each sequencing cycle, natural competition minimizes incorporation bias. The end result is true base-by-base sequencing that enables the industry's most accurate data for a broad range of applications. The method virtually eliminates errors and missed calls associated with strings of repeated nucleotides (homopolymers).

Medical Biofilm Laboratory
The Medical Biofilm Laboratory (MBL) has earned a reputation for being a university lab that focuses on industrially relevant medical research 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 innovativeleaders and managers of this respected, flexible, and adaptable lab group. The MBL team also includes a full-time research professor, three technicians, and one undergraduate research assistant.

Currently, twelve companies, including CBE Industrial Associates, sponsor MBL projects. These projects include evaluating antimicrobial wound dressings, biofilm formation on biomedical polymers, testing novel toothpaste ingredients, and testing biofilm prevention and removal agents. The MBL is also researching the role of biofilms in Lyme disease with funding from a private foundation. 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, validate, and publish quantitative methods for growing, treating, sampling, and analyzing biofilm bacteria. The SBML members work with international standard setting organizations ( ASTM International, IBRG, and OECD) on the approval of biofilm methods by the standard setting community. Under a contract with the U.S. Environmental Protection Agency (EPA), the SBML provides statistical services relevant to the EPA's Office of Pesticide Programs Microbiology Laboratory Branch to assess the performance of antimicrobial test methods—including those for biofilm bacteria. The SBML received funding from the Burroughs Wellcome Foundation to develop a method for assessing the prevention of biofilm on surface modified urinary catheters. In addition, they conduct applied and fundamental research experiments and develop testing protocols for product specific applications. Methods include: design of reactor systems to simulate industrial/medical systems; growing biofilm and quantifying microbial abundances 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 (www.foremanresearchgroup.com) 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 extreme environments, as extremophiles are natural resources for the discovery of pigments, biosurfactants, novel enzymes and other bioactive compounds of industrial relevance.

Microfluidics Laboratory
Dr. Connie Chang runs a soft materials and microfluidics laboratory to study microbes (bacteria, biofilms, and viruses). Dr. Chang is applying drop-based microfluidics—the creation and manipulation of picoliter-sized drops of fluid—for high-throughput screening and assaying in biology. Her lab is developing novel tools for quantifying the behavior of individuals and how they can collectively contribute to large-scale population dynamics. Ongoing projects within her group include the screening of persister and dormant bacteria cells in biofilms and the study of influenza evolution and population dynamics.

Dr. Chang has shared laboratory space in the CBE and an individual laboratory space in the Chemistry and Biochemistry Building (CBB) at MSU. The laboratory spaces include common space for equipment, chemical storage, freezers and reagents. The lab is outfitted with a qPCR machine and also includes a dedicated a room for epifluorescence microscopy and a custom built microscope stand (200 square feet). The lab contains all the equipment and instrumentation necessary for fabrication of new devices, microfluidics handling, PCR, and cell culture.

Microsensor Laboratory
The Microsensor Laboratory provides the capability of measuring microscale chemical and physical parameters within biofilms, microbial mats and other compatible environments. The Microsensor Laboratory has the capability to measure spatial concentration profiles using sensors for oxygen, pH, hydrogen sulfide, nitrous oxide and some custom-made electrodes. All electrodes are used in conjunction with computer-controlled micromanipulators for depth profiling. A Leica stereoscope is used to visualize the sensors while positioning them on the biofilm surface. The laboratory has experience with diverse microsensor applications including biofilms in wastewater, catheters and hollow fiber membrane systems in addition to algal and fungal biofilms.

OTHER MONTANA STATE UNIVERSITY FACILITIES AVAILABLE FOR COLLABORATIVE RESEARCH
Montana Nanotechnology (MONT) Facility
The MONT facility was formed from a $3 million NSF grant awarded to MSU in September of 2015. This collaborative facility includes the Montana Microfabrication Facility (MMF), the Imaging and Chemical Analysis Lab (ICAL), the CBE, the MSU Mass Spectrometry facility, and the Center for Bio-Inspired Nanomaterials. MONT provides researchers from academia, government and companies large and small with access to university facilities with leading-edge fabrication and characterization tools, instrumentation and expertise within all disciplines of nanoscale science, engineering and technology.

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
The CBE maintains several dedicated computational and data storage computer systems including 10 high performance data and image analysis workstations and servers in addition to three large storage servers. The CBE maintains a small to mid-scale computational cluster for modeling and analysis. The center provides personal workstations for staff and graduate students that are connected to the MSU computer network. A student computer laboratory offers nine state-of-the-art PCs along with scanning and printing services.

Additionally, CBE staff and students have access to the centrally maintained computational cluster for data manipulation, analysis, and mathematical modeling. This cluster consists of 77 nodes with a total of 1300 hyper-threaded cores and 22 teraflops of computing power.