A novel sample preparation method is demonstrated for the analysis of agar-based, bacterial macrocolonies via matrix-assisted laser desorption/ionization imaging mass spectrometry.
Understanding the metabolic consequences of microbial interactions that occur during infection presents a unique challenge to the field of biomedical imaging. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry represents a label-free, in situ imaging modality capable of generating spatial maps for a wide variety of metabolites. While thinly sectioned tissue samples are now routinely analyzed via this technology, imaging mass spectrometry analyses of non-traditional substrates, such as bacterial colonies commonly grown on agar in microbiology research, remain challenging due to the high water content and uneven topography of these samples. This paper demonstrates a sample preparation workflow to allow for imaging mass spectrometry analyses of these sample types. This process is exemplified using bacterial co-culture macrocolonies of two gastrointestinal pathogens: Clostridioides difficile and Enterococcus faecalis. Studying microbial interactions in this well-defined agar environment is also shown to complement tissue studies aimed at understanding microbial metabolic cooperation between these two pathogenic organisms in mouse models of infection. Imaging mass spectrometry analyses of the amino acid metabolites arginine and ornithine are presented as representative data. This method is broadly applicable to other analytes, microbial pathogens or diseases, and tissue types where a spatial measure of cellular or tissue biochemistry is desired.
The human microbiome is a highly dynamic ecosystem involving molecular interactions of bacteria, viruses, archaea, and other microbial eukaryotes. While microbial relationships have been intensely studied in recent years, much remains to be understood about microbial processes at the chemical level1,2. This is in part due to the unavailability of tools capable of accurately measuring complex microbial environments. Advances in the field of imaging mass spectrometry (IMS) over the past decade have enabled in situ and label-free spatial mapping of many metabolites, lipids, and proteins in biological substrates3,4. Matrix-assisted laser desorption/ionization (MALDI) has emerged as the most common ionization technique used in imaging mass spectrometry, involving the use of a UV laser to ablate material from the surface of a thin tissue section for measurement by mass spectrometry4. This process is facilitated by the application of a chemical matrix applied homogeneously to the surface of the sample, allowing for sequential measurements to be made in a raster pattern across the sample surface. Heat maps of analyte ion intensities are then generated following data acquisition. Recent advances in ionization sources and sampling techniques have enabled the analysis of non-traditional substrates such as bacterial5 and mammalian6,7,8 cellular specimens grown on nutrient agar. The molecular spatial information afforded by IMS can provide unique insight into the biochemical communication of microbe-microbe and host-microbe interactions during infection9,10,11,12,13,14.
Upon Clostridioides difficile infection (CDI), C. difficile is exposed to a rapidly changing microbial environment in the gastrointestinal tract, where polymicrobial interactions are likely to impact infection outcomes15,16. Surprisingly, little is known about the molecular mechanisms of interactions between C. difficile and resident microbiota during infection. For example, enterococci are a class of opportunistic commensal pathogens in the gut-microbiome and have been associated with increased susceptibility to and severity of CDI17,18,19,20. However, little is known about the molecular mechanisms of the interactions between these pathogens. To visualize small-molecule communication between these members of the gut microbiome, bacterial macrocolonies were grown herein on agar to simulate microbe-microbe interactions and bacterial biofilm formation in a controlled environment. However, obtaining representative metabolic distributions upon MALDI imaging mass spectrometry analysis of bacterial culture specimens is challenging due to the high water content and uneven surface topography of these samples. This is largely caused by the highly hydrophilic nature of agar and the non-uniform agar surface response during moisture removal.
The high water content of agar can also make it challenging to achieve homogeneous MALDI matrix coating and can interfere with subsequent MALDI analysis performed in vacuo21. For example, many MALDI sources operate at pressures of 0.1-10 Torr, which is a sufficient vacuum to remove moisture from the agar and can cause deformation of the sample. These morphological changes in the agar induced by the vacuum environment cause bubbling and cracking in the dried agar material. These artifacts reduce the adherence of the agar to the slide and can cause dismounting or flaking of the sample into the instrument vacuum system. The thickness of the agar samples can be up to 5 mm off the slide, which can create insufficient clearance from ion optics inside the instrument, causing contamination and/or damage to instrument ion optics. These cumulative effects can result in reductions of ion signal reflective of the surface topography, rather than the underlying microbial biochemical interactions. Agar samples must be homogeneously dried and strongly adhered to a microscope slide prior to analysis in vacuo.
This paper demonstrates a sample preparation workflow for the controlled drying of bacterial culture macrocolonies grown on agar media. This multi-step, slower drying process (relative to those previously reported) ensures that the agar will dehydrate uniformly while minimizing the effects of bubbling or cracking of agar samples mounted on microscope slides. By using this gradual drying method, samples are strongly adhered to the microscope slide and amenable for subsequent matrix application and MALDI analysis. This is exemplified using model bacterial colonies of C. difficile grown on agar and murine tissue models harboring CDI with and without the presence of commensal and opportunistic pathogen, Enterococcus faecalis. MALDI imaging mass spectrometry analyses of both bacterial and tissue models allow for the spatial mapping of amino acid metabolite profiles, providing novel insight into bioenergetic microbial metabolism and communication.
NOTE: Animal experiments were approved by the Animal Care and Use Committees of the Children's Hospital of Philadelphia and the University of Pennsylvania Perelman School of Medicine (protocols IAC 18-001316 and 806279).
CAUTION: Clostridium difficile (C. difficile) and Enterococcus faecalis (E. faecalis) are BSLII pathogens and should be handled with extreme caution. Utilize proper decontamination protocols when necessary.
1. Growth of bacterial culture macrocolonies and preparation for overnight shipment
2. Ambient drying of C. difficile + E. faecalis bacterial macrocolonies
3. Vacuum and heat-mediated drying of C. difficile + E. faecalis bacterial macrocolonies
NOTE: A custom-built vacuum drying apparatus (Figure 1) was built to facilitate the removal of excess moisture from the agar samples. This apparatus utilizes a rotary vane vacuum pump connected in line to a HEPA biofilter, a cold trap, and a stainless-steel chamber, where the bacterial samples are placed. A variable voltage transformer is connected to an insulated wire filament, which allows the user to heat the chamber to expedite the drying process.
4. MALDI matrix application via robotic spraying
NOTE: After the agarose samples have been thoroughly dried and the height of the culture sections have noticeably decreased, use a robotic matrix sprayer to homogeneously apply a thin coating of a chemical MALDI matrix compound. This procedure should be performed in a chemical fume hood and with proper personal protective equipment, including gloves, laboratory glasses, and a lab coat.
5. Preparing bacterial macrocolonies for MALDI imaging mass spectrometry data acquisition
NOTE: All imaging mass spectrometry analyses were performed using a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. This instrument is equipped with a Nd:YAG MALDI laser system (2 kHz, 355 nm) .
6. Imaging mass spectrometry data analysis and compound identification
7. Preparation and shipment of noninfected control and C. difficile -infected mouse cecal tissues
8. Cryosectioning of noninfected control verus C. difficile -infected mouse cecal tissues
9. Preparation of noninfected control versus C. difficile -infected mouse cecal tissues for matrix application and MALDI imaging mass spectrometry
We have performed metabolite MALDI imaging mass spectrometry of model bacterial colonies and mice co-colonized with E. faecalis and C. difficile to study the role of amino acids in microbe-microbe interactions. Bacterial macrocolonies grown on agar serve as a well-defined model to analyze distinct biochemical changes in bacterial biofilm formation. It is important to ensure a controlled drying process for bacterial culture macrocolonies grown on agar media to minimize deformations and cracking in the agar surface. This was achieved via a two-step process, involving preliminary drying with desiccant under ambient temperature and pressure, followed by a more rapid vacuum-assisted drying step performed at an elevated temperature. A custom-built drying apparatus was used to facilitate agar dehydration while capturing the release of any bacterial contaminants or spores from the samples (Figure 1). The additional vacuum drying step is necessary to ensure that samples are completely dehydrated and will not undergo deformation upon exposure to the MS vacuum.
Agar sections were initially 2-4 mm in sample height (Figure 2A) and were reduced to ~0.5 mm in height following the drying protocol (Figure 2B). It is important to ensure uniform drying across the sample surface during this sample preparation protocol. Large variations in sample height will result in a defocused MALDI laser beam at the sample surface, which can detrimentally affect ionization efficiency and resulting analyte ion intensity. An example of an unsuccessful drying process is shown in Figure 2D, where the agar surface has bubbled and cracked, rendering the sample unusable for further analysis. Once the bacterial sample has been thoroughly dried, a DAN MALDI matrix layer is applied using a robotic sprayer (Figure 3). This instrument allows for the precise control of spraying parameters, enabling application of a uniform matrix coating (Figure 2C).
Following matrix application, the microscope slides containing the samples are inserted into a slide adapter for analysis on an FTICR MS (Figure 4). Coordinates on the slide are registered between the imaging mass spectrometry acquisition software and the instrument camera by first drawing fiducial marks on the slide around the regions to be measured (denoted by "+" symbols in Figure 4). The slide is then digitally scanned using a flatbed scanner to record an optical image, which is then used to define the acquisition sequence in the flexImaging software. We optimized the mass spectrometry instrument settings for the transmission and detection of metabolite anions. Using CASI gas-phase ion enrichment, an ion isolation window was defined to selectively accumulate ions at m/z 150 ± 50 Da (Figure 5A), which contains an array of compounds observed from the tissue surface. Specifically here for C. difficile infection, we found amino acid pathways to demonstrate interesting and changing roles during bacterial catabolism. Based on accurate mass measurements, the ion signals were identified at m/z 131.083 and m/z 173.104 to be ornithine (0.34 ppm mass error; Figure 5B) and arginine (0.43 ppm mass error; Figure 5C), respectively.
Imaging mass spectrometry of bacterial macrocolonies was performed on C. difficile monocultures and on C. difficile + E. faecalis co-culture colonies (Figure 6). An optical image of the samples following MALDI matrix application highlights the acquisition regions of interest, which extend to the outer areas of colony biofilms (Figure 6A,C). Imaging mass spectrometry of these samples allows for the spatial mapping of both ornithine and arginine amino acid metabolites, which are found to be altered and differentially localized in the presence of E. faecalis (Figure 6B,D). For example, arginine is significantly more abundant in the C. difficile monoculture compared to the C. difficile + E. faecalis co-culture (Figure 6D). We have also extended the imaging mass spectrometry analyses to mouse models of infection using 8-week-old germ-free C57BL/6 female mice that were infected with either C. difficile spores, both C. difficile spores + E. faecalis cells (wt) (5 × 108/mL), or a transposon mutant containing both C. difficile spores + E. faecalis (ArcD mutant) cells (5 × 108/mL) (Figure 7)27. The mouse cecum was harvested and frozen in 25% OCT compound to maintain organ shape and fidelity. The OCT compound also helped support the thin tissue sections during cryosectioning. Similar to the protocol mentioned above, tissue sections are coated with a DAN MALDI matrix layer, scanned using a flatbed optical scanner (Figure 7B), and analyzed by MALDI imaging mass spectrometry (Figure 7C). Histological staining was performed on tissue sections following imaging mass spectrometry analysis, and high-resolution brightfield optical scanning was employed to evaluate tissue morphology (Figure 7A). The epithelial cell layer is clearly inflamed during infection compared to the control tissue.
Figure 1: Home-built vacuum drying apparatus. A custom-built vacuum drying apparatus is used to expedite moisture removal from agar samples. Samples are placed into the vacuum chamber, which is sealed and evacuated using a rotary vane pump, and then heated using a variable voltage transformer. Any contaminant vapors/spores from the sample are trapped in the cold trap and HEPA in-line filters. Pressures of 100-150 mTorr are typically employed. Please click here to view a larger version of this figure.
Figure 2: Drying and preparation of bacterial macrocolonies. Optical images of agar samples (A) prior to and (B) after moisture removal using desiccation and vacuum-assisted drying. (C) A 1,5-diaminonaphthalene MALDI matrix is applied to the bacterial co-culture following drying using a robotic sprayer. (D) Unsuccessful drying typically results in cracking and bubbling within the agar, rendering it unusable for further analysis. Abbreviations: DAN = 1,5-diaminonaphthalene; MALDI = matrix-assisted laser desorption/ionization. Please click here to view a larger version of this figure.
Figure 3: Robotic spraying of MALDI matrix on agarose samples. Images of (A) an HTX M5 TM sprayer and (B) agarose samples loaded into the robotic matrix sprayer for MALDI matrix application. Abbreviation: MALDI = matrix-assisted laser desorption/ionization. Please click here to view a larger version of this figure.
Figure 4: Mass spectrometer adapter with loaded samples for analysis. Matrix-coated agarose samples are loaded into an MTP slide adapter for MALDI analysis. Fiducial markers are visible as black plus signs ("+"). Abbreviation: MALDI = matrix-assisted laser desorption/ionization. Please click here to view a larger version of this figure.
Figure 5: Representative mass spectra of identified metabolite ion peaks. (A) Negative ion mode MALDI analysis of bacterial culture samples allows for the detection of dozens of metabolites. High-resolution accurate mass measurements allow for the tentative identification of (B) ornithine at m/z 131.083 (0.34 ppm mass error) and (C) arginine at m/z 173.104 (0.43 ppm mass error). Mass spectra is acquired using a mass resolving power (FWHM) of 75,000 at m/z 150. Abbreviations: MALDI = matrix-assisted laser desorption/ionization; FWHM = full width at half maximum. Please click here to view a larger version of this figure.
Figure 6: Imaging mass spectrometry analysis of bacterial macrocolonies. (A,C) Optical images of agarose samples shows the measurement regions for imaging mass spectrometry analysis, which are highlighted by white dashed lines. (B,D) MALDI ion images for ornithine (m/z 131.083, 0.038 ppm) and arginine (m/z 173.104, 0.60 ppm) display unique spatial distributions across the bacterial cultures. Note that the absolute intensity scales are different for the co-culture ion images shown in panels B and D. For example, arginine is significantly more abundant in the C. difficile monoculture relative to the co-culture in panels C and D, meaning this intensity scale is relative to arginine's abundance in the monoculture, and it appears that there is no arginine present in this co-culture. Conversely, the co-culture in A and B projects arginine's intensity onto a lower absolute intensity scale, since this is the only sample in the image. Abbreviations: MALDI = matrix-assisted laser desorption/ionization; CASI = continuous accumulation of selected ions. Please click here to view a larger version of this figure.
Figure 7: Imaging mass spectrometry analysis of infected murine cecal tissues. (A) Brightfield microscopy images of hematoxylin and eosin-stained tissues from infected mouse ceca allow for the visualization of tissue morphology. (B) Optical images of mouse ceca tissues show the samples following application of a DAN MALDI matrix layer. (C) MALDI ion images for ornithine (m/z 131.083, 0.88 ppm) and arginine (m/z 173.104, 0.15 ppm) display unique spatial distributions across the tissue samples. Abbreviations: MALDI = matrix-assisted laser desorption/ionization; DAN = 1,5-diaminonaphthalene. Please click here to view a larger version of this figure.
During MALDI imaging mass spectrometry, it is important to have a flat sample surface to provide for a consistent focal diameter of the incident MALDI laser on the sample substrate. Deviations in sample height can cause the MALDI laser beam to shift out of focus, causing alterations in beam diameter and intensity, which can affect MALDI ionization efficiency. These alterations in ionization efficiency can result in differences in analyte intensity across the tissue surface that are not reflective of the underlying tissue biochemistry, but instead reflective of the surface topography. Additionally, most MALDI ionization sources operate at sub-atmospheric pressures, and hydrated agar samples are fragile samples when subjected to in vacuo conditions. The vacuum of these sources can cause dehydration of agarose samples, which can alter the sample surface during drying. Samples containing air pockets or large amounts of water are highly susceptible to these changes. This issue is commonly encountered during MALDI imaging mass spectrometry analysis of bacterial specimens grown on agar. Nutrient agar has a high water content to facilitate bacterial growth; however, it is essential to remove as much moisture as possible before applying a MALDI matrix coating and introducing the sample to the vacuum of the mass spectrometer.
Hydrated agar samples are therefore not conducive for in vacuo analysis. In addition to morphological changes induced by in vacuo conditions, there are also potential issues that exist with sample heights that protrude too far from the MALDI target plate slide adapter, which is used to hold the microscope slide mounted sample and bring it to the proper position for MALDI laser focal point. The MALDI stage mechanism is tightly inferfaced with other mechanical components of the stage, and while in position, there is a clearance of only a few millimeters between the surface of the sample target plate and the front end of the ion source optics. This can cause scraping of the sample, which can move or completely dismount the sample to be analyzed. Moreover, while hydrated agar specimens adhere naturally to a microscope slide surface, this tends to be much weaker than the adherence of a completely dried agar specimen. Therefore, the hydrated agar samples protrude further off of the surface of the sample target plate and have a weaker adherence, which causes a greater risk of the sample dismounting and flaking into the downstream instrument ion optics.
The identity of the MALDI matrix and the selection of the application method are also important variables to consider in an imaging mass spectrometry experiment. The selection of an organic MALDI matrix depends largely on the sample substrate and target compounds of interest. For example, many low-molecular weight metabolites (MW < 300 Da) are comprised of many organic acids, phosphates, and other moieties, which are more readily ionized in the negative polarity28. For negative mode analysis, basic matrices are preferred, such that their chemical structures are more amenable to accepting a proton (e.g., 9-aminoacridine, 1,5-diaminonapthalene), therefore leaving a negative charge on many small-metabolite ions of interest. We performed a matrix screen for the targeted analytes of interest and found that 1,5-diaminonapthalene (DAN) matrix produced the highest signal for amino acid metabolites of interest for control tissue samples. During MALDI matrix application, analytes of interest are extracted from the sample substrate and co-crystallize with the MALDI matrix. In some instances, "wetter" matrix application conditions will facilitate increased extraction and co-crystallization, resulting in improved analyte detection upon MALDI analysis. However, if the matrix application process uses too much solvent, analytes may be delocalized within the tissue. It is important to strike a balance between effective analyte extraction and maintaining the spatial fidelity of analytes in the tissue when selecting the conditions for matrix application. For example, a custom-built sublimation apparatus can be used to apply a homogeneous matrix layer to sample surfaces and produces extremely small matrix crystal sizes29,30. However, this process is quite dry and may not be optimal for analyte extraction31. The robotic sprayer used here provides for reproducible and precise control of the spray pressure, flow rate, nozzle temperature, and other parameters that can allow for optimized methods of analyte extraction and matrix co-crystallization32.
The instrumental parameters on the FTICR MS used here have been optimized for the transmission and detection of metabolite ions in negative ion mode. This includes tuning RF and DC elements in the ion transfer region to preferentially transmit ions of low molecular weight (<m/z 200). Parameters that have a large effect on ion transmission include the funnel RF amplitude (65 V), the Q1 mass setting for quadrupole mass filter ion isolation (m/z 150), and the time-of-flight delay between the hexapole accumulation cell and the ICR cell (0.400 ms). These selected parameter values more efficiently transmit ions of low m/z values at the expense of the transmission efficiencies of ions of larger m/z values. Moreover, we have utilized a continuous accumulation of selected ions (CASI) approach, which allows for the selective enrichment of ions in a defined mass window of interest. In this approach, the quadrupole mass filter is used to selectively transmit a small range of m/z values from the ionization source to the hexapole accumulation cell in the instrument, while ions with m/z values outside this mass window have unstable ion trajectories and are not transmitted through the quadrupole mass filter. This allows for the selective enrichment of lowly abundant analytes of interest to improve the limit of detection and dynamic range by up to three orders of magnitude through the elimination of chemical noise. The high mass resolving power and mass accuracy of the FTICR instrument platform allows for the facile separation of isobaric (i.e., same nominal mass) compounds and the tentative identification of metabolites.
Careful and reproducible control of sample preparation, MALDI matrix application, and imaging mass spectrometry analyses can allow for reproducible views of bacterial biochemistry in colonies and tissue samples. Bacterial macrocolonies serve as simplified models to characterize metabolic cross communication of pathogens during microbial infection, which can then be compared to more complex systems such as animal tissues. For example, arginine is found to be lowly abundant in the C. difficile + E. faecalis co-culture sample compared to the C. difficile culture alone (Figure 6), which is supported by E. faecalis's noted ability to consume arginine as an energy source and export ornithine from the cell through the ArcD antiporter33. The macrocolony results led to the investigation of this regulation of amino acids in an animal model. Mice infected with C. difficile + E. faecalis (wt) showed significantly less arginine than mice infected with C. difficile alone (Figure 7C), recapitulating the colony image results.
To test the hypothesis of arginine metabolism and the involvement of the ArcD antiporter, we have used a transposon mutant that knocks out metabolite transport through E. faecalis's ArcD antiporter. Mice infected with C. difficile + E. faecalis (arcD::Tn) show an increase in the detection of arginine and a decrease in the detection of ornithine relative to the wild-type co-infection model (Figure 7C) and generally rescue the original amino acid levels observed in the model of C. difficile infection alone (Figure 7A). This inverse relationship of metabolite regulation corroborates the findings that E. faecalis cross feeds on amino acid nutrients produced from C. difficile. Furthermore, histological analysis showed extensive cellular damage from mice infected with both C. difficile and E. faecalis, which is primarily observed in the wild-type strain of E. faecalis opposed to the ArcD transposon mutant. These results support our recent findings that arginine and ornithine are key to C. difficile virulence, behavior, and
fitness27. Overall, this workflow demonstrates an effective method for preparing agar-based bacterial macrocolonies for MALDI imaging mass spectrometry and shows that these systems can serve as useful models for examining microbial cooperation during infection.
The authors have nothing to disclose.
This work was supported by the National Institutes of Health (NIH) National Institute of General Medical Sciences (NIGMS) under award GM138660. J.T.S. was supported by the Charles and Monica Burkett Family Summer Fellowship from the University of Florida. J.P.Z. was supported by NIH grants K22AI7220 (NIAID) and R35GM138369 (NIGMS). A.B.S. was supported by the Cell and Molecular Biology Training Grant at the University of Pennsylvania (T32GM07229).
0.2 μm Titan3 nylon syringe filters | Thermo Scientific | 42225-NN | |
1,5-diaminonaphthalene MALDI matrix | Sigma Aldrich | 2243-62-1 | |
20 mL Henke Ject luer lock syringes | Henke Sass Wolf | 4200.000V0 | |
275i series convection vacuum gauge | Kurt J. Lesker company | KJL275807LL | |
7T solariX FTICR mass spectrometer equipped with a Smartbeam II Nd:YAG MALDI laser system (2 kHz, 355 nm) | Bruker Daltonics | ||
Acetic acid solution, suitable for HPLC | Sigma Aldrich | 64-19-7 | |
Acetonitrile, suitable for HPLC, gradient grade, ≥99.9% | Sigma Aldrich | 75-05-8 | |
Ammonium hydroxide solution, 28% NH3 in H2O, ≥99.99% trace metals basis | Sigma Aldrich | 1336-21-6 | |
Autoclavable biohazard bags: 55 gal | Grainger | 45TV10 | |
Biohazard specimen transport bags (8 x 8 in.) | Fisher Scientific | 01-800-07 | |
Brain heart infusion broth | BD Biosciences | 90003-040 | |
C57BL/6 male mice | Jackson Laboratories | ||
CanoScan 9000F Mark II photo and document scanner | Canon | ||
CM 3050S research cryomicrotome | Leica Biosystems | ||
Desiccator cabinet | Sigma Aldrich | Z268135 | |
Diamond tip scriber, Electron Microscopy Sciences | Fisher Scientific | 50-254-51 | |
Drierite desiccant pellets | Drierite | 21005 | |
Ethanol, 200 Proof | Decon Labs | 2701 | |
flexImaging software | Bruker Daltonics | ||
ftmsControl software | Bruker Daltonics | ||
Glass vacuum trap | Sigma Aldrich | Z549460 | |
HTX M5 TM robotic sprayer | HTX Technologies | ||
Indium Tin Oxide (ITO)-coated microscope slides | Delta Technologies | CG-81IN-S115 | |
In-line HEPA filter to vacuum pump | LABCONCO | 7386500 | |
Methanol, HPLC Grade | Fisher Chemical | 67-56-1 | |
MTP slide-adapter II | Bruker Daltonics | 235380 | |
Optimal cutting temperature (OCT) compound | Fischer Scientific | 23-730-571 | |
Peridox RTU Sporicide, Disinfectant and Cleaner | CONTEC | CR85335 | |
PTFE (Teflon) printed slides, Electron Microscopy Sciences | VWR | 100488-874 | |
Rotary vane vacuum pump RV8 | Edwards | A65401903 | |
Tissue-Tek Accu-Edge Disposable High Profile Microtome Blades | Electron Microscopy Sciences | 63068-HP | |
Transparent vacuum tubing | Cole Palmer | EW-06414-30 | |
Ultragrade 19 vacuum pump oil | Edwards | H11025011 | |
Variable voltage transformer | Powerstat | ||
Water, suitable for HPLC | Sigma Aldrich | 7732-18-5 | |
Wide-mouth dewar flask | Sigma Aldrich | Z120790 |