Removal of Exogenous Materials from the Outer Portion of Frozen Cores to Investigate the Ancient Biological Communities Harbored Inside

Summary

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. A protocol is presented to collect and decontaminate permafrost cores of soils and ice. The absence of exogenous colonies and DNA suggest that microorganisms detected represent the material, rather than contamination from drilling or processing.

Abstract

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect conditions over vast time periods and investigation of biological materials harbored inside could provide insight of ancient environments. To appropriately analyze these ecosystems and extract meaningful biological information from frozen soils and ice, proper collection and processing of the frozen samples is necessary. This is especially critical for microbial and DNA analyses since the communities present may be so uniquely different from modern ones. Here, a protocol is presented to successfully collect and decontaminate frozen cores. Both the absence of the colonies used to dope the outer surface and exogenous DNA suggest that we successfully decontaminated the frozen cores and that the microorganisms detected were from the material, rather than contamination from drilling or processing the cores.

Introduction

The cryosphere (e.g., permafrost soils, ice features, glacial snow, firn, and ice) offers a glimpse into what types of organisms persisted under past environmental conditions. Since these substrates can be tens to hundreds of thousands of years old, their microbial communities, when preserved frozen since deposition, reflect ancient environmental conditions. To appropriately analyze these ecosystems and extract meaningful biological information from frozen soils and ice, proper collection and processing of the frozen samples is necessary. This is of utmost importance as climate projections for the 21^st century indicate the potential for a pronounced warming in Arctic and sub-Arctic regions¹. Specifically, Interior Alaska and Greenland are expected to warm approximately 5 °C and 7 °C, respectively by 2100^2,3. This is expected to significantly impact soil and aquatic microbial communities, and therefore, related biogeochemical processes. The warmer temperatures and altered precipitation regime are expected to initiate permafrost degradation in many areas^2-5 potentially leading to a thicker, seasonally thawed (active) layer^6,7, the thawing of frozen soils, and the melting of massive ice bodies such as ground ice, ice wedges, and segregation ice⁸. This would dramatically change the biogeochemical attributes in addition to the biodiversity of plants and animals in these ecosystems.

Glacial ice and syngenetic permafrost sediment and ice features have trapped chemical and biological evidence of an environment representing what lived there at the time the features formed. For example, in Interior Alaska, both Illinoisan and Wisconsin aged permafrost are present and this permafrost in particular provides unique locations dating from modern to 150,000 years before the present (YBP) that contain biological and geochemical evidence of the impact of past climatic changes on biodiversity. As a result, these sediments provide a record of the biogeochemistry and biodiversity over many thousands of years. Since the area has low sedimentation rates and has never been glaciated, undisturbed samples are accessible for collection and analysis, either drilling vertically into the soil profile or drilling horizontally in tunnels. More importantly, extensive records exist that especially highlight the unique biogeochemical features of permafrost in this region^9-14. Specifically, the application of DNA analysis to estimate presence and extent of biodiversity in both extant and ancient ice and permafrost samples enables exploration of the linkage of ancient environmental conditions and habitat to occupation by specific organisms.

Previous studies have identified climatic impacts on mammals, plants and microorganisms from samples dating to 50k YBP^{11, 15-19}, though each study used a different methodology to collect and decontaminate the permafrost or ice cores. In some instances, the drilling cores were sterilized^{16, 20-21}, though the specific methodology did not clarify whether foreign nucleic acids were also eliminated from the samples. In other studies, bacterial isolates¹⁵ (e.g., Serratia marcescens) as well as fluorescent microspheres²² have been used to measure the efficacy of decontamination procedures.

This experiment was part of a larger study investigating microbial communities from permafrost samples dating back to approximately 40k YBP. The specific objective of this portion of the study was to successfully decontaminate ice and permafrost cores. To our knowledge, no methodology has integrated the use of solutions designed to eliminate foreign nucleic acids and associated nucleases from the outer portion of the frozen cores. This is despite the fact that these solutions are commonly used to decontaminate laboratory equipment for molecular experiments.

Once the cores were decontaminated, genomic DNA was extracted using the protocols developed by Griffiths et al.²³ and Töwe et al.²⁴, quantified using a spectrophotometer, and diluted with sterile, DNA-free water to achieve 20 ng per reaction. Bacterial 16S rRNA genes were amplified with primers 331F and 797R and probe BacTaq²⁵ and archaeal 16S rRNA genes were amplified with primers Arch 349F and Arch 806R and probe TM Arch 516F²⁶ under the following conditions: 95 °C for 600 sec followed by 45 cycles of 95 °C for 30 sec, 57 °C for 60 sec, and 72 °C for 25 sec with final extension at 40 °C for 30 sec. All qPCR reactions were conducted in duplicate. The 20 µl reaction volumes included 20 ng DNA, 10 µM of primers, 5 µM of the probe, and 10 µl of the qPCR reaction mix. Standards for bacterial and archaeal qPCR were prepared using genomic DNA from Pseudomonas fluorescens and Halobacterium salinarum, respectively. Both were grown to log phase. Plate counts were conducted and DNA was isolated from the cultures. Genomic DNA was quantified with a spectrophotometer with the assumption of one and six copies of the 16S rRNA gene per genome for H. salinarum and P. fluorescens, respectively^27-28. Copy numbers of the bacterial and archaeal genes were calculated based on the standard curve, log-transformed to account for unequal variances between treatments, and assessed by ANOVA.

Community composition was determined by sequencing the 16S rRNA gene using flow cells and bridge amplification technologies and analyzing the communities with 'quantitative insights into microbial ecology' (QIIME)²⁹. Forward and reverse reads were joined together and then sequences were filtered, indexed, and high quality representatives were selected for de novo operational taxonomic units (OTU) assignment through sequence alignment with a reference database. Aligned sequences were compared to a separate reference database for taxonomic assignment. A phylum level OTU table was created to determine general community composition.

Protocol

1. Equipment Preparation and Permafrost Core Collection

1. Equipment preparation and field sample collection and preservation gear
   1. Assemble auger for sample collection by inserting the drive adapter into the top of the barrel and rotating the lever to lock it in place. Pin the adapter tube onto the drive adapter and pin the motor onto the adapter tube. Insert the cutters on the barrel.
   2. Wear light duty suits, nitrile gloves, and masks to reduce any contamination to the samples. Wear ear protection and a hard hat for safety upon entering the Permafrost Tunnel (Figure 1).
   3. Enter the tunnel and select a location to collect the samples (Figure 1B).
      Note: For vertical or horizontal drilling location, select an area where there is known evidence that the material is frozen (e.g., ice or permafrost), there is no known large root systems, and there is no known gravel. Remove all living plant material from the area before collecting a sample. If drilling vertically or horizontally, select an area that is relatively flat and accessible with the auger.
   4. Prepare a work station by layering the ground with a plastic material that has been sterilized with 70% isopropanol, DNA decontamination solution, and RNase decontamination solution.
   5. Place a 10 cm diameter polyvinyl chloride (PVC) pipe cut in half lengthwise on the workstation to provide a trough to hold the cores as they are removed from the auger. Decontaminate the PVC pipe with 70% isopropanol, DNA decontamination solution, and RNase decontamination solution.
   6. Near the work station, decontaminate the auger by spraying it with 70% isopropanol, DNA decontamination solution, and RNase decontamination solution. Remove solutions from the auger with a wipe.
2. Ice and permafrost core collection and storage
   1. Select an area of the wall to sample (see 1.2.1 Note).
   2. Decontaminate approximately 10 cm diameter of frozen wall area by wiping it with 70% isopropanol, DNA decontamination solution, and RNase decontamination solution.
   3. Elevate the decontaminated auger to the area of interest such that it is perpendicular to the sample area and begin drilling into the cleaned face of the wall (Figure 1C, D).
   4. Carefully remove the auger from sample collection area. Disconnect the auger from the motor and place the auger above the sterile PVC pipe in the clean work station. Carefully tilt auger such that the frozen cores slide out onto the sterile PVC pipe (Figure 1E).
   5. Decontaminate gloves with 70% isopropanol, DNA decontamination solution, and RNase decontamination solution.
   6. Pick up the ice and permafrost cores with sterile gloves and place them into sterile bags.
   7. Place the cores in a cooler or cold room until they are shipped or processed.
   8. Ship the frozen cores using dry ice to maintain them at a temperature below 0 °C.
   9. Immediately store the cores at -80 °C.

2. Permafrost and Ice Core Processing

1. Material Preparation
   1. Prepare sterile, nucleic acid free heavy duty aluminum foil, metal racks, glassware, metal forceps, and glass wool by baking in an oven at 450°C for 4 hr. Set aside these materials until used.
   2. Sterilize 95% ethanol and ultrapure water by passing the solutions through a 0.22 µm filter.
   3. Store ethanol solution at -20 °C and water at 4 °C.
   4. Sterilize a plastic ruler by spraying it with 70% ethanol, DNA decontamination solution, and RNase decontamination solution and immediately wiping after each solution.
2. Bacterial Culture Preparation
   1. Prepare broth and plates to grow Serratia marcescens.
      1. For broth, combine 5 g tryptone, 1 g glucose, 5 g yeast extract, and 1 g potassium phosphate dibasic, and then add distilled water to reach 1 L. To make plates, add 15 g of agar to the above mix. For broth and plates, adjust pH to 7 and autoclave at 121 °C for 15 min.
   2. Prepare 1x phosphate buffed saline solution (PBS) by combining 8 g sodium chloride, 0.2 g of potassium chloride, 1.44 g of sodium phosphate dibasic, and 0.24 g potassium phosphate monobasic and add distilled water to reach 800 ml. Adjust pH to 7.4 and add distilled water to reach 1 L. Autoclave at 121 °C for 15 min.
   3. Inoculate broth with a sterile loop by dipping into the S. marcescens culture that was previously stored frozen at -80 °C. Under aseptic conditions, serial dilute the culture by adding 1 ml of culture to 9 ml of PBS and manually invert the solution. Add 1 ml of this dilution to 9 ml of PBS and manually invert the solution. Repeat eight more times until a 10^-9 dilution is reached.
   4. Spread the 10^-6, 10^-7, 10^-8, and 10^-9 solutions onto agar plates by distributing 1 ml of broth onto the plate and spreading with a cell spreader. Do this in triplicate per dilution. Store the original culture at 4 °C.
   5. Incubate plates at 30 °C for 24 hr. Count the number of colonies to calculate the number of cells in the original culture. Note: The number of colonies in the original culture will depend on the growth rate of the bacteria.
   6. Pipet 250 µl of the original culture into a sterile 1.5 ml microcentrifuge tube. Dilute original culture by adding enough volume of the 1x PBS to obtain approximately 10⁹ cells/ml as determined by colony count in the previous step. Pellet the cells in a microcentrifuge tube by centrifuging at 2,500 x g for 10 min.
      Note: The volume of 1x PBS will vary depending on the growth rate of the bacteria.
   7. In a sterile biohood, pipet off the broth and resuspend the cells in 1 ml 1x PBS buffer. Store at 4 °C until use or approximately two months.
   8. On the day of processing, dilute the S. marcescens culture from step 2.2.7 by adding 1 ml of the original S. marcescens culture to 39 ml 1x PBS buffer in a 50 ml centrifuge tube.
3. Prepare cold room space for core processing and preservation gear
   1. Prior to chilling the cold room, clean walls, floors, and metal table with a 1% bleach solution.
   2. Set temperature of cold room to approximately -11 °C.
   3. Once the cold room has reached the desired temperature, wear light duty suits, nitrile gloves, and masks to reduce any contamination to the cold room and the samples.
      Note: Two properly dressed individuals are required for this procedure.
   4. Bring the sterile materials (e.g., aluminum foil, metal racks, glassware, tray, S. marcescens culture, and microtome blade) and samples into the cold room and place on the sterile table.
4. Permafrost and ice core processing in a cold room facility
   1. Place a permafrost core on a sterile, nucleic acid free sheet of aluminum foil.
   2. Lightly inoculate the outside of the core with the dilute culture of S. marcescens using a sterile foam plug (Figure 2B).
   3. Place the core on the sterile metal rack that sits above a tray.
   4. Sterilize the steel microtome blade with 70% ethanol, DNA decontamination solution, and RNase decontamination solution.
   5. Have Individual A clean nitrile gloves with 70% ethanol, DNA decontamination solution, and RNase decontamination solution and hold core at a 45° angle above the tray.
   6. Have Individual B gently scrape approximately 5 mm of the outside of the entire core, including the ends, using the sterile blade while Individual A turns the core after each scrape (Figures 2C and 3A, B). Wipe the blade with 70% ethanol, DNA decontamination solution, and RNase decontamination solution as needed.
   7. Have Individual B pour filter-sterilized 95% ethanol over the core carefully and quickly, while Individual A turns the core as the solution is poured (Figures 2D, 3C).
   8. Have Individual B immediately rinse the core with filter-sterilized water.
   9. Replace used metal rack on tray with a new sterile metal rack.
   10. Have Individual A clean his or her nitrile gloves with 70% ethanol, DNA decontamination solution, and RNase decontamination solution and hold the core at a 45° angle above the tray.
   11. Have Individual B spray the entire core with DNA decontamination solution.
   12. Have Individual B immediately rinse the core with filter-sterilized water.
   13. Have Individual B spray the entire core with RNase decontamination solution.
   14. Have Individual B immediately rinse the core with filter-sterilized water.
   15. Place the core on a sterile sheet of aluminum foil and lightly wrap.
5. Thaw outer core
   1. Place a sterile metal rack over a sterile glass dish in a sterile biohood with laminar flow.
   2. Place two agar plates specific to S. marcescens in the glass dish below the sterile rack to collect liquid from the core (Figure 2E).
   3. Place the core on the sterile metal rack.
   4. Allow approximately 2-5 mm of the outer surface of core to thaw at 23 °C (on average, this will occur within approximately 10 min). Turn the core approximately 90° every 2 min.
   5. Swab entire surface of core using sterile forceps and sterile glass wool and inoculate two agar plates specific to S. marcescens by swabbing these materials onto the surface of the plates. Inoculate two new plates with the original culture used to dope the outside of the core, which was exposed to the low temperatures of the cold room.
   6. Measure outer dimensions of thawed cylindrical core by placing a sterile ruler near, but not touching the core.
   7. Place the core into large sterile bag and store it at -80 °C.
6. Check for growth
   1. Incubate agar plates at 23 °C for one week.
   2. Examine agar plates for growth of S. marcescens colonies.
      1. If there are no visible colonies on the plate, proceed to step 2.5.3.
      2. If colonies appear on plate, repeat from step 2.1.1 in section 2 "Permafrost and ice core processing".
   3. Obtain the core from freezer and aseptically transfer it to a sterile bag.
   4. Store the core in a sterile bag at 4 °C for approximately 24-48 hr to thaw the entire core.

3. Obtain Subsample for Nucleic Acid Extraction from Ice Cores and Permafrost

1. Nucleic acid extraction from ice cores
   1. Mix the thawed material from the ice core by gently agitating the bag (Figure 2G).
   2. Pour the thawed material into a sterile container with a 0.22 µm filter under vacuum to collect microorganisms.
   3. Aseptically remove the filter with sterile forceps and place it in a sterile 2 ml ceramic bead tube (1.4 mm).
   4. Store the filter at -80 °C.
2. Nucleic acid extraction from permafrost cores
   1. Mix the thawed material from the permafrost core by gently kneading the bag.
   2. After the permafrost has been well mixed, obtain a subsample of approximately 0.5 g bulk soil with a sterile scoopula and place it in a tared sterile 2 ml ceramic bead screw-cap tube (1.4 mm) that sits upright on a balance.
   3. Store subsamples at -80 °C.
   4. Obtain gravimetric water content of samples.
      1. Measure 10 g of the permafrost with a sterile scoopula and place in a tared tin on a balance. Measure wet mass of permafrost and tin.
      2. Incubate permafrost in an oven set at 105 °C for 24 hr. Measure dry mass of permafrost and tin.
      3. Calculate the gravimetric water content by subtracting the wet mass of permafrost by the dry mass of the permafrost and dividing by the dry mass of permafrost.
   5. Store permafrost at -80 °C.

4. Extract Nucleic Acids from Permafrost and Ice Cores

1. Material preparation
   1. Prepare amber vials to hold solutions by treating with 0.1% diethylpyrocarbonate (DEPC), incubating O/N at 37 °C, and autoclaving.
   2. Prepare 240 mM potassium phosphate buffer solution. Add 2. 5 g of potassium phosphate monobasic and 38.7 g of potassium phosphate dibasic. Adjust final volume to 500 ml by adding sterile water.
   3. Prepare hexadecyltrimethylammonium bromide (CTAB) extraction buffer by dissolving 4.1 g sodium chloride in 80 ml water and slowly adding 10 g CTAB while heating and stirring. Adjust final volume to 100 ml by adding sterile water.
   4. Add equal volumes of phosphate buffer solution and CTAB buffer and filter sterilize with a 0.22 µm filter. Cover bottle with aluminum foil and store at RT.
   5. Prepare 1.6 M sodium chloride solution (NaCl) by combining 9.35 g NaCl and 100 ml sterile water. Add 10 g polyethylene glycol 8000 to the 1.6 M NaCl solution and filter sterilize. Store at 4 °C.
2. Nucleic acid extraction according to a modified version of Griffiths et al.²² and Töwe et al.²³
   1. Wear light duty suits, nitrile gloves, and masks. Clean laboratory space and pipets with 70% ethanol, DNA decontamination solution, and RNase decontamination solution.
   2. Remove subsample (filter from ice core or permafrost soil sample) in 2 ml ceramic bead screw-cap tube from -80 °C freezer and thaw at RT.
   3. Add 0.5 ml of hexadecyltrimethylammonium bromide (CTAB) extraction buffer and vortex briefly.
   4. Add 0.5 ml of phenol-chloroform-isoamyl alcohol (25:24:1) (pH 8) and shake tubes horizontally for 10 min using a flat panel adapter on a vortexer.
   5. Following bead-beating, centrifuge tubes at 16,100 x g for 5 min at 4 °C.
   6. Remove aqueous layer to a new sterile 1.5 ml tube and mix with an equal volume of chloroform-isoamyl alcohol (24:1). Centrifuge at 16,100 x g for 5 min at 4 °C.
   7. Remove aqueous layer to a new sterile 1.5 ml tube and add two volumes of 30% polyethylene glycol 8000 and 1.6 M NaCl. Incubate for 2 hr at 4 °C.
   8. Centrifuge at 16,100 x g for 10 min at 4 °C.
   9. Wash nucleic acid pellet with approximately 500 µl of ice-cold 70% ethanol and centrifuge at 16,100 x g for 10 min at 4 °C.
   10. Air dry pellet in a sterile biohood for 2 hr. Resuspend in 50 µl DNase/RNase-free TE buffer (pH 8.0). DNA is ready for downstream applications such as PCR and qPCR.
   11. Determine concentration of DNA with a spectrophotometer.
   12. Dilute DNA with sterile, DNA-free water to achieve 20 ng per reaction.

Results

The presented method could be used to decontaminate frozen samples collected from various cryosphere environments, from glacial ice to permafrost. Here, we present data specifically collected from ice and permafrost samples collected from the Engineering Research and Development Center - Cold Regions Research and Engineering Laboratory (ERDC-CRREL) Permafrost Tunnel located in Fox, AK (Figure 1A and 1B). The Permafrost Tunnel extends approximately 110 m i...

Discussion

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. Though the recovered taxa may not represent the complete historic community, those recovered from analysis of glacial ice and permafrost samples can yield valuable historic information about select time periods^15-16. For instance, meaningful biological information has been drawn from ice studies investigating anaerobic activity in the Greenland ice sheet²⁰ and permafrost studies investigating carb...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Auger	Snow, Ice, and Permafrost Research Establishment (SIPRE), Fairbanks, AK
70% Isopropanol	Walmart	551116880
95% Ethyl Alcohol (denatured)	Fisher Scientific, Pittsburgh, PA	A407-4
DNA decontamination solution, DNA Away	Molecular Bio-Products, Inc., San Diego, CA	7010
RNase decontamination solution, RNase Away	Molecular Bio-Products, Inc., San Diego, CA	7002
Light Duty Suits	Kimberly-Clark Professional, Roswell, GA	10606
Nitrile Gloves	Fisher Scientific, Pittsburgh, PA	FFS-700
Antiviral Masks	Curad, Walgreens	CUR3845
Sterile Sample Bags	Nasco, Fort Atkinson, WI	B01445
Steel Microtome Blade	B-Sharp Microknife, Wake Forest, NC
Metal Rack	Fabricated at CRREL, Hanover, NH
Tray	Handy Paint Products, Chanhassen, MN	7500-CC
Aluminum Foil	Western Plastics, Temecula, CA
500 ml Bottle with 0.22 μm Filter	Corning, Corning, NY	430513
Serratia marcescens	ATCC, Manassas, VA	17991
Biosafety Hood	NuAire, Plymouth, MN	NU-425-400
Petri Dish	Fisher Scientific, Pittsburgh, PA	FB0875712
ATCC Agar 181- Tryptone	Acros Organics, NJ	61184-5000
ATCC Agar 181- Glucose	Fisher Scientific, Pittsburgh, PA	BP381-500
ATCC Agar 181- Yeast Extract	Fisher Scientific, Pittsburgh, PA	BP1422-500
ATCC Agar 181- Dipotassium Phosphate	JT Baker, Phillipsburg, NJ	3252-01
ATCC Agar 181- Agar	Difco, Sparks, MD	214530
NanoDrop 2000 UV Vis Spectrophotometer	Thermo Fisher Scientific, Wilmington, DE
Lightcycler 480 System	Roche Molecular Systems, Inc., Indianapolis, IN
Halobacterium salinarum	American Type Culture Collection (ATCC), Manassas, VA
Pseudomonas fluorescens	American Type Culture Collection (ATCC), Manassas, VA
Microbial DNA Isolation Kit	MoBio Laboratories, Carlsbad, CA	12224-50
Ear Protection	Elvex	EP-201
Hard Hat
Kimwipes	Kimberly-Clark Professional, Roswell, GA	34705
Glass Wool	Pyrex	430330
Ruler
Weighing Tin	Fisher Scientific, Pittsburgh, PA	08-732-100
Sodium chloride	Sigma Aldrich, St Louis, MO	S-9625
Potassium chloride	JT Baker, Phillipsburg, NJ	3040-04
Potassium phosphate, monobasic	JT Baker, Phillipsburg, NJ	3246-01
Potassium phosphate, dibasic	JT Baker, Phillipsburg, NJ	3252-01
Sodium phosphate dibasic, anhydrous	Fisher Scientific, Pittsburgh, PA	BP332-500
50 ml Centrifuge Tubes	Corning, Corning, NY	4558
2 ml Microcentrifuge Tubes	MoBio Laboratories, Carlsbad, CA	1200-250-T
2 ml Ceramic Bead Tubes (1.4 mm)	MoBio Laboratories, Carlsbad, CA	13113-50
Scoopula	Thermo Fisher Scientific, Wilmington, DE	1437520
Balance	Ohaus, Parsippany, NJ	E12130
Diethylpyrocarbonate (DEPC)	Sigma Aldrich, St Louis, MO	D5758
Hexadecyltrimethylammoniabromide (CTAB)	Acros Organics, NJ	22716-5000
Polyethylene glycol 8000	Sigma Aldrich, St Louis, MO	P5413-1KG
Phenol-chloroform-isoamyl alcohol (25:24:1) (pH 8)	Fisher Scientific, Pittsburgh, PA	BP1752-400
Centrifuge	Eppendorf, Hauppauge, NY	5417R
Chloroform-isoamyl alcohol (24:1)	Sigma Aldrich, St Louis, MO	C0549-1PT
TE Buffer	Ambion (Thermo Fisher), Wilmington, DE	AM9860
Pipets	Rainin, Woburn, MA	Pipet Lite XLS, 2, 10, 200, and 1,000 µl pipets
Pipet tips	Rainin, Woburn, MA	Rainin LTS presterilized, low retention, filtered tips, 10, 20, 200, 1,000 µl
Vortexor	Scientific Industries, Bohemia, NY	G-560
Vortex Adaptor	MoBio Laboratories, Carlsbad, CA	13000-V1
Clear Bottle	Corning, Corning, NY	C1395500
Amber Bottle	Corning, Corning, NY	C5135250
Bottle Top Filters, 0.22 µm	Corning, Corning, NY	430513
60 ml Syringe	Becton, Dickenson and Company, Franklin Lakes, NJ	BD 309653
Millex Syringe filters, 0.22 μm	EMD Millipore, Billerica, MA	SLGV033RB
70% Ethanol	Fisher Scientific, Pittsburgh, PA	BP2818-500	diluted & filter sterilized
Isotemp 100 L Oven	Fisher Scientific, Pittsburgh, PA	151030511
Cell Spreader	Fisher Scientific, Pittsburgh, PA	08-100-10
Disposable Inoculating Loops	Fisher Scientific, Pittsburgh, PA	22-363-602