Feb 18, 2025
Performing the Tetrazolium Dye Assay to Assess Substrate Transport in Anaerobic, Thermophilic Microbes
- Isaiah D Richardson1,
- Bishal Dev Sharma1,
- Daniel G Olson1
- 1Dartmouth College Thayer School of Engineering
- Isaiah D Richardson: PhD Student
- Bishal Dev Sharma: PhD Candidate
- Daniel G Olson: Principal Investigator of the Olson Lab
Protocol Citation: Isaiah D Richardson, Bishal Dev Sharma, Daniel G Olson 2025. Performing the Tetrazolium Dye Assay to Assess Substrate Transport in Anaerobic, Thermophilic Microbes. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk8pxdl5r/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: September 15, 2024
Last Modified: February 18, 2025
Protocol Integer ID: 107649
Keywords: Tetrazolium dye assay, Clostridium thermocellum, BIOLOG, Substrate transport, Alternative carbon substrates, Thermoanaerobacterium saccharolyticum, PM MicroPlates
Funders Acknowledgements:
US National Science Foundation
Grant ID: 2313152
Abstract
This experimental protocol describes the first-reported application of the well-established tetrazolium dye assay to qualitatively assess substrate transport in the anaerobic, thermophilic microbes Clostridium thermocellum and Thermoanaerobacterium saccharolyticum. This work was motivated by an effort to expand the substrate range of these microbes to further our understanding of their non-canonical metabolism. To assess substrate transport in these microbes, tetrazolium dye assays were performed using BIOLOG PM1 and PM2A MicroPlates. Each plate contained 96 different carbon substrates to experimentally test. The tetrazolium dye assay results reported here confirmed the transport of known substrates into these microbes, as well as the transport of several previously unknown substrates, such as the ability of wild-type C. thermocellum to transport Arbutin, Propionic Acid, and Turanose into the cell. This protocol is broadly applicable for qualitatively assessing the transport of carbon substrates into any anaerobic, thermophilic microbes of interest.
Attachments
Materials
Potassium hexacyanoferrate (III)
Product Number: P8131-100G; CAS: 13746-66-2 (Sigma Aldrich). https://www.sigmaaldrich.com/US/en/product/sigald/p8131
Menadione (Vitamin K3)
Product Number: M5625-25G; CAS: 58-27-5 (Sigma Aldrich). https://www.sigmaaldrich.com/US/en/product/sigma/m5625
Dimethyl sulfoxide (DMSO)
Product Number: 472301-100ML; CAS: 67-68-5 (Sigma Aldrich). https://www.sigmaaldrich.com/US/en/product/sigald/472301
BIOLOG IF-0a GN/GP Base (1.2x), 125 mL (Anaerobic inoculating fluid [AN-IF])
Product Number: 72268 (Biolog, Inc). https://www.biolog.com/wp-content/uploads/2023/06/1667861199_MS-72268-IF-0a-GNGP-Base.pdf
BIOLOG Redox Dye Mix D (100x), 20 mL
Product Number: 74224 (Biolog, Inc). https://www.biolog.com/wp-content/uploads/2024/02/MS-74224-Redox-Dye-Mix-D.pdf
BIOLOG Phenotype MicroArray PM1 MicroPlate
Product Number: 12111 (Biolog, Inc). https://www.biolog.com/wp-content/uploads/2023/06/1667859492_MS-12111-PM1-MicroPlate.pdf
BIOLOG Phenotype MicroArray PM2A MicroPlate
Product Number: 12112 (Biolog, Inc). https://www.biolog.com/wp-content/uploads/2023/06/1667859533_MS-12112-PM2A-MicroPlate.pdf
ThermalSeal RTS (Sealing Film for 96-well BIOLOG PM MicroPlates)
Catalog Number: TSS-RTQ-100 (Excel Scientific). https://excelscientific.com/products/thermalseal-rts-optically-clear-silicone-adhesive-100/
Millipore Steriflip Vacuum Tube Top Filter (50 mL tubes)
Catalog Number: SCGP00525; Lot Number: MPSF241910 (Millipore Sigma). https://www.sigmaaldrich.com/US/en/product/mm/scgp00525?srsltid=AfmBOorO0aYU9dteho5adstRiO0YitgwYYCFh3Y6Y6bTUOjVlJEl2Rk9
50 mL Polypropylene Conical Screw-Cap Tubes (50 mL Falcon tubes)
Reference Number: 352098 (Corning Science). https://ecatalog.corning.com/life-sciences/b2c/US/en/Liquid-Handling/Tubes%2C-Liquid-Handling/Centrifuge-Tubes/Falcon%C2%AE-Conical-Centrifuge-Tubes/p/352098
AnaeroPack System Pack-Rectangular Jar 2.5 Liter (Anaerobic Box)
Product: Rect.Jar 2.5L(JAR-25) (Mitsubishi Gas Chemical Co., Inc.). https://www.mgc.co.jp/eng/products/sc/anaeropack/containers.html
AnaeroPack-Anaero (Anaerobic sachet)
Product: AnaeroPack-Anaero (Mitsubishi Gas Chemical Co., Inc.).
Protocol materials
Potassium hexacyanoferrate (III)Merck MilliporeSigma (Sigma-Aldrich)Catalog #P8131-100G
MenadioneMerck MilliporeSigma (Sigma-Aldrich)Catalog #M5625-25G
Phenotype MicroArray PM1 MicroPlateBIOLOGCatalog #12111
Phenotype MicroArray PM2A MicroPlateBIOLOGCatalog #12112
Redox Dye Mix D (100x), 20 mLBIOLOGCatalog #74224
IF-0a GN/GP Base (1.2x), 125 mLBIOLOGCatalog #72268
Dimethyl SulfoxideMerck MilliporeSigma (Sigma-Aldrich)Catalog #472301-100ML
Before start
Be sure to wear proper personal protective equipment (PPE), including an appropriately fitting laboratory coat, gloves, goggles, pants, and closed toed shoes at all times while performing this protocol in the lab.
Introduction & Background on the Tetrazolium Dye Assay
Introduction & Background on the Tetrazolium Dye Assay
The tetrazolium dye assay is a well-established experimental assay developed to qualitatively assess the transport of carbon substrates into aerobic or anaerobic microbes via a colorimetric readout. The colorimetric readout of this assay arises from the use of a tetrazolium violet dye. Oxidized tetrazolium violet is colorless and membrane permeable. When a carbon substrate is transported into the cell and modified by a redox reaction in cellular metabolism, the promiscuity of the tetrazolium dye allows the dye to be used as an alternative electron acceptor. Reduced tetrazolium dye forms an insoluble purple compound in the cell that can be observed both by eye and a spectrophotometer at 500 nm.
Figure 1: A conceptual overview detailing how tetrazolium violet dye enables a qualitative assessment of substrate transport in a microbial cell via a purple colorimetric readout. Oxidized tetrazolium violet dye is colorless and reduced tetrazolium violet dye is purple. Abbreviations: Tv = tetrazolium violet dye; ox = oxidized; red = reduced.
This experimental protocol describes the steps for performing and analyzing the results from a tetrazolium dye assay to assess substrate transport in anaerobic, thermophilic bacteria (i.e., Clostridium thermocellum and Thermoanaerobacterium saccharolyticum) using BIOLOG PM1 or PM2A MicroPlates. BIOLOG PM MicroPlates are 96 well plates in which each well contains a different carbon substrate at a specific concentration. When performing a tetrazolium dye assay to examine substrate transport for a specific microbial strain in a BIOLOG PM MicroPlate, both inoculated and un-inoculated (control) plates should be prepared at the same time to: 1) enable comparison between the plates to identify transported substrates, 2) reduce experimental errors, and 3) identify false positives (background). The goal of this assay is to identify wells that are purple in the inoculated plate, but colorless in the un-inoculated plate, as this provides a qualitative indication of substrate transport into the cell. The robustness and versatility of the tetrazolium dye assay arises from its straightforward set up, relatively short incubation period, easy-to interpret results, and broad applicability to any aerobic or anaerobic microbial strain of interest.
Protocol Structure (Table of Contents)
Protocol Structure (Table of Contents)
This experimental protocol is structured into the following sections:
A. Long-Term Storage Conditions for Materials and Reagents (Step 3 [3.1-3.4])
B. Deoxygenating the BIOLOG PM MicroPlates (Step 4 [4.1-4.5])
C. Preparing 1.0 M Potassium hexacyanoferrate (III) (Step 5 [5.1-5.11])
D. Preparing 10 mM Menadione in Dimethyl Sulfoxide (Step 6 [6.1-6.10])
E. Preparing Autoclaved MilliQ Water (Step 7 [7.1-7.8])
F. Deoxygenating Additional Reagents & Materials (Step 8 [8.1-8.5])
G. Preparing & Washing a Microbial Culture (Step 9 [9.1-9.12])
H. Setting up the BIOLOG PM MicroPlates (Step 10 [10.1-10.20])
I. Imaging the BIOLOG PM MicroPlates (Step 11 [11.1-11.8])
J. Analyzing the BIOLOG PM MicroPlate Results (Step 12 [12.1-12.3])
K. Results for Wild-Type C. thermocellum (LL1004) (Step 13 [13.1-13.2])
L. Results for Wild-Type T. saccharolyticum (LL1025) (Step 14 [14.1-14.2])
M. Results for Ethanologen strain of T. saccharolyticum (LL1049) (Step 15 [15.1-15.2])
N. Results for Wild-Type E. coli (MG1655 - LL1812) (Step 16 [16.1-16.2])
Long-Term Storage Conditions for Materials and Reagents
Long-Term Storage Conditions for Materials and Reagents
This section of the protocol describes the proper long-term storage conditions for the BIOLOG PM1 and PM2A MicroPlates prior to setting up a tetrazolium dye assay to assess substrate transport.
Upon receiving the BIOLOG PM1 and PM2A Microplates, store the 96-well plates in their sealed, white BIOLOG packaging at 4°C under aerobic conditions.
Phenotype MicroArray PM1 MicroPlateBIOLOGCatalog #12111
Phenotype MicroArray PM2A MicroPlateBIOLOGCatalog #12112
Upon receiving the BIOLOG Redox Dye Mix D (100x), store this brown, light-proof bottle at 4°C under aerobic conditions.
Redox Dye Mix D (100x), 20 mLBIOLOGCatalog #74224
Upon receiving the BIOLOG IF-0a GN/GP Base (1.2x) solution, wrap each clear 125 mL bottle with aluminum foil to minimize light exposure. Store the BIOLOG IF-0a GN/GP Base bottles at 4°C under aerobic conditions.
IF-0a GN/GP Base (1.2x), 125 mLBIOLOGCatalog #72268
Figure 2: Three photographs detailing several of the reagents used in the setup of the tetrazolium dye assay. (Left): The BIOLOG Redox Dye Mix D (100x) stored in a brown, light-proof bottle . (Center): BIOLOG IF-0a GN/GP Base (1.2x) solution bottle as it is received from BIOLOG. (Right): BIOLOG IF-0a GN/GP Base (1.2x) solution bottle wrapped in aluminum foil to reduce light exposure during storage.
Deoxygenating the BIOLOG PM MicroPlates
Deoxygenating the BIOLOG PM MicroPlates
This section of the protocol describes the steps required to fully deoxygenate the BIOLOG PM MicroPlates prior to setting up a tetrazolium dye assay in an anaerobic glove bag.
Partially open the sealed packaging of two BIOLOG PM1 or PM2A 96-well microplates and place each 96-well MicroPlate (still loosely wrapped in its white BIOLOG packaging) in a clean, dry anaerobic box with a new anaerobic sachet. (Note: Refer to the materials section for the product information for the anaerobic box and anaerobic sachets used for this experimental protocol).
Carefully seal each anaerobic box and allow the BIOLOG PM MicroPlates to deoxygenate for 24 hours at room temperature on a lab bench.
After 24 hours of deoxygenating at room temperature, transfer the deoxygenating BIOLOG PM microplates in their sealed anaerobic boxes to a 4°C freezer and allow them to further deoxygenate for 1-2 days. If desired, the plates can deoxygenate at 4°C for up to a week prior to use.
Do not remove the BIOLOG PM MicroPlates from their sealed anaerobic boxes at any time prior to setting up the plates for a tetrazolium dye assay.
Figure 3: Two photographs detailing how the BIOLOG PM MicroPlate should be placed into the anaerobic box to deoxygenate with an anaerobic sachet prior to setting up a tetrazolium dye assay. (Left): The anaerobic box without the cover. (Right): The anaerobic box with the cover on and sealed.
Preparing 1.0 M Potassium hexacyanoferrate (III)
Preparing 1.0 M Potassium hexacyanoferrate (III)
This section of the protocol describes the steps required to prepare and deoxygenate 10 mL of a 1.0 M solution of potassium hexacyanoferrate (III) for use in setting up a tetrazolium dye assay.
Label a 50 mL screw cap tube (Falcon tube) with "10 mL of 1.0 M potassium hexacyanoferrate (III)". Be sure to include initials of the researcher and the date of preparation.
Equipment
50 mL Polypropylene Conical Screw-Cap Tubes (Falcon Tubes)
NAME
50 mL screw-cap tube
TYPE
Corning Science
BRAND
352098
SKU
LINK
50 mL screw-cap tube (Falcon Tubes)
SPECIFICATIONS
Fill the 50 mL Falcon tube with 5.0 mL of MilliQ water.
Weigh out 3.29 g of potassium hexacyanoferrate (III) in a weigh boat.
Potassium hexacyanoferrate (III)Merck MilliporeSigma (Sigma-Aldrich)Catalog #P8131-100G
Carefully add the 3.29 g of potassium hexacyanoferrate (III) to the 50 mL Falcon tube.
Vortex the solution for 10-15 seconds to start dissolving the potassium hexacyanoferrate (III) in the MilliQ water.
To facilitate further dissolving, fill the 50 mL Falcon tube with MilliQ water up to the 8.0-9.0 mL mark.
Vortex the solution for at least 10-15 seconds to continue dissolving the potassium hexacyanoferrate (III) in the MilliQ water. Continue vortexing until the potassium hexacyanoferrate (III) powder is fully dissolved.
Carefully fill the 50 mL Falcon tube up to the 10 mL mark to produce a 1.0 M solution.
Under aerobic conditions on a lab bench, sterile-filter the 10 mL 1.0 M potassium hexacyanoferrate (III) solution into a Millipore Steriflip Vacuum Tube Top Filter (0.22 μm pore size).
Equipment
Steriflip Vacuum Tube Top Filter
NAME
Sterile-Filter Tube (50 mL volume)
TYPE
Merck MilliporeSigma
BRAND
SCGP00525
SKU
LINK
0.22 uM filter, 50 mL volume tube
SPECIFICATIONS
Transfer the sterile-filtered 1.0 M potassium hexacyanoferrate (III) solution into an anaerobic glove bag. Loosen the cap of the 50 mL Falcon tube to allow the solution to deoxygenate within the glove bag at room temperature for at least 24 hours prior to setting up a tetrazolium dye assay.
Figure 4: Two photographs of the 1.0 M potassium hexacyanoferrate (III) solution after it has been sterile-filtered, but prior to being transferred into the anaerobic glove bag to deoxygenate. Despite potassium hexacyanoferrate (III) being a red, crystalline solid, the resulting 1.0M solution in MilliQ water is a dark golden color in appearance. It is important to note that after sitting in the anaerobic glove bag for 2-3 days, the solution becomes a darker brown color in appearance. It is best to use the 1.0 M potassium hexacyanoferrate (III) solution within 1-2 days of being in the anaerobic glove bag.
Preparing 10 mM Menadione in Dimethyl Sulfoxide
Preparing 10 mM Menadione in Dimethyl Sulfoxide
This section of the protocol describes the steps required to prepare and deoxygenate 10 mL of a 10 mM solution of menadione in DMSO (dimethyl sulfoxide) for use in setting up a tetrazolium dye assay.
Label a 50 mL screw cap tube (Falcon tube) with "10 mL of 10 mM Menadione in DMSO". Be sure to include initials of the researcher and the date of preparation.
Fill the 50 mL Falcon tube with 5.0 mL of DMSO.
Weigh out 17.2 mg of menadione powder using a small weigh boat. Note that the menadione powder is light green in appearance.
MenadioneMerck MilliporeSigma (Sigma-Aldrich)Catalog #M5625-25G
Carefully add the 17.2 mg of menadione powder to the 50 mL Falcon tube.
Vortex the solution for 10-15 seconds to dissolve the menadione in the DMSO.
Fill the 50 mL Falcon tube up to the 10 mL mark with Dimethyl sulfoxide (DSMO) to produce a 10 mM solution.
Dimethyl SulfoxideMerck MilliporeSigma (Sigma-Aldrich)Catalog #472301-100ML
Vortex the solution for 10-15 seconds to finish dissolving the menadione in the DMSO.
Under aerobic conditions on a lab bench, sterile-filter the 10 mL 10 mM menadione in DMSO solution into a Millipore Steriflip Vacuum Tube Top Filter (0.22 μm pore size).
Transfer the sterile-filtered 10 mM menadione in DMSO solution into an anaerobic glove bag. Loosen the cap of the 50 mL Falcon tube to allow the solution to deoxygenate in the anaerobic glove bag at room temperature for at least 24 hours prior to setting up a tetrazolium dye assay.
Figure 5: Two photographs of the 10 mM Menadione in Dimethyl sulfoxide (DSMO) solution after it has been sterile-filtered, but prior to being transferred into the anaerobic glove bag to deoxygenate. The solution is a pale yellow color in appearance and becomes slightly darker yellow in appearance after deoxygenating in the anaerobic glove bag for 2-3 days. It is best to use the 10 mM Menadione in DMSO solution within 1-2 days of being in the glove bag.
Preparing Autoclaved MilliQ Water
Preparing Autoclaved MilliQ Water
This section of the protocol describes the steps required to prepare, autoclave, and deoxygenate a 1.0 L glass screw-cap bottle of MilliQ water for use in setting up a tetrazolium dye assay.
Thoroughly clean with soap and water a 1.0 L glass screw-cap bottle and a screw-cap with a septum. Allow the bottle and the screw-cap to dry overnight at room temperature on a lab bench.
Fill the clean and dry 1.0 L glass screw-cap bottle with 1.0 L of MilliQ water.
Loosely seal the glass screw-cap bottle with the dried cap and septum. Note: This allows air to readily exchange between the headspace of the bottle and the air so pressure does not build up in the autoclave.
Place a piece of autoclave tap on the cap of the bottle to mark it for autoclaving.
Autoclave the 1.0 L of MilliQ water in the 1.0 L glass screw-cap bottle for a 30 min liquid cycle.
Immediately after the autoclave cycle ends, carefully remove the 1.0 L glass screw-cap bottle containing 1.0 L of MilliQ water from the autoclave. Be sure to wear proper personal protective equipment (PPE) and autoclave gloves when handing any autoclaved items as autoclaved items are extremely hot.
Immediately and carefully transfer the warm, 1.0 L bottle of autoclaved MilliQ water into the anaerobic glove bag.
Loosen the cap of the bottle and allow the 1.0 L bottle of autoclaved MilliQ water to deoxygenate for at least 24 hours in the glove bag prior to setting up a tetrazolium dye assay.
Deoxygenating Additional Reagents & Materials
Deoxygenating Additional Reagents & Materials
This section of the protocol describes the required steps for deoxygenating the BIOLOG IF-0a GN/GP Base (1.2x) solution, repeat pipette tips, and serological pipette tips for setting up the tetrazolium dye assay.
Transfer a 125 mL aluminum foil wrapped bottle of BIOLOG IF-0a GN/GP Base (1.2x) from long-term aerobic storage at 4°C into the anaerobic glove bag.
Loosen the white screw-cap of the 125 mL BIOLOG IF-0a GN/GP Base (1.2x) bottle slightly to allow the solution to deoxygenate for at least 24 hours prior to setting up a tetrazolium dye assay.
Upon deoxygenating in the anaerobic glove bag, the 125 mL BIOLOG IF-0a GN/GP Base (1.2x) solution is subsequently referred to as BIOLOG AN IF-0a GN/GP Base (1.2x) solution.
Transfer at least 10-15 1.0 mL, 5.0 mL, and 10 mL repeat pipette tips into the anaerobic glove bag. Partially break the seal of the repeat pipette tips to allow them to deoxygenate in the glove bag for at least 24 hours before use in setting up a tetrazolium dye assay.
Transfer at least 10 25 mL serological pipettes into the anaerobic glove bag. Partially break the seal of the 25 mL serological pipettes to allow them to deoxygenate in the glove bag for at least 24 hours before use in setting up a tetrazolium dye assay.
Preparing & Washing a Microbial Culture
Preparing & Washing a Microbial Culture
This section of the protocol describes the steps required for preparing and washing an anaerobic, thermophilic microbial culture for use in setting up a tetrazolium dye assay. This protocol is applicable to any anaerobic, thermophilic microbial strain, however, the results section included in this protocol details the BIOLOG PM1 and PM2A results for the following anaerobic, thermophilic microbial strains:
1. Wild-type (WT) Clostridium thermocellum (LL1004)
2. Wild-type (WT) Thermoanaerobacterium saccharolyticum (LL1025)
3. An ethanologen strain of Thermoanaerobacterium saccharolyticum (LL1049)
In the anaerobic glove bag, fill a 15 mL screw-cap tube (15 mL Falcon tube) with 10 mL of MTC-5 media (5 g/L Cellobiose).
Transfer the freezer stock of the desired anaerobic, thermophilic microbial strain into the anaerobic glove bag and allow it to partially thaw.
Add 50 μL of partially thawed freezer stock of the desired anaerobic, thermophilic microbial strain to the 10 mL of MTC-5 media in the 15 mL Falcon tube.
Place the inoculated culture in a 55°C incubator in the anaerobic glove bag and allow the culture to grow overnight (for at least 12-16 hours) until it reaches an optical density reading (OD600) of 0.80. Measure the OD600 of the culture using a spectrophotometer device in the anaerobic glove bag.
Upon reaching an optical density reading (OD600) of 0.80, transfer 2.0 mL of microbial culture to two, capped 2.0 mL Eppendorf tubes in the anaerobic glove bag.
Centrifuge the two 2.0 mL Eppendorf tubes for 5 minutes at 13,400 rcf (x g) in the anaerobic glove bag.
Use a pipette to carefully pull off and discard the supernatant (MTC-5 media [5 g/L Cellobiose]) without disturbing the cell pellet in either tube.
Add 2.0 mL of BIOLOG AN IF-0a GN/GP Base (1.2x) from the aluminum-foil wrapped, white-capped 125 mL bottle to each of the two 2.0 mL Eppendorf tubes.
Use a pipette to thoroughly pipette mix and dislodge the cell pellet in each 2.0 mL Eppendorf tube. The purpose of this step is to bathe the cells in the BIOLOG AN IF-0a GN/GP Base (1.2x) solution.
Repeat steps 9.6-9.9 two additional times for a total of 3 washings of the cells using BIOLOG AN IF-0a GN/GP Base (1.2x) solution.
Upon completion of the three washings, transfer the 2.0 mL of microbial cells suspended in BIOLOG AN IF-0a GN/GP Base (1.2x) solution in each Eppendorf tube to a 15 mL Falcon tube labeled "Cells".
To the 4.0 mL of washed cells, add 4.0 mL of BIOLOG AN IF-0a GN/GP Base (1.2x) solution to produce a culture with a final OD600 = 0.40. This solution will subsequently be referred to as "Mix A". Store Mix A in the anaerobic glove bag while setting up the plates for the tetrazolium dye assay.
Setting up the BIOLOG PM MicroPlates
Setting up the BIOLOG PM MicroPlates
This section of the protocol describes the steps for preparing a set of inoculated and un-inoculated (control) 96-well BIOLOG PM MicroPlates in the anaerobic glove bag for subsequent qualitative analysis.
Remove the BIOLOG Redox Dye Mix D (100x) bottle from its long-term storage at 4°C, transfer it into the anaerobic glove bag, and loosen its cap slightly to enable deoxygenation while the plates are being set up. (Note: It is most important to keep this bottle cold and out of the light, which is why it is brought into the anaerobic glove bag immediately prior to use instead of allowing it to deoxygenate overnight at room temperature in the anaerobic glove bag).
Label two fully deoxygenated 50 mL screw-cap tubes (50 mL Falcon tubes) in the anaerobic glove bag as "Master Mix No Cells" and as "Master Mix Cells", respectively. Label each with the researchers initials and date of preparation.
To the 50 mL "Master Mix No Cells" Falcon Tube add 8.0 mL of autoclaved, deoxygenated MilliQ water.
To the 50 mL "Master Mix No Cells" Falcon Tube add 36.4144 mL of BIOLOG AN IF-0a GN/GP Base (1.2x) solution (anaerobic inoculating fluid). (Note: For this step, it is easiest to first add 36.0 mL of BIOLOG AN-IF 0a GN/GP Base via serological pipette and to subsequently add 414.4 μL of BIOLOG AN-IF 0a GN/GP Base via a pipette. Absolute precision is not essential for this step, but it best to be as precise as possible).
Add 480.0 μL of deoxygenated BIOLOG Redox Dye Mix D (100x) solution to a 2.0 mL Eppendorf tube.
Add 57.6 μL of deoxygenated 1.0 M potassium hexacyanoferrate (III) stock solution to the 2.0 mL Eppendorf tube. Use a pipette to thoroughly mix the BIOLOG Redox Dye Mix D and potassium hexacyanoferrate (III) solutions. (Note: These components must be mixed together anaerobically before any additional components are added).
Add 48.0 μL of deoxygenated 10 mM menadione in DMSO solution to the 2.0 mL Eppendorf tube. Pipette mix to ensure that the three components are thoroughly mixed before adding to the "Master Mix No Cells" 50 mL Falcon tube.
Using a 1000 μL pipette set to 585.6 μL, transfer the mixed solution of Redox Dye Mix D, potassium hexacyanoferrate (III), and menadione in DMSO from the 2.0 mL Eppendorf tube to the "Master Mix No Cells" 50 mL Falcon tube.
Thoroughly pipette mix the "Master Mix No Cells" solution. The solution should be light yellow in appearance. (Note: this solution has a final volume of 45.0 mL).
Using a repeat pipette, transfer 22.5 mL of the "Master Mix No Cells" solution to the other 50 mL Falcon tube labeled "Master Mix Cells".
Add 1.5 mL of BIOLOG AN-IF 0a GN/GP Base to the 50 mL Falcon tube labeled "Master Mix No Cells". (Note: the "Master Mix No Cells" solution is now complete and has a final volume of 24.0 mL).
Add 1.5 mL of Mix A (the 8.0 mL of anaerobic, thermophilic microbial cells washed 3x in BIOLOG AN-IF 0a GN/GP Base) to the 50 mL Falcon tube labeled "Master Mix Cells". (Note: The "Master Mix Cells" solution is now complete and has a final volume of 24.0 mL).
Remove one of the BIOLOG PM1 MicroPlates that has been deoxygenating for several days in an anaerobic box at 4°C and transfer it into the anaerobic glove bag. For the cycling process, manually pass only nitrogen into the gas exchanger. (Note: It is important to prioritize minimizing hydrogen exposure to the BIOLOG PM MicroPlates, as exposure of the plates to hydrogen produces undesirable false positives [background noise] in some wells).
Upon transferring the BIOLOG PM1 MicroPlate in the anaerobic box into the anaerobic glove bag, carefully remove the BIOLOG PM1 MicroPlate from its partially opened white packaging within the anaerobic box and place it on the floor of the anaerobic glove bag.
Using a repeat pipette with a 5.0 mL attachment set to dispense 100.0 μL, add 100.0 μL of the "Master Mix No Cells" solution to each of the 96 wells in the BIOLOG PM1 MicroPlate. (Note: this step will require the researcher to refill the repeat pipette after half the plate is filled in order to finish filling all of the wells).
Place a sealing film over the filled BIOLOG PM1 MicroPlate. Ensure that the sealing film is tightly sealed over all the wells, around the wells at the edge of the plate, and on the corners of the plate. This is the finished, uninoculated BIOLOG PM1 MicroPlate.
Equipment
ThermalSeal RTS
NAME
Sealing film for 96-well plates
TYPE
Excel Scientific
BRAND
TSS-RTQ-100
SKU
LINK
Sealing film for 96-well plates
SPECIFICATIONS
Immediately place the sealed, un-inoculated BIOLOG PM1 MicroPlate back into the anaerobic box with an anaerobic sachet and transfer it out of the anaerobic glove bag.
Place the uninoculated BIOLOG PM1 MicroPlate in the anaerobic box into an aerobic 55°C incubator for 24 hours.
In a similar manner to the un-inoculated (control) BIOLOG PM1 MicroPlate, repeat steps 10.13 - 10.18 to set up the inoculated BIOLOG PM1 MicroPlate using the "Master Mix Cells" solution.
Table 1: Table detailing the components needed to prepare Mix A and Mix B in the anaerobic glove bag to prepare the "Master Mix Cells" and "Master Mix No Cells" tubes for the tetrazolium dye assay.
Imaging the BIOLOG PM MicroPlates
Imaging the BIOLOG PM MicroPlates
This section of the protocol describes the steps for imaging the BIOLOG PM MicroPlates after they have been prepared and incubated at 55°C for 24 hours. The obtained images are subsequently used to perform a qualitative analysis to examine the transport of alternative carbon substrates into the cell.
After the inoculated and un-inoculated BIOLOG PM MicroPlates have incubated at 55°C for 24 hours, remove the plates from the incubator and place them on a lab bench.
Remove the inoculated and un-inoculated BIOLOG PM MicroPlates from the anaerobic boxes and dispose of the anaerobic sachets into a biohazard garbage bag.
Carefully and slowly remove the sealing film from each of the BIOLOG PM MicroPlates, ensuring that none of the wells are shook or accidentally spill into an adjacent well during the process.
Separately image both of the plates on an aerobic scanner using the EPSON Scan software. Scan the plate using the color settings specified in Figure 7. Ensure the plate is centered on the scanner while imaging.
Name and save the resulting image file of the inoculated and un-inoculated BIOLOG PM MicroPlates within a labeled folder on the local desktop computer linked to the aerobic scanning device. Use a flash drive to transfer the image files to the researcher's personal laptop or hard drive.
Use Adobe Illustrator or a similar photo editing software program to horizontally invert the image using the following steps (applicable for Adobe Illustrator):
1. Go to and click on the 'Image' tab at the top of the screen
2. Under 'Image' select 'Image Rotation'
3. Under 'Image Rotation' use the side arrow to select 'Flip Canvas Horizontal'
After editing each of the image files using the steps outlined in sub-step 11.6 above, save each of the edited images as a .jpg file to a labeled folder on the researcher's own desktop computer, laptop, flash drive, and/or hard drive for long-term storage.
Figure 6: Photograph of the aerobic scanner used to image the BIOLOG PM MicroPlates with the sealing film removed after 24 hours of incubating at 55°C.
Figure 7: Screenshot of the EPSON Scan software used to image the BIOLOG PM MicroPlates on the aerobic scanner. The EPSON Scan software was installed on a Windows desktop computer.
Analyzing the BIOLOG PM MicroPlate Results
Analyzing the BIOLOG PM MicroPlate Results
Visually compare the uninoculated and inoculated BIOLOG PM MicroPlate images side-by-side in Microsoft PowerPoint or Word to identify differences between the two plates.
If a specific well is purple in the inoculated plate, but not in the un-inoculated plate, this is indicative of the transport of that carbon substrate into the cell. In addition, if a specific well is noticeably darker purple in appearance in the inoculated plate than the un-inoculated plate, this is also indicative of transport of that carbon substrate into the cell.
Due to the inability to entirely eliminate hydrogen exposure to the plates, it is likely that there will be several or many purple or discolored (yellow, brown, or red) wells in both the inoculated and un-inoculated (control) BIOLOG PM MicroPlates. Under the experimental setup conditions described in this protocol, these results are normal.
Use the attached 'Master File of BIOLOG PM1 and PM2A MicroPlate Substrates' to identify the alternative carbon substrates present in each of the 96-wells in both of the BIOLOG PM1 and PM2A MicroPlates to determine what carbon substrates were transported into the cell.
Results for Wild-Type C. thermocellum (LL1004)
Results for Wild-Type C. thermocellum (LL1004)
This section details the BIOLOG PM1 & PM2A MicroPlate results obtained from performing the tetrazolium dye assay to assess substrate transport in WT C. thermocellum (LL1004).
Figure 8: Tetrazolium dye assay results for WT C. thermocellum (LL1004) for the BIOLOG PM1 MicroPlate. The presence of purple color in a well in the inoculated plate (right) and the absence of purple color in the corresponding well in the uninoculated plate (left) indicates that the substrate was transported into the cell. The wells highlighted with a dashed black circle in the inoculated plate (right) indicate the substrates transported into WT C. thermocellum. The transported substrates are listed to the right of the inoculated plate along with the false positives (background). The columns are numbered 1-12 and the rows are labeled A-H. Well A1 is located in the upper left corner of each plate.
Figure 9: Tetrazolium dye assay results for WT C. thermocellum (LL1004) for the BIOLOG PM2A MicroPlate. The presence of purple color in a well in the inoculated plate (right) and the absence of purple color in the corresponding well in the uninoculated plate (left) indicates that the substrate was transported into the cell. The wells highlighted with a dashed black circle in the inoculated plate (right) indicate the substrates transported into WT C. thermocellum. The transported substrates are listed to the right of the inoculated plate along with the false positives (background). The columns are numbered 1-12 and the rows are labeled A-H. Well A1 is located in the upper left corner of each plate.
Results for Wild-Type T. saccharolyticum (LL1025)
Results for Wild-Type T. saccharolyticum (LL1025)
This section details the BIOLOG PM1 & PM2A MicroPlate results obtained from performing the tetrazolium dye assay to assess substrate transport in WT T. saccharolyticum (LL1025).
Figure 10: Tetrazolium dye assay results for WT T. saccharolyticum (LL1025) for the BIOLOG PM1 MicroPlate. The presence of purple color in a well in the inoculated plate (right) and the absence of purple color in the corresponding well in the uninoculated plate (left) indicates that the substrate was transported into the cell. The wells highlighted with a dashed black circle in the inoculated plate (right) indicate the substrates transported into WT T. saccharolyticum. The transported substrates are listed to the right of the inoculated plate along with the false positives (background). The columns are numbered 1-12. The rows are labeled A-H. Well A1 is located in the upper left corner of each plate.
Figure 11: Tetrazolium dye assay results for WT T. saccharolyticum (LL1025) for the BIOLOG PM2A MicroPlate. The presence of purple color in a well in the inoculated plate (right) and the absence of purple color in the corresponding well in the uninoculated plate (left) indicates that the substrate was transported into the cell. The wells highlighted with a dashed black circle in the inoculated plate (right) indicate the substrates transported into WT T. saccharolyticum. The transported substrates are listed to the right of the inoculated plate along with the false positives (background). The columns are numbered 1-12. The rows are labeled A-H. Well A1 is located in the upper left corner of each plate.
Results for Ethanologen strain of T. saccharolyticum (LL1049)
Results for Ethanologen strain of T. saccharolyticum (LL1049)
This section details the BIOLOG PM1 & PM2A MicroPlate results obtained from performing the tetrazolium dye assay to assess substrate transport in an ethanologen strain of T. saccharolyticum (LL1049).
Figure 12: Tetrazolium dye assay results forT. saccharolyticum strain LL1049 for the BIOLOG PM1 MicroPlate. The presence of purple color in a well in the inoculated plate (right) and the absence of purple color in the corresponding well in the uninoculated plate (left) indicates that the substrate was transported into the cell. The wells highlighted with a dashed black circle in the inoculated plate (right) indicate the substrates transported into T. saccharolyticum LL1049. The transported substrates are listed to the right of the inoculated plate along with the false positives (background). The columns are numbered 1-12. The rows are labeled A-H. Well A1 is located in the upper left corner of each plate.
Figure 13: Tetrazolium dye assay results forT. saccharolyticum strain LL1049 for the BIOLOG PM2A MicroPlate. The presence of purple color in a well in the inoculated plate (right) and the absence of purple color in the corresponding well in the uninoculated plate (left) indicates that the substrate was transported into the cell. The wells highlighted with a dashed black circle in the inoculated plate (right) indicate the substrates transported into T. saccharolyticum LL1049. The transported substrates are listed to the right of the inoculated plate along with the false positives (background). The columns are numbered 1-12. The rows are labeled A-H. Well A1 is located in the upper left corner of each plate.
Results for Wild-Type E. coli (MG1655 - LL1812)
Results for Wild-Type E. coli (MG1655 - LL1812)
This section details the BIOLOG PM1 & PM2A MicroPlate results obtained from performing the tetrazolium dye assay to assess substrate transport in wild-type (WT) E. coli (MG1655 - LL1812).
Figure 14: Tetrazolium dye assay results for WT E. coli (MG1655 - LL1812) for the BIOLOG PM1 MicroPlate. The presence of purple color in a well in the inoculated plate (right) and the absence of purple color in the corresponding well in the uninoculated plate (left) indicates that the substrate was transported into the cell. The transported substrates are listed to the right of the inoculated plate along with the false positives (background). The significantly smaller number of false positives in the uninoculated plate arises from reduced exposure of the plates to hydrogen. These plates were prepared under aerobic conditions and were incubated aerobically at 37°C for 24 hours. The columns are numbered 1-12. The rows are labeled A-H. Well A1 is located in the upper left corner of each plate.
Figure 15: Tetrazolium dye assay results for WT E. coli (MG1655 - LL1812) for the BIOLOG PM1 MicroPlate. The presence of purple color in a well in the inoculated plate (right) and the absence of purple color in the corresponding well in the uninoculated plate (left) indicates that the substrate was transported into the cell. The transported substrates are listed to the right of the inoculated plate along with the false positives (background). The significantly smaller number of false positives in the uninoculated plate arises from reduced exposure of the plates to hydrogen. These plates were prepared under aerobic conditions and were incubated aerobically at 37°C for 24 hours. The columns are numbered 1-12. The rows are labeled A-H. Well A1 is located in the upper left corner of each plate.
Protocol references
1. Bochner, B.R., Gadzinski, P. & Eugenia Panomitros. Phenotype MicroArrays for High-Throughput Phenotypic Testing and Assay of Gene Function. Genome Research 11, 1246-1255 (2001).
2. Bochner, B.R. & Savageau, M.A. Generalized Indicator Plate for Genetic, Metabolic, and Taxonomic Studies with Microorganisms. Applied and Environmental Microbiology 33(2), 434-444 (1977).
3. Bhupathiraju, V.K., Hernandez, M., Landfear, D. & Alvarez-Cohen, L. Application of a tetrazolium dye as an indicator of viability in anaerobic bacteria. Journal of Microbiological Methods 37, 231-243 (1999).
4. Bochner, B.R. (1980). Method for Testing and Identifying Microorganisms (U.S. Patent No. 4,235,964). U.S. Patent and Trademark Office.
5. BIOLOG. PM Procedures for Anaerobic Bacteria. Biolog, Inc. Received through direct correspondence with the manufacturer. (2023).
6. BIOLOG. AN MicroPlate Instructions for Use. Biolog, Inc. (2008).
Acknowledgements
This work was supported by the National Science Foundation under Grant Number 2313152.