Oct 04, 2023
XTT Assay for Detection of Bacterial Metabolic Activity in water-based Polyester Polyurethane
Peer-reviewed method
- 1Departamento de Microbiología Molecular, Instituto de Biotecnología, UNAM, Av. Universidad #2001, Col. Chamilpa, 62210 Cuernavaca, Morelos, Mexico
- Nallely Magaña-Montiel: nallely.magana@ibt.unam.mx
- Luis F Muriel-Millán: luis.muriel@ibt.unam.mx
- Liliana Pardo-López: liliana.pardo@ibt.unam.mx
Protocol Citation: Nallely Magaña-Montiel, Luis F Muriel-Millán, Liliana Pardo-López 2023. XTT Assay for Detection of Bacterial Metabolic Activity in water-based Polyester Polyurethane . protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l27zbjg1y/v1
Manuscript citation:
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
We use this protocol and it's working
Created: March 22, 2023
Last Modified: October 04, 2023
Protocol Integer ID: 79267
Keywords: XTT, plastic biodegradation, marine bacteria, Gulf of Mexico, polyurethane
Funders Acknowledgement:
Conacyt PhD Scollarship
Grant ID: 417065
PAPIIT-DGAPA
Grant ID: IG200223
Abstract
In microbial biodegradation assays, the detection of bacterial growth in water-based plastic dispersions can be difficult to measure using traditional methods because of the turbidity of culture media and the formation of flocculi. Here, we present a protocol for the detection of bacterial growth in Impranil®DLN, a polyester polyurethane (PU) water-based dispersion. By measuring bacterial metabolic activity, as an indicator of cell viability with the water-soluble 2, 3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) salt. Viable growing cells, i.e., those cells that can utilize PU as a carbon source, will reduce the yellow-colored XTT to a water-soluble orange formazan by the action of dehydrogenase enzymes of the respiratory chain. For the standardization of the protocol, we used Pseudomonas putida KT2440 and Escherichia coli BL21 strains as positive and negative controls, respectively. We determined the metabolic activity of the strains grown with citrate or both citrate and impranil as carbon sources. P. putida KT2440 showed higher XTT-detected metabolic activity in the presence of PU than when it was grown only with citrate, indicating that the strain also used PU as a carbon source. In contrast, the negative control did not show differences in metabolic activity between the growth conditions. Our protocol can be adapted to different bacterial strains and culture media.
Image Attribution
Laboratory of Marine Biotechnology, Institute of Biotechnology (IBT) UNAM
Guidelines
Practical considerations
- The assay can be adapted for other bacterial strains.
- Also, you can use an automated microplate spectrophotometer to directly incubate and measure the transformation of XTT to formazan continuously (i.e., every hour).
- Glassware preparation: before use in this procedure, clean glassware is washed with the solvents to extract contaminants and avoid plastic residues.
Materials
LEGEND
1 for preparation of bacterial culture
2 for prior preparation of glass material
3 for XTT assay
Reusable materials:
- Erlenmeyer 50 mL flasks1
- Erlenmeyer 250 mL flasks1
- 50 mL measuring cylinders1
- Glass container for storing solution 1:1 Methanol:Chloroform2
- Bacteriological loops1
- 10 mL pyrex tubes1
- 50 mL graduated cylinder1
- Ice buckets1
Disposable materials:
- Sterile 96 well culture microplates3
- Sterile polystyrene petri dishes1
- Sterile 50 mL polypropylene centrifuge tubes1
- Neoprene & Nitrile Chemical resistant Gloves2
- Pipette tips1,3
- Nitrile gloves1,3
Equipment:
- Multichannel pipette 20-200 µL with 12 chanels3
- Fume hood2
- Digital clock1,3
- Vortex1,3
- Epoch™ 2 Microplate Spectrophotometer Biotek®1,3
- Centrifuge 5415 R Eppendorf1
- Incubated shaker Lab Companion1
Chemicals:
- XTT sodium salt powder (Sigma-Aldrich) Catalog #X46263
- Basal mineral medium (BM) (Composition in step 8)1
- Impranil®DLN1,3
- Luria-Bertani (LB) agar (NaCl 10 g•L-1, peptone 10 g• L-1 and yeast extract 5 g •L-1, ACS grade)1
- Chloroform ACS grade2
- Methanol ACS grade2
- MgSO4 ACS grade1
- Sodium citrate ACS grade1,3
Strains:
- Pseudomonas putida KT2440 (positive control)1,3
- Escherichia coli BL21 (negative control)1,3
Software:
- Microsoft excel3
- Gen5 Data Analysis Software from Epoch™ 2 3
Protocol materials
XTT sodium saltMerck MilliporeSigma (Sigma-Aldrich)Catalog #X4626
Step 9
Safety warnings
This procedure involves the use of hazardous chemicals (chloroform and methanol) for the glassware preparation.
XTT contains no substances with occupational exposure limit values.
- Read the corresponding safety data sheets for each chemical in the procedure.
- Use personal protective equipment throughout the procedure: nitrile gloves, wear a lab coat and safety glasses.
- Dispose of all chemical waste in appropriately labeled containers.
Before start
- Washing glassware All glassware should be washed twice using 2 mL of methanol-chloroform mixture (1:1) and allowed to dry in a fume hood.
Preparation of bacterial strains
Preparation of bacterial strains
Scrape some of the frozen surface of the glycerol stock using a sterile loop and streak the bacteria onto a Luria-Bertani (LB) agar plate.
Incubate the culture at 30 °C during 24:00:00
Note
Use adecuate incubation temperature for your strain.
1d
Take one isolated colony and inoculate it into 5 mL LB broth.
Incubate Overnight at 30 °C with shaking at 180 rpm .
Measure the optical density (OD) at 600 nm of cultures using a spectrophotometer.
Note
If needed, prepare a dilution of the culture (e.g., 10-1) using fresh LB broth and measure its OD600. The OD600 of the original culture is calculated by multiplying the obtained OD by the dilution factor.
Inoculate 250-mL flasks containing 50 mL of Basal Medium standardize supplemented with Instant Ocean Sea Salt (0.06 g•L-1) (BM), peptone (10 g•L-1) and yeast extract (5 g•L-1) (BM-PY broth) with an aliquot of the previous culture to obtain an OD600 of approximately 0.1.
Note
Our research group uses marine salts to standardize the protocol and screen marine bacteria for subsequent toxicity assays of bacterial culture in zebrafish embryos. For your specific requirements, feel free to use an appropriate minimal medium and carbon source. Composition of Basal Medium in g•L-1: 0.8 K2HPO4, 0.2 KH2PO4, 0.3 NH4Cl, 0.19 Na2SO4, 0.07 CaCl2, 0.005 FeSO4⋅7H2O, 0.16 MgCl2, and 0.0002 Na2MoO4 (all J.T. Baker® ACS grade).
CITATION
Incubate the new culture at 180 rpm, 30°C for the time needed to reach the exponential growth phase: approximately 04:00:00 (see the note below).
Note
This 4 hour timeframe applies to our experimental and cultural conditions. It is important to be knowledgeable of the exponential growth rate of your bacterial strains.
4h
Collect the cells by centrifugation at6000 rpm, 4°C, 00:20:00 and discard the supernatant.
20m
Wash twice the cellular pellet by suspension in 20 mL of sterile 10mM MgSO4 followed by centrifugation at 6000 rpm, 4°C, 00:10:00 to remove all traces of the old culture medium.
10m
Resuspend the cells in5 mL of fresh sterile 10mM MgSO4 and reserve to be used as inoculum for the next steps. Keep the washed cells on ice to facilitate their handling and preparation.
Culture media preparation
Culture media preparation
The strains' ability to grow using the commercial PU coating Impranil‱DLN as a carbon source is evaluated in Basal Medium, which is always supplemented with Instant Ocean Sea Salt (0.06 g•L-1).
XTT solution
Prepare a solution of XTT sodium saltMerck MilliporeSigma (Sigma-Aldrich)Catalog #X4626 at 2 mg•mL-1 in BM.
BM-citrate solution
Prepare a solution of 20 mM sodium citrate in BM.
Note
The presence of easily metabolizable carbon sources such as citrate has been shown to promote the degradation of xenobiotic compounds (Fonseca 2011, Johnsen et al. 2002).
CITATION
BM-citrate-Impranil solution
Prepare a BM-citrate solution added with Impranil®DLN (1 mg•mL-1)
XTT experiment for detection of bacterial growth
XTT experiment for detection of bacterial growth
XTT experiment standardization
Add 150 µL of the BM solution and 50 µL of the XTT solution into a 96-well microplate and measure the UV-Vis spectrum in a range of 300 to 700 nm with a microplate spectrophotometer. Also, obtain the spectrum of BM-citrate and BM-citrate-Impranil in the same UV-Vis range. Perform in triplicate.
Note
Perform experiment standardization for each condition, i.e., for each medium. The signal intensity is subject to several parameters, including concentration of formazan salts, incubation time, number of viable cells, and cellular metabolic activity. Optimization of test conditions requires consideration of all these parameters (Ghasemi et al. 2021, Riss et al. 2023).
CITATION
CITATION
To obtain the maximum absorbance range of XTT with viable cells:
Prepare Pseudomonas putida KT2440 (positive control) cultures in BM-citrate, BM-citrate-Impranil, and BM without any carbon source (biotic control), by inoculating 20 mL of each medium on 50-mL flasks up to obtain an OD600 approximately of 0.3 (~1x 108 cells) in triplicate.
To obtain the maximum absorbance range, add 150 µL of each culture medium of Pseudomonas putida KT2440 and50 µL of the XTT solution into a 96-well microplate and measure UV-Vis spectrum in a range of 300 to 700 nm with a microplate spectrophotometer, immediately after the addition of XTT and again after 1-3 hours of incubation at 180 rpm, 30°C in dark.
Note
The assay can be adapted for other bacterial strains.
Also, you can use an automated microplate spectrophotometer to directly incubate and measure the transformation of XTT to formazan continuously (i.e. every hour).
Expected result
Cells with metabolic activity reduce XTT (yellow) to formazan (orange), which has a wavelength range of maximum absorbance between 450-500 nm (Cell Counting & Health Analysis, 2023; Maldonado et al. 2017).
CITATION
CITATION
Detection of Bacterial Growth in Polyester Polyurethane
Detection of Bacterial Growth in Polyester Polyurethane
To evaluate microbial growth, measure the absorbance of each treatment every hour with an automated microplate spectrophotometer at Absmax (range of 450nm - 500nm) with a reference wavelength of 630nm immediately after the addition of XTT and again every hour.
Note
The use of a reference wavelength considerably reduces the noise of the particles in the medium (background subtraction at 630–690 nm). Also, subtract the absorbance at time zero (Absi ) from subsequent readings to obtain the change in absorbance.
Absorbance= [Absmax - Abs630] - Absi
Culture media:
| A | B | C | D | E | |
| Abiotic control | Pos. control | Neg. control | Test microorganism | ||
| BM | x | x | x | x | |
| BM-citrate | x | x | x | x | |
| BM-citrate-Impranil | x | x | x | x |
"x" indicates that must be included.
Note
Note that BM in positive control, negative control, and test microorganism correspond to biotic controls without any carbon source.
Add 150 µL of the different cultures into a 96-well microplate containing 50 µL of XTT in each well.
We tested the metabolic activity of P. putida KT2440 (Franklin et al. 1981) and E. coli BL21 as positive and negative controls, respectively.
CITATION
Note
The assay can be adapted for other bacterial strains.
Immediately after XTT addition, incubate the plate24:00:00 at 30. °C
1d
To evaluate microbial growth, measure absorbance every hour with an automated microplate spectrophotometer reader EPOCH2 (BioTek Instruments Inc.) both at 470nm and 630nm (reference wavelength for background subtraction).
Expected result
Expected results for XTT assay. Viable cells transform the yellow XTT to formazan (orange). The signal intensity depends both on the number of viable cells and the cellular metabolic activity. BM-C: BM-citrate, BM-IC: BM-citrate-Impranil, C-: negative control, C+: positive control. The numbers indicate biological replicates each one performed in duplicate (technical replicates).
Citations
Step 10
Johnsen Anders R., Bendixen Karen, Karlson Ulrich. Detection of Microbial Growth on Polycyclic Aromatic Hydrocarbons in Microtiter Plates by Using the Respiration Indicator WST-1
doi: 10.1128/AEM.68.6.2683-2689.2002Step 12
Terry L Riss, PhD, Richard A Moravec, BS, Andrew L Niles, MS, Sarah Duellman, Hélène A Benink, Tracy J Worzella, and Lisa Minor. Cell Viability Assays
https://www.ncbi.nlm.nih.gov/books/NBK144065/Step 13
Maldonado Cubas, Juan & Casañas Pimentel, Rocio & Merlin, Iván & San Martín-Martínez, Eduardo.. La espectroscopia UV-Vis en la evaluación de la viabilidad de células de cáncer de mama
https://www.researchgate.net/publication/340915352_La_espectroscopia_UV-Vis_en_la_evaluacion_de_la_viabilidad_de_celulas_de_cancStep 13
Cell Counting & Health Analysis, Merck guides, online 2023. Cell Viability and Proliferation XTT Assay Protocol Guide
https://www.sigmaaldrich.com/MX/es/technical-documents/protocol/cell-culture-and-cell-culture-analysis/cell-counting-and-health-Step 15
F. C. Franklin, M. Bagdasarian, M. M. Bagdasarian, and K. N. Timmis. Molecular and functional analysis of the TOL plasmid pWWO from Pseudomonas putida and cloning of genes for the entire regulated aromatic ring meta cleavage pathway.
https://doi.org/10.1073/pnas.78.12.7458Step 5
Muriel-Millán LF, Rodríguez-Mejía JL, Godoy-Lozano EE, Rivera-Gómez N, Gutierrez-Rios RM, Morales-Guzmán D, Trejo-Hernández MR, Estradas-Romero A, Pardo-López L.. Functional and Genomic Characterization of a Pseudomonas aeruginosa Strain Isolated From the Southwestern Gulf of Mexico Reveals an Enhanced Adaptation for Long-Chain Alkane Degradation.
https://doi.org/10.3389/fmars.2019.00572