This is a protocol preprint. Preprints are preliminary reports that have not undergone peer review. They should not be considered conclusive, used to inform clinical practice, or referenced by the media as validated information.
Method Article
In vitro antimicrobial assay of nano-iron sulfide (nFeS)
Lizeng Gao
Gao Lab (Yangzhou University)
Corresponding Author
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
This protocol describes a detailed process of antibacterial (Streptococcus mutans UA159) activity by nanometer-sized iron sulfide (nFeS). Streptococcus mutans has long been considered to be a critical factor in caries-causing plaque development. The pathogens are enriched in 3D matrix of extracellular polymeric substances and lead to dental caries by producing highly acidic niches. We find that nano-iron sulfide improves the killing efficacy of Streptococcus mutans UA159 by more than 500 times compaired with garlic-derived organosulfur compounds. Here the Fe3O4 nanoparticles and nFeS( Cys-nFeS, GSH-nFeS, NaHS-nFeS, Cyss-nFeS, and DADS-nFeS ) converted from different organosulfur sources were used to evaluate the antimicrobial (Streptococcus mutans UA159) activity within 30 minutes.
Keywords
pathogens; Streptococcus mutans UA159; bactericidal; dental caries
Figure 1
ethyl alcohol, tryptone, yeast extract, glucose, 0.1 M sodium acetate (pH 4.5)
Bacteria culture and inoculum preparation
Cryopreserved Streptococcus mutans UA159 were thawed from a frozen stock, and spread on tryptone-yeast extract ager broth with 1% glucose. Then the plate was incubated at 37°C and 5% CO2 for 48 h.
- Transfer several colonies with inoculation loop and place into 15 mL of sterile tube, resuspend, and incubated the pre-culture at 37°C and 5% CO2 for 12-16 h.
- Transfer 200 µL of overnight cultured bacteria solution to 3.8 mL fresh tryptone-yeast extract broth with 1% glucose and cultured for 4 h when the optical density (OD) at 600 nm was about 1.0. CAUTION: Streptococcus mutans UA159 on ager broth stored at 4°C was easy to lose activity. Choose the bacteria on the ager plate stored within two weeks, and pick several colonies for activating bacteria overnight.
- Harvest the bacteria by centrifugation at 5000 g for 5 min.
- Wash the bacteria two times by resupending the pellet into 0.1M NaAc (pH 4.5) and centrifugr two times at 5000 g for 5 min.
- Resuspend the pellet into 0.1M NaOAc (pH 4.5)
Adjust the bacteria population to 108 CFU/mL.
Antimicrobial preparation
- Wash the Cys-nFeS, DADS-nFeS nanoparticles with ethyl alcohol 3 times, respectively.
- Resuspend the nanoparticles into ddH2O and magnetic separation for 3 times.
- Autoclave the nanoparticles by 121°C, 20 min.
Dilute the autoclaved nanoparticles to 5 mg/mL.
In vitro antimicrobial assay
- Add 100 µL of bacteria into 900 µL of 0.1 M sodium acetate (pH 4.5) and resuspend the mixture. Dilute and plate (at least 3 dilutions per sample) accordingly.
- Add 100 µL of bacteria and 100 µL of 5 mg/mL Cys-nFeS nanoparticles into 800 µL of 0.1 M sodium acetate (pH 4.5), and resuspend the mixture. Dilute and plate (at least 3 dilutions per sample) accordingly.
- Add 100 µL of bacteria and 100 µL of 5 mg/mL DADS-nFeS nanoparticles into 800 µL of 0.1 M sodium acetate (pH 4.5), and resuspend the mixture. Dilute and plate (at least 3 dilutions per sample) accordingly
- Count colonies 24-48 h later and plot them.
- Colony counts of less than 20 colonies or more than 350 colonies per plate were not considered.
- Streptococcus mutans UA159 can be killed significantly by iron sulfide nanoparticles in 0.5 mg/mL within 30 min.
Version History
CURRENT STATUS: Posted
Version 1
Posted 03 Dec, 2018
Metrics
Comments: 0
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Subject Areas
Microbiology
Associated Publications
Converting organosulfur compounds to inorganic polysulfides against resistant bacterial infections
by Zhuobin Xu, Zhiyue Qiu, Qi Liu, +15
Nature Communications (22 November, 2018)
Method Article
In vitro antimicrobial assay of nano-iron sulfide (nFeS)
Lizeng Gao
Gao Lab (Yangzhou University)
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 03 Dec, 2018
Metrics
Comments: 0
HTML Views: ...
Subject Areas
Microbiology
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
This protocol describes a detailed process of antibacterial (Streptococcus mutans UA159) activity by nanometer-sized iron sulfide (nFeS). Streptococcus mutans has long been considered to be a critical factor in caries-causing plaque development. The pathogens are enriched in 3D matrix of extracellular polymeric substances and lead to dental caries by producing highly acidic niches. We find that nano-iron sulfide improves the killing efficacy of Streptococcus mutans UA159 by more than 500 times compaired with garlic-derived organosulfur compounds. Here the Fe3O4 nanoparticles and nFeS( Cys-nFeS, GSH-nFeS, NaHS-nFeS, Cyss-nFeS, and DADS-nFeS ) converted from different organosulfur sources were used to evaluate the antimicrobial (Streptococcus mutans UA159) activity within 30 minutes.
Figure 1
ethyl alcohol, tryptone, yeast extract, glucose, 0.1 M sodium acetate (pH 4.5)
Bacteria culture and inoculum preparation
Cryopreserved Streptococcus mutans UA159 were thawed from a frozen stock, and spread on tryptone-yeast extract ager broth with 1% glucose. Then the plate was incubated at 37°C and 5% CO2 for 48 h.
- Transfer several colonies with inoculation loop and place into 15 mL of sterile tube, resuspend, and incubated the pre-culture at 37°C and 5% CO2 for 12-16 h.
- Transfer 200 µL of overnight cultured bacteria solution to 3.8 mL fresh tryptone-yeast extract broth with 1% glucose and cultured for 4 h when the optical density (OD) at 600 nm was about 1.0. CAUTION: Streptococcus mutans UA159 on ager broth stored at 4°C was easy to lose activity. Choose the bacteria on the ager plate stored within two weeks, and pick several colonies for activating bacteria overnight.
- Harvest the bacteria by centrifugation at 5000 g for 5 min.
- Wash the bacteria two times by resupending the pellet into 0.1M NaAc (pH 4.5) and centrifugr two times at 5000 g for 5 min.
- Resuspend the pellet into 0.1M NaOAc (pH 4.5)
Adjust the bacteria population to 108 CFU/mL.
Antimicrobial preparation
- Wash the Cys-nFeS, DADS-nFeS nanoparticles with ethyl alcohol 3 times, respectively.
- Resuspend the nanoparticles into ddH2O and magnetic separation for 3 times.
- Autoclave the nanoparticles by 121°C, 20 min.
Dilute the autoclaved nanoparticles to 5 mg/mL.
In vitro antimicrobial assay
- Add 100 µL of bacteria into 900 µL of 0.1 M sodium acetate (pH 4.5) and resuspend the mixture. Dilute and plate (at least 3 dilutions per sample) accordingly.
- Add 100 µL of bacteria and 100 µL of 5 mg/mL Cys-nFeS nanoparticles into 800 µL of 0.1 M sodium acetate (pH 4.5), and resuspend the mixture. Dilute and plate (at least 3 dilutions per sample) accordingly.
- Add 100 µL of bacteria and 100 µL of 5 mg/mL DADS-nFeS nanoparticles into 800 µL of 0.1 M sodium acetate (pH 4.5), and resuspend the mixture. Dilute and plate (at least 3 dilutions per sample) accordingly
- Count colonies 24-48 h later and plot them.
- Colony counts of less than 20 colonies or more than 350 colonies per plate were not considered.
- Streptococcus mutans UA159 can be killed significantly by iron sulfide nanoparticles in 0.5 mg/mL within 30 min.
Associated Publications
Converting organosulfur compounds to inorganic polysulfides against resistant bacterial infections
by Zhuobin Xu, Zhiyue Qiu, Qi Liu, +15
Nature Communications (22 November, 2018)