Jan 06, 2023
Hestrin-Schramm (HS) medium
- 1University of Oslo
Protocol Citation: Andreas Sagen 2023. Hestrin-Schramm (HS) medium. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly7xyelx9/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
We use this protocol and it's working
Created: January 05, 2023
Last Modified: January 06, 2023
Protocol Integer ID: 74795
Keywords: Komagataeibacter, bacterial cellulose, cellulose, growth of komagataeibacter, komagataeibacter, yeast extract, schramm medium, medium hestrin, yeast, primary nitrogen source, bacteria, hestrin, schramm, common medium composition, usable energy in aerobic organism, nitrogen, magnesium sulphate, phosphate, medium composition, glucose, citrate, sodium phosphate, magnesium, intermediate compound, aerobic organism
Abstract
Hestrin-Schramm medium is a common medium composition to grow Komagataeibacter. The medium contains glucose as a source for energy and carbon, while yeast extract and peptone are the primary nitrogen sources. Both sodium phosphate and magnesium sulphate is added to benefit the bacteria, but it is not necessary to support growth of Komagataeibacter. Citrate is used as an intermediate compound in the tricarboxylic acid cycle and an essential element to produce usable energy in aerobic organisms.
Guidelines
Follow step by step, unless stated otherwise. Equipment needed should be standard to a microbiology lab.
Materials
Analytical scale, autoclave, bottle(s), weight vessel, LAF bench
Protocol materials
DextroseMerck MilliporeSigma (Sigma-Aldrich)Catalog #G7021
Yeast ExtractMerck MilliporeSigma (Sigma-Aldrich)Catalog #Y0875
Magnesium sulfateMerck MilliporeSigma (Sigma-Aldrich)Catalog #M7506
CitrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S4641
PeptoneMerck MilliporeSigma (Sigma-Aldrich)Catalog #P5905
Disodium phosphateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S9763
AgarMerck MilliporeSigma (Sigma-Aldrich)Catalog #A1296
Safety warnings
You can mix Dextrose from the beginning with the other compounds, and autoclave together. While this is more time efficient and easier, it is important to take into account the possibility of toxic byproducts produced by the Millard reaction when autoclaving, producing Acrylamide, a probable human carcinogen (IARC Group 2A).
Furthermore, when removing autoclaved components, be sure to take care as these can be very hot. If using antibiotics, use sufficient PPE to protect yourself, as some can be toxic to humans.
Before start
Prepare glassware by cleaning it, and ensure that scale is sufficiently calibrated
100 mL Dextrose solution
20m
All compounds are measured using a high precision analytical scale from powdered compounds. Each compound is measured to within 1% of the target weight. All compounds are mixed in a Duran bottle
Fill the bottle with 60 mL double-distilled water
Measure 10000 mg Dextrose
Powdered compounds:
DextroseMerck MilliporeSigma (Sigma-Aldrich)Catalog #G7021
Add powdered solids into bottle, and use a magnetic mixer with a stir bar to mix for 00:05:00
5m
Adjust pH while mixing to 5.7 using concentrated sodium hydroxide
Add distilled water to a total of 100 mL
Autoclave liquid at 121 °C for 00:15:00
15m
500 mL HS (broth) medium
20m
All compounds are measured using a high precision analytical scale from powdered compounds. Each compound is measured to within 1% of the target weight. All compounds are mixed in a Duran bottle
Fill the bottle with 300 mL double-distilled water
Measure 2500 mg Peptone, 2500 mg Yeast extract, 1350 mg Disodium phosphate, 750 mg Citrate and 600 mg Magnesium sulfate
Powdered compounds:
PeptoneMerck MilliporeSigma (Sigma-Aldrich)Catalog #P5905
Yeast ExtractMerck MilliporeSigma (Sigma-Aldrich)Catalog #Y0875
Disodium phosphateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S9763
CitrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S4641
Magnesium sulfateMerck MilliporeSigma (Sigma-Aldrich)Catalog #M7506
Add powdered solids into bottle, and use a magnetic mixer with a stir bar to mix for 00:05:00
5m
Adjust pH while mixing to 5.7 using concentrated citric acid
Autoclave liquid at 121 °C for 00:15:00
15m
In a LAF bench, add 100 mL sterile Dextrose solution
Add sterile water to a total of 500 mL
Note
Cool to 50°C and supplement with antibiotics as appropriate
500 mL HS (agar) medium
20m
All compounds are measured using a high precision analytical scale from powdered compounds. Each compound is measured to within 1% of the target weight. All compounds are mixed in a Duran bottle
Fill the bottle with 300 mL double-distilled water
Measure 2500 mg Peptone, 2500 mg Yeast extract, 1350 mg Disodium phosphate, 750 mg Citrate, 600 mg Magnesium sulfate and 7500 mg agar
Powdered compounds:
PeptoneMerck MilliporeSigma (Sigma-Aldrich)Catalog #P5905
Yeast ExtractMerck MilliporeSigma (Sigma-Aldrich)Catalog #Y0875
Disodium phosphateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S9763
CitrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S4641
Magnesium sulfateMerck MilliporeSigma (Sigma-Aldrich)Catalog #M7506
AgarMerck MilliporeSigma (Sigma-Aldrich)Catalog #A1296
Add powdered solids into bottle, and use a magnetic mixer with a stir bar to mix for 00:05:00
5m
Adjust pH while mixing to 5.7 using concentrated citric acid
Autoclave liquid at 121 °C for 00:15:00
15m
In a LAF bench, add 100 mL sterile Dextrose solution
Add sterile water to a total of 500 mL
Note
Cool to 50°C and supplement with antibiotics as appropriate
Agar can be stored, then reheated to 50°C to be poured