Jan 06, 2023

Public workspaceHestrin-Schramm (HS) medium

DOI
dx.doi.org/10.17504/protocols.io.eq2ly7xyelx9/v1
  • 1University of Oslo
Andreas Sagen
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DOI: dx.doi.org/10.17504/protocols.io.eq2ly7xyelx9/v1
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
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
ReagentYeast ExtractMerck MilliporeSigma (Sigma-Aldrich)Catalog #Y0875
In 2 steps
ReagentMagnesium sulfateMerck MilliporeSigma (Sigma-Aldrich)Catalog #M7506
In 2 steps
ReagentCitrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S4641
In 2 steps
ReagentPeptoneMerck MilliporeSigma (Sigma-Aldrich)Catalog #P5905
In 2 steps
ReagentDisodium phosphateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S9763
In 2 steps
ReagentAgarMerck MilliporeSigma (Sigma-Aldrich)Catalog #A1296
Step 3.2
ReagentDextroseMerck MilliporeSigma (Sigma-Aldrich)Catalog #G7021
Step 1.2
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
100 mL Dextrose solution
20m
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 Amount60 mL double-distilled water
Measure Amount10000 mg Dextrose

Powdered compounds:
ReagentDextroseSigma AldrichCatalog #G7021
Add powdered solids into bottle, and use a magnetic mixer with a stir bar to mix for Duration00:05:00

5m
Adjust pH while mixing to Ph5.7 using concentrated sodium hydroxide
Add distilled water to a total of Amount100 mL

Autoclave liquid at Temperature121 °C for Duration00:15:00
15m
500 mL HS (broth) medium
500 mL HS (broth) medium
20m
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 Amount300 mL double-distilled water
Measure Amount2500 mg Peptone, Amount2500 mg Yeast extract, Amount1350 mg Disodium phosphate, Amount750 mg Citrate and Amount600 mg Magnesium sulfate

Powdered compounds:
ReagentPeptoneSigma AldrichCatalog #P5905
ReagentYeast ExtractSigma AldrichCatalog #Y0875
ReagentDisodium phosphateSigma AldrichCatalog #S9763
ReagentCitrateSigma AldrichCatalog #S4641
ReagentMagnesium sulfateSigma AldrichCatalog #M7506
Add powdered solids into bottle, and use a magnetic mixer with a stir bar to mix for Duration00:05:00
5m
Adjust pH while mixing to Ph5.7 using concentrated citric acid
Autoclave liquid at Temperature121 °C for Duration00:15:00
15m
In a LAF bench, add Amount100 mL sterile Dextrose solution
Add sterile water to a total of Amount500 mL
Note
Cool to 50°C and supplement with antibiotics as appropriate


500 mL HS (agar) medium
500 mL HS (agar) medium
20m
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 Amount300 mL double-distilled water
Measure Amount2500 mg Peptone, Amount2500 mg Yeast extract, Amount1350 mg Disodium phosphate, Amount750 mg Citrate, Amount600 mg Magnesium sulfate and Amount7500 mg agar

Powdered compounds:
ReagentPeptoneSigma AldrichCatalog #P5905
ReagentYeast ExtractSigma AldrichCatalog #Y0875
ReagentDisodium phosphateSigma AldrichCatalog #S9763
ReagentCitrateSigma AldrichCatalog #S4641
ReagentMagnesium sulfateSigma AldrichCatalog #M7506
ReagentAgarSigma AldrichCatalog #A1296
Add powdered solids into bottle, and use a magnetic mixer with a stir bar to mix for Duration00:05:00

5m
Adjust pH while mixing to Ph5.7 using concentrated citric acid
Autoclave liquid at Temperature121 °C for Duration00:15:00
15m
In a LAF bench, add Amount100 mL sterile Dextrose solution
Add sterile water to a total of Amount500 mL
Note
Cool to 50°C and supplement with antibiotics as appropriate

Agar can be stored, then reheated to 50°C to be poured