Hestrin-Schramm (HS) medium

Andreas Sagen

Published: 2023-01-07 DOI: 10.17504/protocols.io.eq2ly7xyelx9/v1

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.

Before start

Prepare glassware by cleaning it, and ensure that scale is sufficiently calibrated

Steps

100 mL Dextrose solution

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

1.1.

Fill the bottle with 60mL double-distilled water

1.2.

Measure 10000mg Dextrose

Powdered compounds:

1.3.

Add powdered solids into bottle, and use a magnetic mixer with a stir bar to mix for 0h 5m 0s

1.4.

Adjust pH while mixing to 5.7 using concentrated sodium hydroxide

1.5.

Add distilled water to a total of 100mL

1.6.

Autoclave liquid at 121°C for 0h 15m 0s

500 mL HS (broth) medium

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

2.1.

Fill the bottle with 300mL double-distilled water

2.2.

Measure 2500mg Peptone, 2500mg Yeast extract, 1350mg Disodium phosphate, 750mg Citrate and 600mg Magnesium sulfate

Powdered compounds:

2.3.

Add powdered solids into bottle, and use a magnetic mixer with a stir bar to mix for 0h 5m 0s

2.4.

Adjust pH while mixing to 5.7 using concentrated citric acid

2.5.

Autoclave liquid at 121°C for 0h 15m 0s

2.6.

In a LAF bench, add 100mL sterile Dextrose solution

2.7.

Add sterile water to a total of 500mL

Note

Cool to 50°C and supplement with antibiotics as appropriate

500 mL HS (agar) medium

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

3.1.

Fill the bottle with 300mL double-distilled water

3.2.

Measure 2500mg Peptone, 2500mg Yeast extract, 1350mg Disodium phosphate, 750mg Citrate, 600mg Magnesium sulfate and 7500mg agar

Powdered compounds:

3.3.

Add powdered solids into bottle, and use a magnetic mixer with a stir bar to mix for 0h 5m 0s

3.4.

Adjust pH while mixing to 5.7 using concentrated citric acid

3.5.

Autoclave liquid at 121°C for 0h 15m 0s

3.6.

In a LAF bench, add 100mL sterile Dextrose solution

3.7.

Add sterile water to a total of 500mL

Note

Cool to 50°C and supplement with antibiotics as appropriateAgar can be stored, then reheated to 50°C to be poured

Hestrin-Schramm (HS) medium

Abstract

Before start

Steps

100 mL Dextrose solution

500 mL HS (broth) medium

500 mL HS (agar) medium

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