TITLE: Hestrin-Schramm (HS) medium
AUTHORS: Andreas Sagen

[DESCRIPTION]
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

[GUIDELINES]
Follow step by step, unless stated otherwise. Equipment needed should be standard to a microbiology lab.

[STEPS]
SECTION: 100 mL Dextrose solution
1. 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
SECTION: 100 mL Dextrose solution
1.1. Fill the bottle with 60 mL double-distilled water
SECTION: 100 mL Dextrose solution
1.2. Measure 10000 mg Dextrose

Powdered compounds:
  
SECTION: 100 mL Dextrose solution
1.3. Add powdered solids into bottle, and use a magnetic mixer with a stir bar to mix for 5 min

SECTION: 100 mL Dextrose solution
1.4. Adjust pH while mixing to pH 5.7 using concentrated sodium hydroxide
SECTION: 100 mL Dextrose solution
1.5. Add distilled water to a total of 100 mL

SECTION: 100 mL Dextrose solution
1.6. Autoclave liquid at 121 °C for 15 min
SECTION: 500 mL HS (broth) medium
2. 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
SECTION: 500 mL HS (broth) medium
2.1. Fill the bottle with 300 mL double-distilled water
SECTION: 500 mL HS (broth) medium
2.2. Measure 2500 mg Peptone, 2500 mg Yeast extract, 1350 mg Disodium phosphate, 750 mg Citrate and 600 mg Magnesium sulfate

Powdered compounds:
 
 
 
 
 
SECTION: 500 mL HS (broth) medium
2.3. Add powdered solids into bottle, and use a magnetic mixer with a stir bar to mix for 5 min
SECTION: 500 mL HS (broth) medium
2.4. Adjust pH while mixing to pH 5.7 using concentrated citric acid
SECTION: 500 mL HS (broth) medium
2.5. Autoclave liquid at 121 °C for 15 min
SECTION: 500 mL HS (broth) medium
2.6. In a LAF bench, add 100 mL sterile Dextrose solution
SECTION: 500 mL HS (broth) medium
2.7. Add sterile water to a total of 500 mL
 


SECTION: 500 mL HS (agar) medium
3. 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
SECTION: 500 mL HS (agar) medium
3.1. Fill the bottle with 300 mL double-distilled water
SECTION: 500 mL HS (agar) medium
3.2. 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:
 
 
 
 
 
 
SECTION: 500 mL HS (agar) medium
3.3. Add powdered solids into bottle, and use a magnetic mixer with a stir bar to mix for 5 min

SECTION: 500 mL HS (agar) medium
3.4. Adjust pH while mixing to pH 5.7 using concentrated citric acid
SECTION: 500 mL HS (agar) medium
3.5. Autoclave liquid at 121 °C for 15 min
SECTION: 500 mL HS (agar) medium
3.6. In a LAF bench, add 100 mL sterile Dextrose solution
SECTION: 500 mL HS (agar) medium
3.7. Add sterile water to a total of 500 mL