ANAEROBIC MEDIUM Preparation

For culturing anaerobic microorganisms, you have to maintain Oxygen-free conditions during cultivation. The main things that you have to keep in mind are: • Make all the solutions in anaerobic water • Put overpressure in the head space of your cultures, flushing with N2/CO2 (replacing the oxygen) • Check your medium protocol, how to prepare it.

Anaerobic Water Boil 1 liter of demi water for 20-30 minutes in an erlenmeyer flask Close with a loose lid, (e.g. aluminium foil) Cool to room temperature flushing with N2 or 90%N2:10%CO2

Sterile bottles (for culturing): Sterilize serum bottles (covered with aluminium foil) and septum stoppers in a beaker for 20 min 120°C

Prepare stock solutions Prepare all the stock solutions with anaerobic water, flush the solutions before sterilization with N2 or 90%N2:10%CO2 . • Non-sterilisable components Solve the components in anaerobic water, filter sterilize it into a sterile anaerobic bottle sealed with a sterile cap and flush the headspace through a filter for 15 min. • Sterilisable components Solve the components in a serum bottle, cap it and flush with gas, then autoclave the solutions.

Media preparation Mix all the growth media ingredients, except heat-sensitive materials (e.g., vitamins), precipitate-causing materials (e.g., bicarbonate), and the reducing agents (e.g., sulfide and cysteine hydrochloride) in distilled water, adjust the pH to the desired level, and dispense this mixture into the culture vessels (at 10 ml medium/20 ml vessel). If the pH is affected by autoclaving or by the subsequent addition of reagents, the pH should be adjusted later. Resazurin, a blue dye, is included in the media (1 mg/L) to monitor the redox potential of the media. On reduction, its color changes from blue to pink and from pink to colorless

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ANAEROBIC MEDIUM Preparation

Oxygen scavenger: Sodium sulphide or L-cysteine

Na2S x 9 H2O (3%, w/v), 100 ml

Filter-sterilise into a sterile anaerobic bottle Use gloves and be careful!!! Work in fume hood. Use Na2S x 9 H2O (3%, w/v) 10ml / liter After adding the sulfide solution, the medium becomes colorless after a while. If the liquid is pink it means that: - there are traces of oxygen in the bottle or - indicates wrong pH

Use sulphide solution only with batch cultures, don´t put into the chemostat!!! Sulphide can cause corrosion in the fermentor!!

Reductant compound for continuous culture use L-Cysteine: 0.4g/l final concentration

0.8 M L-cysteine (9.7 g L-Cysteine in 100 ml anaerobic water)

Flush to remove traces of oxygen Autoclave Keep at room temperature, will otherwise precipitate. Use L-Cysteine (0.8 M) 4 ml / liter medium

Resazurin solution

Solve 500 mg/liter resazurin (7-Hydroxy-3H-phenoxazin-3-one 10-oxide) is a blue dye (oxygen indicator) You can prepare 50ml and store it in a Falcon tube at 4oC Use 1ml / liter growth medium

Culture in serum bottles:

• Use bottles which are suitable for your experiment. • Dispense the medium (frequently we use 20ml or 50ml) into 100 ml bottle (leave at least 10% headspace). • Flush the medium in the serum bottles with 90%N2/10%CO2 for 5 minutes (to remove possible oxygen during the transfer)

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ANAEROBIC MEDIUM Preparation

Caps: butyl rubber normally (grey and soft ones) plus aluminum crimp caps, but if the experiment is with PCE or Benzene use the hard one (Viton) as they are not permeable or sorb. • After closing the bottle Flush again with 90%N2/10%CO2 ( overpressure 0.5 atm)

Complete the media: Working sterile: work near flame, connect filter (if it is needed) and needle next to it, use cotton with 97% of ethanol to flame the stopper. Let first cool down to room temperature after autoclaving the medium, this is important before adding bicarbonate and the vitamins.

Complete the medium using syringes and continuous N2 flushing to keep anaerobic conditions. Add vitamins (sterilized by filtration), sulfide (from sterile, anoxic stock solution) in case of batch cultures (use cysteine for chemostat, see above) and bicarbonate (from a sterile, anoxic stock solution prepared under 90%N2 and 10% CO2)

Sodium bicarbonate (1 M) stock solution for anaerobic cultures: 4.2 g NaHCO3 in 50 ml Prepare as above; flush with N2:CO2 (90:10) Filter sterilize, keep at room temperature. Use 1.5 ml / 50 ml (final conc. 30 mM)

Sodium carbonate (Na2CO3) solution (1 M) 5.25 g Na2CO3 in 50 ml Prepare as above; flush with N2:CO2 (90:10) Filter sterilize, keep at room temperature. Use 1.5 ml / 50 ml (final conc. 30 mM)

- Inoculum: Add 5% of inoculum in your medium (1ml in ~20 ml of medium).

- Incubation: Incubate at 37 ºC usually it takes 2-5 days to get a fully-grown culture depending the substrates.

More information in these papers:

Establishing anaerobic hydrocarbon-degrading enrichment cultures of microorganisms under strictly anoxic conditions. Nature Protocols volume 13, pages 1310–1330 (2018) https://www.nature.com/articles/nprot.2018.030?platform=oscar&draft=collection

A Simple Preparation of Liquid Media for the Cultivation of StrictAnaerobes https://www.omicsonline.org/open-access/a-simple-preparation-of-liquid-media-for-the-cultivation- of-strict-anaerobes-2157-7463.S3-001.php?aid=2806

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