Avoid significant or sudden changes in temperature. Retain some fluid that can be filtered as used as trhe basis of a medium. Keep a record of where you sampled. Typical sampling gear:

• buckets
• 50 - 500 ml wide-mouthed flasks / jars with tightly sealing caps.
• sealable plastic bags
• sealable plastic containers - such as sandwich boxes
• forceps
• scissors
• trowel
• corers
• tape - for containers
• indelible pens - to write on tape
• notebook

All sediments and organically ernriched sites tend to become anoxic with depth. In anoxic regions (also referred to as reduced), metabolic pathways tend to involve the production of reduced sulphur or m,ethane, and where the reduced region has an interface with overlying oxygen rich environment, oxidation of reduced sulphur compounds may take place.

Anoxic sites with significant amounts of reduced sulphur compounds tend to be black - because of metal sulphides - and smell of rotten eggs.

Anoxic sites in which methane production occurs tends to release bubbles of methane.

Most areas will have microhabitats that are anoxic or reduced and so any habitat can be treated as an anoxic site.

Intertidal sediment (mangroves). The upper 2 millimetres or so are light colored. Deeper regions are grey or black. The dark regions anoxic. Image and copyright D. J. Patterson.

Anoxic sites contain organisms that are obligate anaerobes and may die if exposed to oxygen. Most species that occur in anoxic sites seem to be facultative anaerobes, and can tolerate some of extended exposure to oxygen. When collecting, the precautionary principle is not to allow a free air space or air bubbles into the sample.

Collect samples from regions which either are grey or black and/or smell of rotten eggs

Put into a sealable container, fill to the brim with fluid from the habitat, and seal tightly with no air space above.

Remember to collect some site water for use as a basemedium in early cultures.

Cultures can be grown in serum vials (20-100 mls) with boiled or autoclaved site-water, one to four boiled wheat grains and a small quantity of the redox indicator Resazurine . Medium is added to the vials, flushed with oxygen-free nitrogen gas, the bottles closed with silicon stoppers and sealed with crimp caps. The headspace can aso be flushed by pushing two syringe needles through the silicon stopper, and forcing nitrogen gas through one. The vessels are allowed to stand until the Resazurin changes color to pink - or clear (which will happen because the microbial activity will cause anoxia to develop). . Cultures are best inoculated after the color change and can be inoculated by syringe needles. Observations are also carried out only resazurine decolouration indicates the onset of anoxia. A mixed bacterial flora was present in all cultures.