E31 Medium

This medium is made up in 2 parts to avoid precipitation:

Soil extract (SE1)50.0 ml
KNO30.10 g
K2HPO40.01 g
MgSO4·7H200.01 g
Cyanocobalamin100.00 ng
Thiamine HCl50.00 g
Biotin100.0 ng
Distilled water to500.0 ml

Filtered natural seawater 500.0 ml

Autoclave Parts 1 and 2 separately at 15 psi, allow to cool then mix aseptically.

Soil Extract (SE1):
Site selection for a good soil is very important and for most purposes a soil from undisturbed deciduous woodland is best. Sites to avoid are those showing obvious signs of man's activity and particular care should be taken to avoid areas where fertilizers, crop sprays or other toxic chemicals may have been used.

A rich loam with good crumb structure should be sought. Stones, roots and larger invertebrates should be removed during an initial sieving through a 1 cm mesh. The sieved soil should be spread to air dry and hand picked for smaller invertebrates and roots. It should be turned periodically and picked over again. When dry it may be sieved through a finer mesh (2-4 mm) or stored as it is prior to use.

Air-dried soil and twice its volume of supernatant distilled water are autoclaved together at 15 psi for 2 hours and left to cool. The supernatant is then decanted and filtered through Whatman No. 1 filter paper, then distributed to containers in volumes suitable for making up batches of media. The aliquots and their containers are autoclaved for an appropriate length of time (eg., 1 L or less for 15 minutes) and are then kept in a cool place (eg., a refrigerator) until required.

1:1 mixture of
E31 and ANT media.

Autoclave separately. Mix aseptically when cool.

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