An additional anaerobic layer, this time of unsupplemented mud, brings the container to two thirds full. Alternatively, some procedures call for sand to be used for the layer above the enriched sediment as to allow for easier observation and sampling of resulting populations.
An additional anaerobic section, composed of unsupplemented mud, raises the container's level to two-thirds full. Furthermore, some protocols specify using sand for the upper layer to enhance observation and sampling of resulting populations.