Plant specimens, air dried in the field and averaging 700 GMS, are first finely ground into a powder resembling sawdust using a variety of mills including bandsaw, hammermill or knife mill.   The powder is added to a 10cm diameter borosilicate column and covered with dichloromethane/methanol (1:1) and allowed to steep overnight at room temperature inside a chemical fume hood.   The solvent is drained, and the column filled with 100% methanol. After 30 minutes the methanol is drained into the same flask and the combined organic solvent is concentrated by rotary evaporation, then transferred into a 120ml borosilicate bottle for final high vacuum drying and permanent storage.   To the extracted plant material in the column high purity water is added, and this is allowed to steep overnight.  We have determined that this solution contains typically 5 to 8% methanol, which improves extractability of molecules into the water.  The aqueous extract is transferred into a borosilicate dish, frozen and lyophilized. The dry powder is transferred into a 120 ml borosilicate bottle for long term freezer storage.  Though highly variable, we have observed that it is not unusual for 10% of the weight of a specimen is organic solvent soluble and an additional 10% is water soluble.

Marine specimens, most of which come from the South Pacific were quickly frozen on board the ship and are air freighted to Frederick on dry ice, then at -20 deg until processed .   Frozen specimens are broken into chunks with a hammer and chisel, then mixed with dry ice.   We have found that most marine specimens, when chilled to dry ice temperature, become brittle, and so can be finely ground using a Hobart hamburger grinder, just like a grocery store uses.  Since grinding is done while frozen, the rapid decomposition and odor often encountered with marine organisms is avoided.   After the dry ice sublimes, (3 days storage at the -20 deg.) the finely ground sample is mixed with high purity water, mechanically stirred to create a uniform aqueous slurry, and dumped into a basket rotor in a centrifuge, where particulates are trapped on filter paper, and the water soluble molecules pass through and are collected in a flask.   Both aqueous soluble and particulate portions are lyophilized.  The dry particulates are then weighed and extracted with dichloromethane/methanol (1:1) , followed by methanol, to produce an organic extract.

More than 7,000 fungal cultures have been acquired and are in liquid nitrogen storage.  Thus far, about 3,500 cultures have been grown in a variety of media.  Fungal ferments consisting of broth and cells, typically 800 MLS, are homogenized after the addition of 10% V/V methanol, then, in a separatory funnel, an equal volume of dichloromethane is added.   After agitation and separation of the organic layer, the cell debris is removed by filtration and the broth is lyophilized, thus giving both organic soluble and water soluble extracts for screening.   Extracts of fungi selected for regrow at a larger scale, up to 80 L, are also prepared.

If you'd like to ask a question via email to Tom, Chris, Shoshanna, or Matthew just click on the center of their picture.  Thank you!

Return: http://npsg.ncifcrf.gov/