COLLECTION PROCESSING

Many biomedical teams travel around the globe to collect samples from a variety of environments. In shallow habitats (0-50 m) they wade, snorkel, or scuba dive. In the open ocean, teams use towed nets and samplers deployed from research vessels.

Some research teams have access to more specialized tools. Florida's Harbor Branch Oceanographic Institution, for example, has regular access to the deep sea via the institution's two Johnson Sea-Link (JSL) manned submersibles, which allow direct observation and sampling from previously inaccessible domains. The submersibles are equipped with multi-function manipulator arms that can grab, scoop, or slurp up specimens as well as still and digital video cameras and a data recorder that logs depth, temperature and salinity throughout a dive.

Teams collect a diverse array of marine organisms for analysis of the potential biological and pharmaceutical activity of their natural products. Typically sessile organisms such as sponges, coral, and tunicates are collected as well as algae and in some cases sediment samples. All samples will, of course, also include an array of microorganisms such as bacteria growing in and on them.

Most Harbor Branch collections are conducted from a ship such as the Research Vessel Seward Johnson. Other groups may conduct scuba operations from small boats or in remote areas of the South Pacific and elsewhere from chartered live-aboard dive boats.

The following article is based on methods and protocols used by the Harbor Branch Oceanographic Institution's Biomedical Marine Research group. Similar techniques are used by most groups that collect samples for marine natural products research.

The well-choreographed shipboard sample processing system involves a number of scientists working together to ensure that materials are properly prepared and available for a variety of analyses.

First, samples are sorted and then given labels with information about when and where they were collected. Once samples have been properly identified, subsampling begins. This information is correlated in logs with all available data regarding the sample, including the temperature and salinity at the location where it was found, physical descriptions of the location and the sample itself, and the weather conditions at the time of collection.

Photos

All samples are photographed intact along with their identification label to create an easily accessible permanent record before samples are broken up during processing.

Vouchers

Vouchers of each sample are frozen and eventually stored in the Harbor Branch museum collection for later taxonomic work to identify a species. A small voucher is stored in a vial, while a larger voucher is usually placed in a plastic bag. Scientists typically complete taxonomy using the small voucher, which is the most easily accessible, with the large voucher available if identification is not possible using that sample.

Extracts

Except in the case of very small samples, pieces of each specimen are blended into what some refer to as a "sponge daiquiri." To make the daiquiri, specimen pieces are first chopped and mixed with ethanol or other solvent using a laboratory grinder. The grinding breaks up cells and suspends a sample's chemical products within the solvent. The resulting solvent-chemical mixture is filtered to remove solid pieces and yield a crude extract, so named because it contains a crude mix of all the chemicals in the original sample with no refinement. Limited work may be done with the crude extracts onboard the ship, but for the most part they are stored for later use in biological activity screening assays on land.

HPLC

A similar extract is made using ethyl acetate and then analyzed onboard the ship using a chomatographic technique called High Pressure Liquid Chromatography (HPLC). Individual chemical compounds within an extract will produce signature peaks on the instrument that can be compared to signatures from known compounds to determine if a given sample has produced something not yet seen. When that occurs, scientists may decide to try and collect more of the sample to ensure they will have plenty of material to work with to determine any biomedical potential of the new compound.

Microbial Analysis

Some of the bioactive natural products ultimately isolated from marine invertebrates are actually produced by microorganisms such as bacteria associated with them, meaning that the microorganisms themselves have great potential biomedical value. For this reason, the scientists maintain samples of the microorganisms they are able to culture from collected invertebrates. They also work to culture microorganisms from marine sediment samples.

This process typically involves mixing sediment or ground up bits of invertebrate sample in seawater and then diluting this mixture. Small portions of this seawater, which then includes a mix of all the microorganisms found in the sample, are spread on sterile Petri dishes with growth media such as brain-heart infusion agar, an enticing (to microorganisms, if not humans) blend of cow parts, which promote growth of microorganism colonies. More recently, the Harbor Branch team has also been performing this procedure and then storing plates in oxygen-free containers to examine anaerobic microorganisms, which can be killed by oxygen and so will not grow in open air. Anaerobic species have been studied far less than have aerobic ones and so may represent a nearly untapped source of important compounds.

Scientists incubate all the dishes, each of which may produce colonies of several different organisms, for periods from days to months. Back in laboratories on land, colonies of individual species are isolated to determine whether they produce chemicals with pharmaceutical potential. For later microbial studies, bits of sample are also frozen in seawater with glycerol added as a preservative that prevents ice crystal damage to the cells.

DNA Analysis

Small pieces of each sample are cut up and added to vials with a commercial preservative known as 'RNA Later' and stored for later DNA analyses.

The Rest

All sample material remaining after the above subsamples have been taken is bagged and frozen at -80°C so that it will be available for any additional work that may be needed.