The following are four simple techniques for capturing bacteria and protists from marine and freshwater environments.

Uhlig Extraction

The spaces between sand grains and other sediments can harbor abundant protists and bacteria. Protists, especially ciliates, can be extracted from their interstitial environment for observation by the following technique. This modified and simplified "Uhlig ciliate sandy sediment separator" is primarily for the extraction of ciliates from marine and estuarine fine grain wet sandy sediments.

Materials needed: Two plastic cups that can be cut with scissors or a razor blade, Nytex nylon mesh or other loosely woven fabric or mesh, small petri plate in which bottom of cup can fit, standard size petri plate.

1. Take two plastic cups of the same size and remove the bottom with scissors or a razor blade. In one cup, cut two small (approx. 5mm in size) triangle-shaped notches into the bottom edge, directly opposite each other.
2. Place a square of Nytex nylon mesh, or other loosely woven fabric or mesh, tightly over the bottom opening of the unnotched cup. Place this cup and the mesh into the other plastic cup.
3. Place a moist sand sample within the inner cup. Place the whole plastic cup assembly into the bottom of a small petri plate. Add water from the sample site into the bottom of the petri dish so that the water is in complete contact with the bottom of the nylon. Add more of the same water to the upper surface of the sand sample until the inner cup is full. If using this method, an overflow for the water will be needed. Place a larger (standard) size petri plate under the smaller petri plate. The other half of the standard size petri dish can be used to cover the inner cup so as to prevent dehydration.
4. Extraction sampling is accomplished by carefully removing the small petri dish and observing under a dissecting microscope. Individual ciliates can be removed with a micropipette and placed on a microscope slide with coverslip for high powered observation (this requires some patience and skill). Another petri dish can be placed under the device and more water poured through the sediment (or the overflow water can be poured through the sample again.

• Sand samples from various depths can be sampled by slicing cored samples and placing each into its own Uhlig extractor.
• Ciliates are generally positively geotropic and will migrate out of the sand at different rates of speed and at different times. Therefore it is best to sample the separator at time intervals (e.g. 6, 12, 24, and 36 hours). The method works well for freshwater, brackish, and marine silicate and carbonate sandy sediments.
• Ciliates will also move in reaction to salinity and temperature gradients. Another method of extracting organisms is to place frozen sea water ice cubes onto the surface of the sediment. As they melt, the ciliates will move out of the sediment and into the water below.

Coverslip Traps

Many organisms, including bacteria, ciliates and diatoms will colonize bare surfaces in an aqueous environment. By providing a substrate one can encourage the formation of these biofilms. Coverslips are a suitable substrate for colonization, and can easily be observed under the microscope.

1. Take a 4" piece of thick walled rubber tubing and slice four slits across the tubing, taking care not to cut completely through the wall of the tubing. The slits will be perpendicular to the long axis of the tubing.
2. You will need eight coverslips. Place two coverslips, back to back, into each of the four slits. so that the tubing holds them in place.
3. Tie string through the tube and attach fishing weights to keep the device from floating.
4. Suspend the trap by tying it to a stick, rock or post in a suitable location such as a pond, estuary or marine environment. Be sure that the string or monofilament is long enough so that the trap is not exposed at low tide.
5. After at least 24-36 hours, the traps can be removed. Place the whole device (tubing and coverslips) into a covered jar or ziploc bag containing water from the environment the traps were in. Be sure the coverslips are submerged; do not let them dry out at any time.
6. To observe the organisms on the coverslips, gently remove a pair of coverslips and separate them. Place one of them onto a microscope slide so that the surface that was on the inside of the pair is facing up. If the coverslip starts to dry out, place a drop of water between the coverslip and the microscope slide. Observe first under low power and then move up to higher power. Make drawings of the different species you may see and the numbers of each in a given area of the coverslip.

• The longer the traps stay in the water, the more they will be colonized. Diatoms and attaching (sessile) ciliates are the most common protists that will colonize the glass surface. Experiments can be set up to sample and compare organisms from different environments. To study the patterns of colonization from a single environment, traps can be removed and observed at different times over the course of many days or weeks.

Plankton Nets

Plankton refers to organisms that spend all or some of their lives living at the water surface. Viruses, bacteria, protists, fungi and animals ranging in size from nanometers to several centimeters can all be found in the plankton of fresh or marine waters. An easy way to sample planktonic organisms is with a variety of inexpensive, easy to assemble nets. There are many ways to make a plankton net (e.g. see Microcosmos Curriculum Guide, Kendall Hunt Publishers). Here is another variation that we will try.

Materials needed:

• Nylon stocking or knee high hose
• Strong twine
• Thin rope (e.g. clothesline)
• Duct tape
• Small can (e.g. tuna can, coffee can, cat food can) or
• strong plastic cup that can be cut with a razor blade
• Light weight jar (e.g. spice bottle) or
• disposable plastic test tube (50 ml size)
• Strong rubber band

1. Find a can small enough to fit inside the rim of the nylon stocking or knee high being used. Cut off both ends of the can with a can opener (or cut off bottom end of plastic cup). Make sure edges are smooth. Put the stocking through the can and fold the edge of the stocking over the outside rim.
2. Take three equal lengths of string, approximately 12 inches long, gather in a bundle and tie with a single knot at one end. Spread the three pieces over the top of the can so they are equidistant from each other and hang below the folded edge of the stocking. The knot should be above the center of the can opening.
3. Tightly wrap duct tape all around the can so that the strings and stocking are held securely in place. Enough string should hang below the bottom edge of the duct tape so that each can be flipped up and tied to itself with a small knot at the top of the duct tape.
4. Drop an uncovered, 50 ml plastic test tube into the toe of the stocking. Secure this in place with a rubber band or tightly tied piece of string.
5. A long tow rope (clothesline at least one yard long) can be attached to the knot holding the three strings together at the top center of the plankton net.

This net can be used to sample the surface of the water, simply by slowly pulling it along the top of the water, or it can be dropped to a certain depth in the water (from a pier or boat for example) and dragged vertically upward to sample the water column.

As water washes through the stocking, organisms will be caught in the mesh and washed down into the bottom tube. To insure that all organisms are washed off the sides of the net, a squirt bottle full of water from the environment can be used to wash organisms into the bottom test tube.

To transport organisms back to the lab, empty the concentrated sample from the test tube into a slightly larger jar, add water from the environment sampled, and cover. The temperature of the sample should not be allowed to change very much. Covered jars can be kept in a large bucket and covered with water from the environment to keep the temperature from changing too much.

Samples can be observed under a dissecting scope, or pipetted onto a microscope slide and observed under high power.

Styrofoam Traps

The tiny spaces in styrofoam peanuts and blocks and polyurethane foam can also be colonized by microorganisms and can be used to fish bacteria and protists out of marine and freshwater environments.

Gather several pieces of string or monofilament fishing line (approximately eight inches long) together in a bundle and tie in a single knot at one end. To the end of each piece of string except one, tie a styrofoam peanut or a small piece of soft foam. Add a fishing weight to last piece of string and add a long string or fishing line leader to the top of the tied bundle. This array of traps can be tied to a pole and suspended in the water, or tied to a stone or brick and allowed to sink to the bottom of the water being sampled. Be sure that the foam is not exposed at low tide. The length of string leading to each foam piece should be short so that the foam does not float above the water surface.

As with the coverslip traps, the longer they are allowed to sit, the more different organisms will be observed. Allow traps to sit at least 24 hours before removing. Collect traps in the water the are suspended in and place into a ziploc bag or covered jar. To observe organisms, simply squeeze the styrofoam or polyurethane foam pieces into a dish. Observe dishes with a dissecting scope or pipette a drop of water onto a microscope slide and cover with a coverslip to observe under the light microscope.