Presentation on theme: "Kavitha Govindasamy Jimmy R.Rich Maria L .Mendes"— Presentation transcript:
1Kavitha Govindasamy Jimmy R.Rich Maria L .Mendes Nematode Extraction Methods: Baermann Funnel and Modified Baermann TechniquesKavitha GovindasamyJimmy R.RichMaria L .MendesRespectively, Formerly Graduate Assistant, Professor, and Senior Biological Scientist, Department of Entomology and Nematology, University of Florida, Gainesville, FL 32611, U.S.A.
2A Plant-Parasitic Nematode Nematodes are very small, worm-like animals that may damage plants but are often difficult or impossible to detect with the unaided eye." The word "nematode" when literally translated means "thread-like." Other names commonly used include "roundworm," "eelworm," or "nema.“ Plant parasitic nematodes cause economic damage to all annual and perennial crops grown for food as well as landscape ornamentals and turfgrass.For more information on plant nematodes, go to: and
3Sampling for Nematodes Sampling and extracting of nematodes serves two purposesDiagnosis a current problemPredict a future problemNematodes cannot be detected or quantified without being extracted from the soil and identified under a microscope.
4Objectives of sampling and extraction Nematode identificationPopulation estimationDiagnose a nematode diseaseMake management decisionsRegulatory purposesResearch and surveysNematodes cannot be detected or quantified without being extracted from the soil and identified under a microscope.
5Some Common Nematode Extraction Methods Soil samplesBaermann funnelModified Baermann funnelRoot SamplesJar incubation and sievingBlending and sievingFoliar samplesPunching and incubatingScissors techniqueOther extraction methods are also available and used including the common ‘Centrifugation-Sugar Flotation’ methods.
6Soil Extraction Techniques: Baermann Funnel PrincipleExtraction based on the active movement of the nematodes (motility dependent method)The Baermann funnel technique is used to extract live nematodes from soil or plant tissue samples. Nematodes move out of the soil or plant tissue into the water, then fall to the bottom of the funnel from where they are collected.
7Materials required Funnel Funnel stand Wire mesh Tissue paper/ coffee filterRubber or plastic tubeClamp or spring clipBeaker or vialPlastic drink cup coverA variety of materials may be used: Funnels can be glass or polyethylene. The wire mesh used to support the soil or plant tissue on the filter paper is usually handmade. The rubber or plastic tubes needs to be flexible enough for clamps to securely close to prevent water leakage.
8Baermann Funnel set up Funnel Soil sample over wire mesh Clamp to hold funnelRubber tubingThe original Baermann funnel technique used a funnel which is connected to a rubber or plastic tube. The other end of the tube is placed inside a beaker to collect the nematode sample.ClampBeaker
9Begin Baermann funnel setup A wire mesh is placed into the glass or plastic funnel. Attach a piece of rubber tube to the funnel stem to prevent leakage of water.Close the end of tube with the clamp. Place funnel in the ring stand or similar apparatus for support.Adjust the height of the ring on stand so the tubing hangs free.
10Add the soil sample to the coffee filter or tissue paper Most coffee filters, coarse mesh laboratory filter paper or even facial tissue can be used. The quantity of soil used can vary but 50 cm3 is an appropriate amount to place in the 10 cm diameter funnels illustrated in this presentation.
11Place the sample on the funnel The funnel is filled with water to a level that is slightly over the wire mesh prior to placing the tissue paper containing soil on the wire mesh. Air pockets may form in the funnel stem or under the tissue paper and these must be dislodged. The tissue paper is used to prevent the movement of the soil particles into the funnel.
12Cover the tissue over the soil Make sure that the wire mesh touches the water in the funnel. Add more water to the funnel until the soil in the tissue paper is barely submerged. Ensure that the rubber tubing clamps are tight and do not allow leakage. Also, fold the paper over the soil to help prevent ‘wicking’ of water from the funnel.
13Incubate for 48 hours and collect the nematodes Addition of soft drink covers slows water evaporation and should reduce the need to add water to the funnels during the hour extraction period. Nematodes leave the soil, pass through the tissue paper, accumulate at the constriction of the tube created by the clamp.The clamp is loosened slightly to allow a few milliliters of solution to pass into a container, leaving a clean solution to view under a microscope.
14Collect nematodes in a beaker After hours, nematodes are present near the clamp at the bottom of the tube. The nematodes are concentrated in a few ml of water and may be collected in a small container for direct viewing. Do not collect too much water or the sample will become cloudy from the soil above.
15Advantages and Limitations Recovery of clean, live samplesSieves not neededInexpensive to constructLimitationsNot effective for inactive nematodesRecovery percentages may be lowInactive nematodes such ring nematodes are rarely collected with this method.
16Modified Baermann Funnel Baermann trays (Whitehead & Hemming’s modification, 1964) PrincipleExtraction based on the active migration of the nematodes (motility dependent method)The original Baermann Funnel Method used funnels and funnel holders that requires extra space in the laboratory. The Modified Baermann Method uses the same nematode extraction principles but materials that can be stacked and more easily handled than funnels.
17Materials required Pan or tray Wire mesh Tissue paper, coffee filter Squeeze bottle500 mesh sieveThe wire mesh shown above is actually two rings of PVC pipe with a window screen material (hardware cloth) glued between them.
18Add water to the dishAdd water to the dish until it slightly covers the wire mesh. The dish should be placed on a level surface for even nematode extraction.
19Place soil on tissue paper Most coffee filters, coarse mesh laboratory filter paper or even facial tissue will work here. Shown here is 100 cm3 soil placed in a double fold facial tissue paper.
20Place tissue paper on the wire mesh The tissue paper is superimposed on a wire mesh in the pan.
21Fold the tissue over soil, add water The tissue paper is folded over the soil to make sure that the soil sample doesn’t leak or over flow into the dish through the tissue paper.Allow a few minutes and add more water if needed but not directly on the soil.
22Completed technique, ready for incubation A cover is needed for completion of the technique. The cover prevents water evaporation and also allows stacking the dishes to conserve space.
23Incubate at room temperature for 3 days Pans can be arranged one above the other and incubated at 21-24ºC for 3 days. This allows enough time for the nematodes to migrate from the wire mesh to the water in the dish.
24Following incubation, remove wire mesh from the dish Slowly and gently remove wire mesh from the dish. The filter or tissue paper may break releasing soil particles into the water.
25Pour water into sieveThe nematodes are collected in the water in the pan. Pour the water into a 325 or finer mesh sieve to collect the nematodes.
26Rinse nematodes from the sieve The nematodes are collected in a tube for observation.
27Advantages and Limitations Recovery of clean, live samplesInexpensive to constructMaterials readily availableLimitationsFine soil particles cloud sampleLack of aeration can be a problemSieves are needed to concentrate nematodes
28AcknowledgmentsJanete Brito, Department of Plant Industry, Gainesville, FloridaFrank Woods, Nematode Assay Lab, University of Florida, Gainesville, FloridaJoey Orajay, Entomology and Nematology Department, University of Florida, Gainesville, FL
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