Great Lakes Worm Watch

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Multiple Plot Studies

  1. Introduction to Conducting a Multiple Plot Study
  2. Choosing a Site
  3. Sample Plots
  4. Multiple Plot Sampling Strategies
  5. Generating Multiple Geographic Coordinate
  6. Using a GPS Unit
  7. Using iTouchMap
  8. Sampling Earthworms
  9. Preservation
  10. Identification
  11. Sending your Findings to the Great Lakes Worm Watch
  12. soon!)
  13. Analysing Your Data

1. Introduction to Conducting a Multiple Plot Study

This guide follows the structure of the “Multiple Plot Study” data sheet, each of these sections on this page will provide you the information you need to collect all the required data and ultimately complete your study. We have also developed a study protocol which provides a checklist for the studies.

Download the Multiple Plot data sheet

Collecting earthworms from multiple (i.e. more than 3) plots requires a little more time than both single plot studies and document and occurrence style studies, and are therefore usually more appropriate for small groups of people or more dedicated individuals. This level of study requires you to document the occurrence of earthworms using the mustard sampling method for over MULTIPLE plot, collecting earthworms (if apparent) and provide a geographic coordinate of that site using a GPS unit or online mapping software. Once you have collected earthworms you have to preserve them in formalin and isopropyl alcohol place them in vials and then send this preserved earthworms to the GLWW.  Once you have collected earthworms you have to preserve them in formalin and isopropyl alcohol, placing them in vials and then send this preserved earthworms to the GLWW.  The equipment and chemicals required for preservation can be purchased from local pharmacists and food cooperatives, a full equipment list can be seen in the “Preservation” section of this page. This collection and preservation process is the only thing that differentiates the document an occurrence study and the single plot study.  From a our research perspective, collecting and sending your earthworm samples (in single and multi plot studies) is very useful as allows us to identify the species and determine the distribution of exact species.

Multiple Plot Studies are the must useful study style in terms of statistical analysis. It allows you to make more accurate generalizations about an area as a whole. Multiple samples lead to much better representations of the level earthworm invasion in your site. You can carry out multiple plots between habitats along a transect in order to calculate earthworm community change over a special scale e.g. between habitat types (more information on transects and other methods is covered in the sections below).

Depending on what you would like to get out of a GLWW study, you may be interested in the ways you can analyze the data you have collected. A vast majority of our GLWW contributors get involved as part of educational projects with specific analytical aims, so it may be important to understand how each study level affects what you can do with the data collected. It is important to remember that data analyses is only OPTIONAL, the GLWW does not require you to analyze you data in any form before you submit your data.  All additional information on how to analyze your data can be found in the ‘Earthworm Analysis’ section of the Great Lakes Worm Watch site or in the ‘Analyzing Your Data’ section at the bottom of this page.

Although preservation increases the complexity of a Multiples Plot Study once you have collected and preserved your earthworms it opens up opportunities to do multiple types of data analysis. As you have stored the earthworms you can calculate biomass by measuring earthworm length. Also, because you use mustard sampling you have a representative sample of all species of earthworms in your plot you can easily calculate earthworm density as a factor of the number of earthworms and your plot size. The most exciting aspect of analysis that is made available by preservation is the opportunity to identify the species in you sample site.

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2. Choosing a Site

The first thing you need to do is find a site where you would like to sample. A site is a particular habitat in which you would like to sample, such as a forest or field. Many people already have an area in mind, but natural ecosystems including forests, woodlands, and prairies are especially interesting to us at the Great Lakes Worm Watch. However, data from habitats dominated by human activity are also of value, such as farm fields, pastures, and parks. As you are using multiple sampling points you may want to cover more than one of these habitat types, this can be done by using different sampling strategies (see Multiple Plot Sampling Strategies).

Once you have a found an appropriate site you are ready to begin you study! It is useful to find out the name of your site for future studies, if your sample site does not have a given name (e.g. Superior National Forest, MN) you can always name it yourself with something relating to its surrounding features (e.g. Boulder Lake Forested Area, MN).

We always ask for a little contact information about the study coordinator this is only used by Great Lakes Worm Watch staff.

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3. Sample Plot

A sample plot is the section of soil in which you sample your earthworms inside of your site. Multiple Plot Studies look for earthworms in MULTIPLE sample plots, which allows it to calculate earthworm occurrence over a larger scale (using transects etc.).

You have complete control over the number of plots you want to use in your study; if you have a lot of time or people in your group you can do lots of points. If you think multiple plots may be too difficult you may want to look at the Single Plot Study or the Document an Occurrence study (which does not use plots).

All plot studies use a set area of ground for your sample in (both Multiple and Single Plot Studies). Sampling from an area of a particular size (33 x 33 cm plot) can allow you to work out the density of earthworms. These ‘fixed’ plots’ have a set area from which you sample and the most basic plot is a 33 x 33 cm square. Using a fixed plot makes is an essential if you are doing multiple plots, it makes it easier to analyze and replicate your plots and it allows you (and us) to calculate lots of interesting statistics like earthworm density!

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4. Multiple Plot Sampling Strategies

Generally, the goal of doing a study is to determine the average condition for a given site. As you cannot sample every square inch of your study site, using multiple sample plots is the best alternate available. You can always just use a arbitrary system i.e. you just pick points yourself but sampling strategies are often simple to use and are much more useful in higher level educational projects. Arbitrary plot locations (hey, this looks like a good spot!), and if you are doing a study to simply get a feeling for what is there, that can be fine (scientists call this “qualitative data”). But if you want use your data to answer a more specific question, you may want “quantitative data” that you can say with more confidence actually represents the conditions at your study site. To get “quantitative data” you would want to use some kind of sampling strategy. Two sampling strategies commonly used include locating your sample plots randomly or along transects.

Randomly located plots

Randomly located plots are your best bet if you are interested in determining the average condition for your study area. If you are simply looking around for a “good” place to put your sample plots, you will probably NOT place them in brushy, hard to get to locations. Many methods of randomization exist, Overlay Grids, Random Walks and Random Number Tables, they all are methods of placing sampling plots in order to make them representative of the habitat you’re working in. Basically they take the placing plots out of your decision and into the hands of mathematics; they are useful at limiting sample bias.

The image above shows a simple grid can be laid over a map of your site. The grid can have as many cells as you need to get adequate coverage of your site. Label the cells on two sides with numbers and then use a random number table or calculator to identify which cells you will sample. There are many other random sampling strategies you can use.

Read more about random sampling


Transects are simply a line that spans the gradient of interest, and then you locate your sample plots along this line. Transects with plots are your best bet if you have an obvious gradient in your habitat that you are interested in exploring, such as the transition from a lakeshore to a forest, or from the edge of a forest to the interior of the forest.

The length of your transect would be determined by the gradient you are sampling. You can place these plots in equal intervals across your transect (eg. Every 10 meters) or by choosing points along the line yourself.

random stratification

Read more about transect sampling

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5. Generating Multiple Geographic Coordinates

The First and probably most important piece of data you need to find out is the geographic coordinate of your site. This geographic coordinate allows us to place your findings on a map in order to document the distribution of earthworms. Providing accurate geographic coordinates is essential! It may sound extreme but earthworm sightings are basically useless without an accurate geographic coordinate. You only need to provide a single geographic coordinate for your site.

There are two methods that you can use to calculate the geographic coordinate of your site: a GPS unit or the mapping website iTouchMap (we have developed guides for using either). If you already have access to a GPS unit then use this to find your sites geographic coordinate. If you don’t have access to a handheld GPS unit don’t worry iTouchMap can easily produce coordinates for your site using a Google Maps style webpage.

There are different formats of writing longitude and latitude coordinates, for consistency we use the Decimal Degree format (i.e. 44.98127° latitude, -93.34479° longitude). The more numbers reported after the decimal point, the better your accuracy. We ask for at least 5 decimal places to give a reading with at least 50-100 meter accuracy. By convention, the latitude value is listed first, then the longitude value, separated by a comma.

There are several other commonly used and similar looking coordinate formats that may look similar (all numerical) but are actually very different. Just as 5 feet is very different to 5 meters, a coordinate given in Decimal Degrees-Seconds is very different than a coordinate in Decimal Degrees.

Read more about the coordinate systems, and the fundamentals mapping technology

Detailed instructions on how to locate map points

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6. Using a GPS Unit

If you have a handheld GPS unit you will need to be able to create a waypoint in decimal degree format, read the accuracy of your GPS unit and change the format settings. You will need to note the:

  • Geographic Coordinate - set ‘Coordinate Units’ to decimal degrees (hddd,ddddd°)
  • Accuracy reading - setDistance/speed’ to metric units
  • Datum - set Map DATUM to NAD83

You need to check your GPS unit and make sure each is set to their correct respective units. You can use the guide bellow to check and change your GPS settings.

How to format the options and settings of your GPS unit .

To find the geographic coordinate simply stand over your sample area whilst holding your GPS unit. The accuracy of your GPS unit is found on the map screen and is usually indicated quite clearly in a box entitled ‘ACURACY’ with an M for meters. This value should be written down on your data sheet before you calculate your waypoint (ideally below 5 meters for best reading).

Once this is noted down you can make a waypoint. This is most commonly done by pressing and hold the ‘Enter’ button on the map screenuntilanewwaypoint’screen appears. The ‘LOCATION’ box will contain the geographic location of your sample area (e.g. N 44.98127° W -93.34473°). You should write down this value but you can also save your waypoint coordinate information onto the internal memory of your GPS unit.

Guide for creating waypoints using a GPS unit

You can check the Datum of your GPS unit at any point by finding the ‘Units’ screen in your GPS internal settings and writing down the value in the‘Map Datum’ option.

Introduction to GPS and using GPS devices

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7. Using iTouchMap

If you do not have access to a handheld GPS unit you can use the mapping website iTouchMap. There are MANY other mapping websites but we have chosen to provide instruction on using iTouchMap because it is easy to find the latitude and longitude in the format we request (decimal degrees). You will need to record the:

  • Geographic coordinate - in the Get the Latitude and Longitude of a Point’ section
  • Datum - always WGS84 for iTouchMap

If you are experienced with using Google Maps, using iTouchMap should be very straightforward. You can find for the approximate location of your site by typing its address (or a surrounding landmarks) ‘Address’ search box and clicking the enter key. This magnifies and moves your map screen around to the area you searched. You can then pan to your site and zoom into the exact location of you individual sample area. If it is difficult to see you site try switching to ‘hybrid’ mode to see aerial images as well as labeled roads etc.

Once you have zoomed in as much as possible you can left click on your mouse to create a blue marker over you sample location. This will produce two values in the ‘Latitude’ and ‘Longitude’ box directly belowGet the Latitude and Longitude of a Point’ title, these are the geographic coordinates of your sample area, whichyou can write down onto your data sheet (and/or copy these into a word document) making sure you write them down to 5 decimal places. If you want to replace or move marker delete it by simply right clicking your mouse over the marker.

Don’t worry if you can’t exactly find your sample area from the aerial images, just give the coordinate to the best detail you can. Using iTouchMap is a good way to find a map coordinate but it allows large amounts of room for human error so is rarely as good as a GPS unit. If you want a more detailed guide to using iTouchMap we have developed a guide that follows as lesson like tutorial (see link below).

Click here for a tutorial on using iTouchMap.

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8. Sampling Earthworms

 The Multiple Plot Study uses mustard sampling as it is the most effective and comprehensive method of earthworm sampling. It allows the sampling of all earthworm species in the soil.

Liquid Mustard Extraction

Liquid Extraction

In this method you pour a solution of mustard water on the soil allowing it to percolate down. The mustard solution irritates the skin of earthworms and they come to the surface to avoid it, where they can be collected, preserved and identified. This technique works well for all species of earthworms but only when the earthworms are active. If it has been very dry, very hot or very cold in the week(s) prior to sampling they may not respond as very well since they may be in aestivation (earthworm version of hibernation). To make the solution, mix 4 liters (~1 gallon) of water with 40 grams (~1/3rd cup) ground yellow mustard seed. This is the same non organic yellow mustard you will find in the spice rack in any grocery store. In contrast, if air temperatures have been moderate and it has rained recently they are likely to be active and respond well to the liquid extraction. AN EXCEPTION – if the soil is very compacted and/or has a poor structure (heavy clay, fields, roads, etc.) the extractant doesn’t move well through the soil and the earthworms will not respond because the liquid doesn’t reach them. BUT, in most hardwood forests of our region the liquid extraction method works very well. Try it, it’s fun!


  1. Slowly pour about half of the jug of mustard solution over the area allowing it to soak in as much as possible. If worms are present and active they should begin coming up almost immediately. If they don’t come up immediately, wait 2 minutes or so and then pour again. A forceps is handy for picking the earthworms up off the surface.
  2. After a few minutes, and the initial flush of earthworms slows down, pour more of the solution over the sample area. You will probably get another flush of earthworms coming to the surface. Continue this until the whole gallon is used up (2 or 3 pourings). Deeper dwelling species take longer to surface than those close to the surface to don’t be too impatient. Total sample time is usually 5-10 minutes.
  3. As earthworms appear at the surface wait until they are completely out to the ground before grabbing them with forceps. If they are not completely out of the ground they may break. Place them into a collection tray with isopropyl alcohol for temporary preservation(we recommend Tupperware with a lid). You can always use gloves instead of forceps when collecting earthworms.

Read more about earthworm collection methods and their limitations.

You can also collect simple soil information this is not essential for us but may be useful or interesting for you to investigate. The data sheet has an optional section for habitat and soil data.

A given site (nature center, school forest, etc.) may have several different habitat types (hardwood forests, conifer forests, restored prairie, wetlands, etc.). In general, you want to report your data for each habitat separately so the data sheet includes a section to help you describe your habitat in a general but valuable manner, including habitat type, current and past disturbance, habitat size, surrounding land use and some basic soil features.

The level of impact of an earthworm invasion can, in part, be seen through the affects on the forest soils. When there are lots of earthworms the formerly thick forest floor (duff layer) composed of slowly decomposing leaves is eaten and mixed into the mineral soil below. So, for plot based surveys, we ask you to tell us how much exposed bare soil there is, some features of the forest floor, and to do a simple soil texture test. To read more about how, click here. If you want to explore other aspects of the forest soils, click here check out the soil sampling methods section.

Read more about collecting habitat and soil data.

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9. Preservation

The next step after collecting your earthworms is to preserve them for postage. By preserving earthworms you can save your specimens for later identification and analysis. Collection and preservation allows you to look at the earthworms in greater detail looking at there physical characteristics like color and size which allows you to work out things like species and biomass (see identification and analysis sections below). This does require you to have certain items of equipment.

You will need:

  • Isopropyl alcohol
  • Alcohol For immediate preservation
  • Leak Proof vial(s) For transportation
  • Formalin For final preservation
  • Gloves for handling earthworms and chemicals

1. Kill the earthworms in alcohol (isopropyl – rubbing alcohol works great)

  • Get as much dirt off the earthworms as possible. Put the earthworms in the alcohol one at a time (rather than a whole handful all at once) so they don’t get tangled up into a big mess of earthworms. After a few seconds, the earthworm will become anesthetized and relax into a mostly extended position.
  • Place the anesthetized earthworms into a vial. You can keep the earthworms in alcohol for up to 24 hours (keeping them cool and out of direct sunlight). But don’t wait to long, after a day or so in alcohol they start to get mushy.

2. Put the earthworms in Formalin (within 24 hours).

  • Place the earthworms in a leak proof vial and cover them in Formalin (a cellular fixative that will prepare the specimens for long-term storage).
  • Once the earthworms have been in formalin for at least 24 hours, they can be placed back into alcohol (70-100% isopropyl, NOT ethyl which bleaches any pigmentation) for long-term storage. This allows you to re-use the formalin over and over again.
  • CAUTION: Formalin is a dangerous chemical and safety precautions must be taken. It should be used only in a well ventilated area. It should not be breathed, swallowed or come in contact with bare skin or eyes. If external contact does occur wash the area with large amounts of water. If ingested contact a poison control center immediately. Formalin has been shown to cause cancer in laboratory animals.

Download the full preservation equipment list and protocol
Read more about earthworm preservation
Guides and shops to buying the equipment

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10. Identification

Identification is only OPTIONAL, if you are concerned that you do not have the time to identify the worms but would still like to know what species are in your site don’t worry, when you send your samples to the Great Lakes Worm Watch we will identify them for you and send can send you back a list of the species present! The identification process itself is an advanced process; requiring you need to look for the presence or absence of a certain key characteristics.

Identification can be done at different levels; simple identifications may only identifying juveniles and adults, oruse more advanced identification methods to identify exact genus or species. Once you have sorted these earthworms into juveniles, genus or species you can calculate species diversity as well as density and biomass values for each species and for all the earthworms community as a whole. As the GLWW checks all earthworm samples sent to us you can check your identification results against the GLWW results to see how effective you are at identifying worms!

Identification can be quite tricky for someone who is doing it for the first time and because of this we have created a whole section of the Great Lakes Worm Watch website to identification. Don’t be scared to take part in identification yourself, although this requires a bit more work it is very rewarding and provides you a much better understanding of earthworm biology. Lots of people have used our identification guide before, so don’t worry if you are interested you can check out the link below to read more about earthworm identification.

Read about earthworm identification

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11. Sending your Findings to the Great Lakes Worm Watch

You can ether send you printed data sheet vial mail or by emailing a copy of the digital data sheet to the Great Lakes Worm Watch (

Mail and packing instructions.

Label, pack and ship all sample vials (with location information!) to:
Great Lakes Worm Watch
Natural Resources Research Institute, University of Minnesota,
5013 Miller Trunk Hwy,
Duluth, MN  55811

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12. is a website that acts as a base for many citizen science projects around the US. We are currently altering the structure of the Great Lakes Worm Watch CitSci page to improve its usability. Check back soon! 

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13. Analysing Your Data

Using multiple plots over your sample site indicates the earthworm community in the wider area (e.g. on a habitat scale). The more pints you do the more accurate description you can give of your whole site, if you are interested in sampling a large area you may want to use more plots e.g. 25 plots may not be able describe the earthworm distribution in an area of 100 meters squared than 3 would! Therefore, if you want to find information earthworm occurrence on a larger scale you will need to use a multiple plot study.

More information on earthworm analysis.

If you have collected earthworms from a set area and have list of earthworm species you can carry out many statistical tests. Both Single and Multiple Plot Studies require the collection of earthworms. When you send your earthworms to the Great Lakes Worm Watch our scientists indentify the species in your sample(s) for you. You can use our species list to check how effective you are at identifying earthworms! If this identification aspect sounds enjoyable to you, you may want to look at conducting a Single and Multiple plot study. Mustard sampling on the other hand provides a fully representative sample of the earthworm species in the soil, which is why it is the only earthworm sampling method used in Single and Multiple plot studies.
Other simple modifications to your study like using a set sampling area (or plot) can drastically increase your data analysis opportunities. Counting the number of earthworms alongside a set plot area allows you to calculate earthworm density. If you have collected and identified worms you can then use this plot size to work out and compare the densities of a particular species.

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