The preservation and study of microinvertebrates, specifically specimens of the phylum Tardigrada, has been stifled by the lack of an adequate and attainable microscope slide mounting medium. In the past, Hoyer's medium has been used to fix specimens and make slides. However, it is difficult to obtain some of the ingredients in this solution e.g. chloral hydrate. It is especially difficult for individuals and high schools to purchase these chemical compounds. In order to get high school students involved in studying these microinvertebrates, a safer and easier method was needed.

Collection:
Members of the Phylum Tardigrada may be found among several different kinds of habitat. Mosses, leaf litter, and lichens are the most common places to investigate. Simply collect these kinds of material, dry them and then store in paper bags until it is convenient to begin dissecting the samples. Searching for specimens among lichens and other substrates is accomplished by soaking the material in distilled water contained in glass containers approximately 2 inches in depth by 4 inches in diameter (pic 1). After allowing samples to soak for 8 to 24 hours the glass containers can be examined with an observation scope (pic 2). It may be necessary to remove larger substrate pieces so the bottom of the container can be observed unobstructed. Most of the specimens will settle to the bottom of the jar and can be removed with a small eye dropper while viewing through the 30 or 40 power observation scope (pic 3). Tardigrades often attach themselves to micro flora and become difficult to persuade from the bottom of the jar. If that happens, carefully dislodge the critter with an Irwin loop or small sharp dissecting tool. [Previously published method using micro-pipette or Irwin Loop]

Preservation (Optional):
The first step in preserving microinvertebrates is asphyxiating the organism without shrinking, distorting, or otherwise altering the overall morphology. After pulling the specimen and a tiny quantity of distilled water into the dropper place the contents of the dropper into a glass container of FAA solution. The organisms should immediately die, relax, and extend body parts well. FAA solution is a mixture of formalin, isopropyl alcohol, glacial acetic acid, and water. It can be purchased from Wards Natural Science, Rochester, NY. Leave the specimen in the solution overnight. Specimens can be left in the solution for longer periods of time without damage. Glass containers that can be placed under an observation scope are essential in order to find the specimen so it can be moved from subsequent solution to solution. To reduce evaporation, small glass plates can be used to cover the containers. Observations can be made through the glass covers and the covers also reduce exposure to the unpleasant vapors.

To prepare specimens for mounting they will be passed through a series of alcohol solutions and finally xylene and then piccolyte. Move the specimens first from the FAA into 90% alcohol (isopropyl) solution. This can be accomplished by using a small glass pipette or small eyedropper. A small amount of FAA will be transferred into the alcohol solution at this time and does not adversely affect the process. If at anytime during this process the specimens shrink or distort adversely, they can be placed back into FAA which will usually distend the organism. It is also possible to rehydrate the organism by lacing in clean water and then going to FAA again.

After specimens have been in the 90% alcohol for at least an hour they can be moved into a 95% alcohol solution. Specimens should remain in this solution for at least one hour also. Next transfer the specimens to a 99.5% alcohol solution. Once again the specimens should be left in this solution for at least an hour. Specimens can be left in these solutions for longer periods of time if need be.

The next solution specimens should be transferred to is 50% alcohol and 50% xylene. The final solution specimens should be transferred to is 100% xylene. After at least an hour the specimens can be transferred to a microscope slide. Place a drop of Piccolyte where you want the specimen to be located. A 40% Piccolyte/60% Xylene solution also available from Ward's and other scientific supply companies is an acceptable mounting medium. Using a small glass pipette or glass eyedropper place the specimen on the slide near the piccolyte. A small quantity of xylene will mix with the piccolyte on the slide as you tease the organism into the correct postion. This is best achieved with the use of an Irwin loop or a small, sharp dissecting probe and an observation scope (pic 4). Glass utensils must be used in moving organisms due to the fact that these organic solvents will dissolve most types of plastic pipettes.

Once the specimen is centered in the drop of piccolyte, a cover slip can be placed over the drop. Slides should be allowed to dry laying flat for several days. They can be observed under a microscope immediately. Care should be taken to avoid putting pressure on the cover slip for several days. Piccolyte tends to remain semi-solid and often never hardens completely, therefore care should be taken to store slides flat. Often the specimens tend to look better after being stored for several days. After a week, clear fingernail polish maybe used to seal the edges of the cover slip. This helps seal the mounting medium and will sustain the specimens for later examination.

Identification and Reporting:
After tradigrades have been extracted from the substrate, use a 40 power (or better) microscrope to make visual identifications. The following resources will help to better identify genera and species.

1. Use the PathFinder Science key to identify the genus of the animal.
   1. PFS Key to Genus
2. A few texts are available for assisting with species identification, including:
• The Biology of Tardigrades by Ian M. Kinchin
Reporting:
1. Record the substrate type (tree lichen, moss, etc.) you are collecting your tardigrades from, such as tree lichen, rock lichen, moss, water, etc.
2. Collect the latitude and longitude for each sample and record in the table below [Available Options for lat/long].
3. Record the Tardigrade Genus and species if available.
4. The students schould upload the recorded data through the Data Submission Area. This could include one or more digital photos of the tardigrade.

   The table below may be helpful for recording data prior to submitting it at PathFinder Science.

   School Name and Number
   Sample Date
   Identifying Sample number
   Substrate sample collected from
   Latitude
   Longitude
   Tardigrade Genus
   Tradigrade Species if possible



Additional help:
A Quicktime Tardigrade video introducing tardigrades and basic sampling techniques is online. This is a large file so anticipate a long download time.