US Long-Term Ecological Research Network
Jar Preparation
Label 4-ounce jars with computer-printed labels for the Hypsometrically Pooled and Wisconsin Net samples. Add the sample date to the label using a soft pencil. Cover the label with a strip of clear contact paper, making sure the contact paper completely covers the label and encircles the jar. Jar labels should contain the following information:
Lake Name         Station #                              Lake Name         Station #
Day  Month         YYYY                                 Day  Month         YYYY
Hypsometrically pooled: surf.-bottom            Wisconsin Net 80um mesh
2-m, 45 L. Schindler Patalas Trap                  Vertical Tow from        Meters
53um mesh,   Preserv: 95%EtOH                  Preservative: 95% EtOH       
Weigh the empty jars, including lids, recording these weights on the 'Volume by Weight' data form. 
Fill the Schindler field collection and Wisconsin Net jars with 80 ml cold 95% EtOH, making sure that the Schindler collection jars have the correct lids.  Leave the HP jar empty. Keep preservative-filled jars cold until the zooplankton samples are collected.
Sample Collection: Schindler-Patalas Trap
For LTER lakes use the 2-meter high, 45L Schindler-Patalas trap with 53um mesh net and cup. The volume of the trap used should be indicated on the Volume by Weight form.
Collect samples from the target depths at the deep sampling station in each lake. Sample depths are measured from the middle of the trap.
            Target Depths:
TR: 1, 3, 5, 7, 9, 15, 20, 27, 31 meters
CR/BM: 1, 3, 5, 7, 9, 11, 13, 15, 18 meters
SP: 1, 3, 5, 7, 9, 11, 13, 15, 17 meters
AL/TB: 1, 3, 6 meters
CB: 1 meter
Take samples starting at the surface and working down. Lower the trap slowly so that it remains vertical in the water. Pause at the target depth long enough to allow both trap doors to close completely, and check when it reaches the surface that both did close. Drain the trap through the net and cup, swirling the cup until the liquid level is below the mesh windows. Remove the cup from the net and pull out the center pin to drain the sample into jar, then rinse cup and pin several times with 95% EtOH into the sample jar.
Sample Collection: Wisconsin Net
Lower the net to the bottom sample depth. Pull it up slowly, at a rate of about 3 seconds per meter. A slow haul prevents the net from pushing water and plankton away from the mouth of the net. Drain the cup until the water level is below the lower window, then pour contents into the sample jar. Rinse the cup with 95% EtOH several times, adding the rinse to the sample jar.


Hypsometric Pooling
Rationale and Definition
In March 1986 the LTER Zooplankton Group decided to pool the discrete depth Schindler Patalas trap samples into one pooled sample per lake-date for counting. Counting pooled samples rather than all of the depth samples reduces the time to produce zooplankton count data. The group hoped to count pooled samples from the entire backlog of uncounted samples and eventually to count samples shortly after collection.
Samples are pooled considering lake hypsometry and, therefore, represent the entire lake. Previously, unpooled samples (2-9 samples per lake-date) or samples pooled considering only a water column were counted. Hypsometric pooling allows us to consider the zooplankton community as representing the entire lake, as our other limnological methods do, instead of just a column of water.
Lake hypsometry is a three dimensional image of a lake or basin. In a simplified example of hypsometry, a lake is similar to a cone filled with water. If the cone were divided into three layers by two equidistant horizontal planes, the volumes in those layers would be very different from each other. The uppermost layer would contain the most water. Similarly, the upper depths typically contain most of the volume of a lake.
Pooling is the creation of a new sample from subsamples of the Schindler trap samples collected from one lake-date. The volume of each subsample used to make the pooled sample reflects the depth range the sample represents and the volume of water that range represents relative to the entire lake volume. Samples pooled in this manner are called HP samples, for “hypsometrically pooled”, and are referred to as “volume weighted” because the volume of lake water each depth sample represents determines the subsample size.
In sum, the advantages to this method of pooling are quicker turnover time and representation of the entire lake in a volume weighted fashion. Disadvantages of this method include the time required to pool subsamples, errors introduced during pooling, and the loss of more specific depth information.
Pooling Procedure
Allow the sample jars to air dry for a day or two. Weigh the Wisconsin Net sample and record the weight on the Volume by Weight form. Mark the liquid level on the jar with a ‘Sharpee’ brand permanent marker.
Add 95% EtOH to each Schindler trap sample to bring liquid volume up to a weight of 105g, measured by weighing the sample jar with lid on the balance. If sample jars already contain more than 105g of liquid, allow some of the volume to evaporate in the hood, and then bring up to 105g.    Record the final weight of jar + sample + EtOH on the Volume by Weight form.
Calculate the subsample volumes, called ‘target’ volumes, using the hypsometric table for each lake. Record these volumes on the Volume by Weight form.
Mix the first sample gently and thoroughly by tilting the jar from side to side. Measure the target volume into a plastic graduated cylinder. Pour the subsample quickly and smoothly because the plankton settle out quite rapidly. Choose the smallest size graduated cylinder that can measure the target volume in one aliquot. Add the subsample to the labeled HP jar. Repeat with all other depth samples. When all of the subsamples have been added to the HP sample, rinse each graduated cylinder into the HP jar with several small volumes of EtOH.
Place the HP sample in the hood to evaporate the excess volume of EtOH. The final weight of the HP sample should be 105g. Mark the liquid level on the jar with a Sharpee brand permanent marker. If the samples are from the August quarterly, pour the remainder of each Schindler sample into a labeled jar for archival. For all other sample dates, discard what is left of the Schindler samples. Rinse and air dry the field sample jars.
Sample Storage and Record Keeping
Store samples in cardboard records boxes obtained from UW Stores, storing samples from each lake in a separate box. Approximately one year of samples will fit in one box. Fill out the forms for each sample and sample box, as noted below.
Box Inventory Form:   A record of box contents. It remains in the sample storage box.
Volume by Weight Form:   A record of samples collected for any one lake-date and their volumes, storage box number, and history of sample usage. Filed in 3-ring binders, one copy at the Zoology Museum and one copy with Corinna Gries.
Samples Stored Form:   A record of all samples collected and storage box number for each. Current forms are kept in a binder at Trout Lake; archived forms are kept in the UW Zoology Museum. The data are eventually entered into the electronic LTER Museum Catalog.
When boxes become full, check the contents against the Inventory Form and Samples Stored Form, and transfer them to the sample storage room in the garage. LTER samples and related paperwork are eventually transferred to the Zoology Museum at UW-Madison.
Zooplankton Counting
Before removing a subsample from any zooplankton sample jar, weigh the sample to check for evaporation. If the weight is within 0.1 gram of the last weight recorded on the Volume by Weight sheet, no fluid replacement is necessary. If the weight is more than 0.1 gram low, add 95% EtOH to the sample to bring it up to the correct volume.
Mix the sample well by turning the jar on its side and tipping back and forth gently. We use a Hensen-Stempel pipet with a 5-ml plunger for subsampling zooplankton samples. After mixing the sample, take the subsample as quickly as possible to avoid biasing the subsample as organisms begin to sink. There should be no air bubbles inside the Hensen-Stempel pipet. If there are, replace the subsample into the jar, completely dry the pipet, and begin again with the mixing. When you have a bubble-free subsample, dry the outside of the pipet and dispense the subsample into a cup with 53µ mesh bottom. Rinse the pipet into the cup with RO water, and continue rinsing the sample in the cup, washing the ethanol out of the sample through the mesh.  Rinse the subsample into the counting tray with RO water, washing the mesh thoroughly to transfer all organisms into the tray.
After removing subsample(s) from the jar, weigh the sample jar, and record this weight in a new column of the Volume by Weight form. Record the balance used, your initials, and the date at the top of the column, and add a column header such as 'Column C minus subsamples removed for counting'. Do not put the subsample back into the sample jar after counting. Mark the new liquid level on the jar with a permanent marker. Replace the sample jar into the proper storage box.
Count copepods and cladocerans first, identifying individuals to species wherever possible, and staging all copepodids. Measure a subset of each species.  Then add a few drops of Lugol's solution to the subsample to stain it, and count the rotifers and nauplii. Count two subsamples for copepods and cladocerans. Count one subsample for rotifers and nauplii. If there are less than 100 of the dominant rotifer in one subsample, count a second subsample for rotifers and nauplii. Add milli-RO water to the tray as necessary to keep the surface of the subsample level. If the surface becomes concave as the subsample evaporates, it is difficult to focus clearly, and measurements may become distorted.
Count all eggs attached to any species. For copepods and cladocerans, keep track of the number of individuals with eggs as well as the total number of eggs. Total number of eggs is sufficient for rotifers.
Measurements are done as follows: Measure copepods from the tip of the head to the end of the urosome, excluding the caudal rami. Measure cladocerans from the tip of the head to the posterior of the carapace, excluding tailspine. However, measure helmeted Daphnia species from the anterior edge of the eye to the posterior of the carapace.
Rotifers are not routinely measured, but where they have been, the total body length excluding spines is measured.  Body width rather than length is measured for Conochilus, Conochiloides, and Collotheca.
Describe, measure, and draw any unknown species on a separate sheet of paper. If possible, take a photograph of the unknown.
LTER Keywords
Protocol Format
Protocol ID
Protocol Type