- Water wash bottle - Deionized
- Ethanol wash bottle
- Zooplankton wheel and stand
- Zooplankton cup with mesh
- 110 ounce zooplankton sample cup
- Small Spatula
- Henson Stemple
- Metal probe
- Empty sample bottle
- Zooplankton sample to be counted
Take the entire zooplankton sample and pour it, ethanol and all, through the mesh cup with the empty sample bottle below to catch and save the ethanol. Once all the ethanol is drained from the mesh cup, use the water wash bottle to clean the zooplankton and to gather them on side of the mesh cup. Gently tip the mesh cup upside down and use the water to rinse the entire sample into the empty 110 ounce sample cup. After the zooplankton are in the 110 ounce cup you may have to fill the sample with deionized water to either 50 or 100 ml, depending on the density of zooplankton in the sample. At this point it may be advantageous to add a little soap to the sample with the metal probe in order to break some of the surface tension.
Mix the sample with the small spatula to ensure that the zooplankton are evenly dispersed throughout the 110ml container. As quickly as possible, to make certain that the zooplankton do not settle out, take the Henson Stemple, push the plunger down, and submerge the end into the sample. Allow the spring to compress without the end of the stemple leaving the sample. You now have a 1ml sample of zooplankton from the original sample. Next, take the empty zooplankton wheel and place the end of the stemple into the wheel’s groove. Slowly open the stemple and allow the 1ml sample to fill the groove. If there are zooplankton on the head of the stemple use a small amount of deionized water to wash them off and add a little more water to the wheel, if necessary, in order to spread the sample evenly throughout the groove.
Finally use a small amount of soap on the metal probe to break the surface tension on the sample in the wheel. When you are done counting a subsample return it to the main sample and start this process over for your second and third subsamples. When you are finished with a sample, dump the sample back through the mesh container and gather the zooplankton to one side of the container. Using the ethanol wash bottle, get the zooplankton back to their original sample container making sure to get them all. Top the container off with the ethanol that was originally in the sample.
Microscope and Leica Program
Turn the microscope light on by flipping the switch in the back left of the microscope. Make sure the microscope is set on 1.6 magnification. Place the zooplankton wheel on the base plate and use the fine adjust knob toward the back of the scope until the zooplankton come into focus. You may need to adjust the light knob and lever on the left side of the scope until the proper amount of light is emitted. Next, double click on the Leica Application Suite V3.0.0 icon on the desktop. Once the application is done loading, click on the Mic1 tab, and set the zoom drive (manual) to 16.0 x. Next click the acquire tab on the upper left of the screen, click on the camera sub-tab, and then open the calibration settings beneath that. Make sure the type is calculated, and the configuration is set on LTER. The Actual length of line shown on image should now be close to 6.0mm. If it is not, click on one side of the measuring line in the window and drag it until it equals 6mm. Minimize this window then work with the zoopomatic program.
Double click on the Zoopomatic3 icon on the desktop and wait for it to load. Once the program is loaded, place the Z3, Measurement Progress, and Data windows on the left side of the screen and the Controls window on the right side. After moving all of these windows, bring the Leica application back up. The zoopomatic program windows should stay in front of the leica application, so both programs should be visible. Next take the video overlay and adjust it so that the entire overlay is perfectly placed over the Leica screen. After the video overlay is placed, left click on the left end of the 6mm line (as close as you can to the end) and then left click on the right end of the line. There should be a red line that overlaps the 6mm line and it should be the same length as the original 6mm line. When this is complete type 6.0 in the length section of the Controls window for the zoopomatic program and then click calibrate.
After all windows are placed in the correct spot and everything is calibrated, look under File in the Z3 window and click on New Workspace. The program will ask you if you want to use the same workspace and click yes. In the next window make sure Full taxonomy data and Report types are checked under the optional section then click ok. Next a create new workspace window will appear. You will want to name the workspace something different from the previous (for example: Z3 LTER 1,2,3….) After you have named your workspace click save and your workspace will be saved and opened. Next, click on Data on the Z3 window and click on New Data Set. Once the data set window opens up it should be blank. Right click in this empty window and click on New Data Set. After you click this, a new window should pop up entitled New Container. The first thing you want to set up here is a sample, so make sure Sample is selected under Type and click ok. The next window that pops up should be entitled Edit. Fill in the Name of the sample (ex. Mendota 3/10/2009,) and the volume of the sample (either 50 or 100) then click ok. The name of you sample should now pop up in the Select Data Set window.
Next, right click on the new sample you just created and click on New Data Set. This time when the New Container window pops up, you should select subsample under Type, and the sample you just created should be listed under Parent, click ok. When the Edit window pops up, name the subsample under the Value column (ex. ME 3/10/09 #1) and enter 1 for the SubSampleVolume under the Value column and then click ok. Now the Select Data Set window should have the sample you created listed on it with a plus sign by it. Click this plus sign and the subsample you created will drop down below the sample. Click on the name of the new subsample and then click ok. You are now ready to count the first of three subsamples for this sample. After you count this first subsample, you should start again at the beginning of this paragraph for subsamples 2 and 3.
Each of the species listed under the Daphnia, Diaphanasoma, Sinobosmina, Chydorus, Leptodora, Aglodioptomis, and Bythotrephes must be measured and counted for the first 30 individuals, and then only counted for the rest of the sample. The species under Dioptomus, Diacyclops, Acanthocyclops, Mesocyclops, Tropocyclops, and Nauplii only need to be counted for the first 1/3 of the sample and never measured. Copepodites species need to be measured for the first 30 and then counted thereafter for only the first 1/3 of the sample.
After calibrating the Leica program and setting up the sample and subsample you are ready to begin counting. Before you start counting make sure the image of the zooplankton is visible on the computer screen. If it is not, make sure the knob on the upper left of the microscope is set to DOC not VIS.If you need to get a better look at the sample when you are counting you can switch the scope to VIS which will allow you to see the sample through both eye pieces. If the scope is set to DOC you will only be able to see the sample through one eye piece. Throughout the counting process you can adjust the magnification of the microscope if you need to look more closely at and individual zooplankton but make sure the major adjustment knob is placed back at 1.6 before a measurement is taken.
At this point all windows for the Zoopomatic program and the Leica program should be visible. The zoopomatic Controls window on the right side of the screen will have the names of all the Genus’ of zooplankton to be counted. Make sure that you click the plus signs in front of each of the Genus’ in order to expand its whole list of species. Once the whole list is expanded and you are ready to count your subsample, click on the Start Counting button on the Controls window of the Zoopomatic program. As you start counting the sample and begin to measure zooplankton, measure each individual from tail (end of body) to head. In order to do this select (click on) the species you wish to count from the Controls window on the Zoopomatic program. Next, right click at the tail of the individual you wish to count and then left click at its’ head. After you left click to finish measuring the individual, the species should pop up in the Measurement Progress window, and the species name and length of that individual should show up in the Data window. You should do this for all of the individual species that need to be counted. Each individual species listed on the Controls window have a letter in parentheses listed next to them (ex. Dioptomus spp. (W),) this is called a hot key. For the individuals that do not need to be measured, you can click this hot key and the individual will automatically be counted. After you are done counting the subsample be sure to click the Stop Counting button on the Controls window in the Zoopomatic program.
After you have counted all three subsamples under you primary sample you will need to export the data into a folder on the computer. In order to do this look under the file tab on the Z3 window in the Zoopomatic program and click on Export Measurements. Name the file (ex. Mendota 3-10-2009) and then save it into the folder you created to hold you data. (You cannot save dates using forward slashes and must you dashes instead.) When you have exported your data you can close the program and it will save the last step you took. After you are done with a sample you will want to create a new workspace and start the whole procedure over again.