US Long-Term Ecological Research Network
A. Nutrient Sampling: Refer to the Field Sheet to see which bottles need to be sampled at which depths and the 'Southern Lakes LTER Bottle Codes’ for preservation, filtering, and coding information.
1.     Purge the lines: Whenever sampling from a new depth, the peristaltic pump tubing must be purged of the water from the previous depth. After reaching the proper sampling depth, use a graduated cylinder to measure the volume of water purged before beginning the sampling. Purge at least 1200 mL of water for each 20 meters of tubing. Start sampling from the lake surface and work towards the bottom.
2.     Arrange the sample bottles: All bottles from a particular depth should have been placed in a cooler in the same bag. Be sure to pay attention to filtered and unfiltered bottles.
3.     Rinse each sample bottle 3 times: After purging the tubing, add a small amount of the sampling water to a sample bottle, cap the bottle, shake the bottle to coat the inside with the sampling water, and discard the water. Repeat 2 more times.
4.     Fill the the large pH and the H sample bottles up to the neck. Fill the unfiltered S bottle with 100 mL of sample. The air-excluded unfiltered samples should now be filled, capped, inverted, and tapped to see if any air bubbles rise in the bottle. If there is air in a sample, fill it again until no air exists in the bottle. The air excluded samples are more easily filled at a lower pump speed. All samples should be placed in a cooler with ice.
5.     For sample bottles which need to be filled with filtered water, first insert a nutrient filter holder onto the end of the pump tubing. Turn the pump to its lowest setting. Purge a new filter by letting a small volume of sample water run through the filter before adding water to a bottle. After rinsing a filtered sample bottles 3 times, fill it to the proper level (see step 4). Switch to a new nutrient filter when the one in use becomes clogged. Also switch filters when starting a new depth and when starting the blind depth.
6.     Before proceeding to the next depth, double check the field sheet to make sure that all nutrient samples (and discrete depth chlorophyll) at a particular depth have been sampled.
B. Chlorophyll Sampling - Discrete Depth: All chlorophyll sampling is done in the field
1.       Discrete depth chlorophyll should be sampled directly before or after the nutrients from the same depth are sampled.
2.       Calculate the correct volume to filter based on the secchi reading, and on each specific lake. A chart is kept in the metal field clipboard. Discrete depth chlorophyll is analyzed fluorometrically (more sensitive than the spectrophotometer) and the filter should only be slightly discolored (otherwise dilution of the sample is required during analysis). DO NOT check the filter color in the field (risk of chlorophyll degradation).
3.     Pump through the chlorophyll filter at a moderate speed into a graduated cylinder. Connect one end of the pump tubing to the sampling hose and the other to the filter cartridge. Pump designated amount of water through the filter into a graduated cylinder. Record the filter cartridge number and the volume filtered onto the field sheet. Place the filter cartridge into the same cold cooler as the other samples.
C. Collecting water for integrated samples: The water should be collected shortly before it is filtered
1.     0 to 2 meter integrated samples are collected for chlorophyll, TPM, and phytoplankton (except Lake Mendota). 0 to 8 meter integrated samples are collected for chlorophyll on Lakes Mendota and Monona, and phytoplankton on Lake Mendota
2.     Rinse the large jugs (covered with duct tape) 3 times with surface water. Attach the line (also used for secchi depth measurement) onto the integrated depth sampling tube.   Unwind enough line to lower the tube to the desired depth.  Place the tube in the water and lower it to the desired depth. If the tube is lowered 8 meters, step on the end of the tube to prevent water from flowing out. If the tube is only lowered 2 meters, it is not necessary to plug the other end of the tube.
3.     Raise the sampling tube quickly out of the water by pulling up the line. It may be necessary to raise the tube in the boat simultaneously to prevent water from running further into the tube. 
4.     Place the end of the tube coming out of the water into the sampling bottle. Raise the tube to drain all of the water from the tube into the bottle.  
5.       Repeat until the bottle is full.   Cap the bottle and avoid exposing the sample to light as much as possible to avoid chlorophyll degradation.
D. Sampling for Phytoplankton
1.     Rinse the phytoplankton bottles out once with surface water. Gently shake the jug containing 0 to 2 meter integrated sample and fill the phytoplankton bottles up to the neck (Lake Mendota phytoplankton samples are taken with 0 to 8 meter water).
E.   Sampling Integrated Depth Chlorophyll
1.     Connect the short, weighted tube to the pump and insert it into the large duct taped sampling bottle containing 0-2 or 0-8 meter water. Place the cover on the bottle to avoid chlorophyll degradation.
2.     To sample spectrophotometric chlorophyll, gently shake the sample jug (to mix up the water) and turn the pump to a low speed until water starts to come out the end of the tube. Turn off the pump. Connect the filter cartridge to the end of the tube and turn the pump to a low speed. Filter the water into a graduated cylinder until designated amount of water is filtered (a chart is kept in the metal field clipboard). Record the filter holder number and volume filtered and place the holder in a cooler.  
3.     To sample fluorometric chlorophyll, determine the volume to be filtered based on the secchi reading, and specific lake (a chart is kept in the metal field clipboard). Perform the filtering as described above remembering to periodically shake the sample bottle to mix up the sample water. Record the filter holder number and volume filtered and place the holder in a cooler.  
F.   Sampling TPM (Total Particulate Matter)
1.     To sample TPM, filter the TPM sample from the 0 to 2 meter integrated water sample after the chlorophyll has been sampled.   Gently shake the sample jug (to mix up the water) and turn the pump to a low speed until water starts to come out the end of the tube. Turn off the pump. Connect the filter cartridge to the end of the tube and turn the pump to a low speed. The water should be filtered into a graduated cylinder until there are only drops of water coming out of the filter holder (generally between 50 and 500 mL is filtered depending on the lake and date).   Record the filter cartridge number and volume filtered, and place the filter holder in a cooler.  
G. Reading Temperature and Dissolved Oxygen 
1.     Before leaving to sample a lake, check to make sure that there are no air bubbles under the probe membrane of the YSI Temperature/Dissolved Oxygen meter. If there are air bubbles or if it has been several months since changing the membrane (or if the instrument does not calibrate well or the oxygen readings wander), change the membrane as explained in the manual. Note: We have always used the Standard membranes. If adding water to new membrane fluid bottle (KCl), make sure to add Milli-Q water and not the CFL distilled water.  
2.     Be sure to always store the probe in 100% humidity surrounded by a wet sponge or paper towel.
3.     Turn on the temperature/dissolved oxygen meter at least 30 minutes before using it. It is best to turn it on before leaving to sample a lake as it uses up batteries slowly.              
4.     Calibrate the meter using the chart on the back of the instrument (adjusted to the Madison altitude - 97% oxygen saturation).    Leave the plastic cap on the probe (at 100% humidity). The temperature should not be changing during the calibration. Zero the instrument. When the temperature equilibrates, adjust the oxygen to correspond to the chart.   After calibrating the instrument, switch the knob to % oxygen saturation to make sure it is close to 97%.
5.     Take readings at 1 meter intervals making sure to gently jiggle the cord when taking the oxygen readings (to avoid oxygen depletion). Jiggling the cord is not necessary if using a cable with a stirrer.   Take half meter readings in the metalimnion (when temperature and/or oxygen readings exhibit a greater change with depth). A change of temperature greater than 1°C warrants half-meter intervals.
6.     Record the bottom depth using the markings on the temp./oxygen meter cord and take a temperature and dissolved oxygen reading with the probe lying on the lake bottom. Don’t forget to jiggle the probe to remove any sediment.
7.     If any readings seem suspicious, check them again when bringing the probe back up to the surface. You can also double check the calibration after bringing the probe out of the water (and putting the cap back on).


H.  Taking the zooplankton samples: Each zooplankton sample bottle is filled with one tow from the specified depth. Tows are taken with a Wisconsin net which has 80 um mesh, is 1 meter long and has a 30 cm diameter ring.


1.     Without attaching the zooplankton cup, rinse the net in the lake water. This is especially important if another lake was sampled earlier in the day (without rinsing the net with tap water).

2.     Unravel enough line to get to the desired depth (note that it is occasionally necessary to untwist the line while unraveling it as it tends to get badly twisted)
3.     Screw on the zooplankton cup and gently lower the net into the water. Allow the net to sink freely to the desired depth by throwing line into the water.
4.       After feeling the weight of the net at the desired depth, bring up the net at a constant rate of 1 foot per second (or 1 meter per 3 seconds). It is useful to silently count out 3 seconds between meter measurements.
5.       Before removing the zooplankton cup, allow the water to drain from the net and cup until the cup is less than half full of water. Unscrew the cup and continue to twirl it until there is only a small volume of water left.
6.       Pour the sample from the zooplankton cup into the sample bottle. Using a squirt bottle containing ethanol, rinse all of the visible zooplankton inside of the zooplankton cup into the sample bottle.
7.       Fill the remaining volume of the sample bottle with 95% ethanol.
8.       Repeat the above steps if the field sheet lists a second zooplankton tow (to be put into a different sample bottle).
I.    Taking the Secchi Depth
1.       Attach the rope to the secchi disc and move to the side of the boat that has the most shade (the direction of your shadow).
2.       Kneel down so that your head is hanging over the side of the boat (kneel on a boat cushion).
3.       Slowly lower the secchi disc until it is no longer visible. Mark the depth with your fingers. Raise the secchi until it becomes visible again. Also mark that depth. The average of those two depths is the reading to record (they should be within approximately 0.1 meters of each other depending on the secchi depth). Record the reading to 2 significant figures (e.g. 1.1 meters, 0.88 meters).
J.    Measuring Air Temperature, Wind Speed, Wind Direction, % Cloud Cover, and Wave Height
1.       Estimate air temperature and check forecast before and after going into the field.
2.       Estimate the wind speed. Record a range if the wind is variable (e.g. 10-15 mph).
3.       Face the direction that the wind is coming from. Use lake maps to determine which direction the wind is flowing.
4.       Estimate and record the % cloud cover.  
5.       Estimate and record the wave height in cm (record the vertical distance from wave crest to the wave trough). 

K.   Record Comments
1.       Comments which should be recorded are those which directly or indirectly affect the samples or measurements taken. Examples of appropriate comments are ‘heavy rain’ or ‘dissolved oxygen readings at 5 through 8 meters double checked on the way up’.

L.   Look Over the Field Sheet
1.     Make sure that all of the parameters on the field sheet have been completed and recorded (including Time Off Station)


Protocol ID