Laboratory Standard Sample Sorting Procedures
The general procedures we follow for processing samples are similar to those
described by the USGS NAWQA program (Cuffney, T.F., Gurtz, M.E. and Meador,
M.R.. 1993. Methods for collecting benthic invertebrate samples as part of
the National Water-Quality Assessment Program. United States Geological Survey
Open-File Report 93-406 and Moulton, S.R. II, J.L. Carter, S.A. Grotheer,
T.F. Cufney and T.M. Short. 2000. Methods of analysis by the U.S. Geological
Survey National Water Quality Laboratory - processing, taxonomy, and quality
control of benthic macroinvertebrate samples. United States Geological Survey
Open-File Report 00-212). Our procedures were also described in Vinson
and Hawkins (1996. Effects of sampling area and subsampling procedures on
comparisons of taxa richness among streams. Journal of the North American
Benthological Society 15:393-400).
The following is a step-by-step description of how we typically process quantitative benthic macroinvertebrate samples:
1) Pour the preservative from the sample
through an appropriate sized 500 micron sieve 
into the laboratory waste container. 
2) If the sample contains a lot of sand and gravel
you will need to separte the organic matter from these inorganic particles.
To do this, pour the entire sample from the sieve into a bucket. 
Partially fill the bucket with water. Swirl the bucket so that the organisms
and organic matter become suspended in the water column and the heavier sand
and gravel fall to the bottom. Carefully decant the water and floating organisms
back through a 500 micron sieve. 
Continue to add water to the bucket and swirl and decant until no organic
matter remains in the bucket. When finished, closely examine the remaining
material in the bucket and pick out any caddis flies, snails, clams, or other
organisms that remain. Add these organisms to those on your sieve.
3) Keep the sample in the sieve and rinse the sample under the faucet to wash additional fine particles and silt into the sink.
4) Place the sieve in an enamel pan or bucket
that is partially filled with water and "float" the sample so that it becomes
level within the sieve. 
Once leveled, carefully remove the sieve from the enamel pan. Place
an appropriately sized separator bar (see photo of sieves with separators
above) into the sieve to split the material on in the sieve in half.
Make sure it appears that there is an equal amount of sample material in each
half.
5) Flip a coin to determine which half of the
sample is to be processed; heads = right or top, tails = left or bottom. 
Keep the portion of the sample to be processed in the sieve. 
Place the other half into a cup using a spoon and/or rinse the material into
the cup using an alcohol filled squeeze bottle. 
Cover the cup with ParaFilm 
and write the portion or split of the sample on the lid, e.g., 50%.
If you judge that you will start with less than 50% of the sample, place
the sieve back in the enamel pan and re-float the material to level it. Re-flip
the coin and divide this portion in half again. Place the material you
are not going to immediately sort through in a different cup, cover with ParaFilm,
and label with the split percentage, e.g., 25%. 
Repeat this process until it appears that approximately 600 organisms remain
in one-half of the sieve. It is best to start with small splits to assess
how many bugs you will find (3.125%, 6.25%). The goal is to
sort at least 600 organisms from the sample. Once you start a split
you must finish it in its entirety. If you start to sort through a split
and realize that this split will contain considerably more than 600 organisms,
e.g., 1000 or more critters; stop and pour all the material, including the
bugs you have already removed from the sample back into a sieve and split
it down to a more appropriate percentage.
6) The material to be sorted is placed little-by-little
into a petri dish and all organisms within the petri dish are removed under
a dissecting microscope at 7x magnification. 
The material under the microscope is illuminated by a fiber optic light source. 
Petri dishes are divided into sections by lines drawn on the bottom of the
dish with a permanent marker. The lines subdivide the dish so that you can
systematically move across the dish. 
Move along these "guides" removing all organisms that you encounter. 
7) As you remove the organisms count
them using a clicker 
and drop them into vials separated by the major taxonomic
orders, i.e., Diptera, Coleoptera, Ephemeroptera, Plecoptera, Trichoptera,
other insects, and non-insects. 
You need to remove all of the organism from the debris, but not all organisms
will be counted. Remove, but do not count the following
in your 600 minimum count (follow this link for a more in-depth description
of NO COUNT organisms):
1. damaged and immature organisms
2. molt skins (exuviae)
3. adult insects [excluding water-dwelling adults such
as beetles (Coleoptera) and true bugs (Hemiptera)]
4. eggs
5. empty snail shells
6. brooding juveniles
7. zooplankton
8. Collembola
9. pupae
10. worms
Additional portions of the sample (splits) are sorted until at least 600 organisms are found. Once you start a split, remove all the organisms within it. It is fine if you exceed 700 organisms, but beware of exceeding 1000.
8) When you have removed a minimum of 600 bugs,
perform a "Big/Rare" search. This search is called a "Big/Rare" search
because we tend to find larger individuals, but the goal is to collect rarer
taxa that may not have been present in the split samples. Spread the
entire un-sorted portion of the sample into an appropriately sized white enamel
pan. Place this pan under the lighted magnifier lamp. Systematically
search the pan and remove any organisms that you did not find in your split
samples. Perform this search for 10 minutes. If you are in doubt
that a critter is new, it is much better to pick up duplicates than to miss
a bug. Put these bugs into a separate vial labeled "B/R" for "Big/Rare". 
9) Print a sheet of labels off our website (www.usu.edu/buglab/forms/vials.pdf) onto waterproof paper. The label will contain the following information:
| Sample #, Sample Set/Customer name
Site name/Station I.D., Date collected County, State Sorter, Date sorted, Total split percent, Total # bugs sorted I.D.er, Date I.D.ed # of each Chironominae, Orthocladinae, Tanypodinae counted |
#3, Wasatch-Cache National Forest
Wood Camp- Logan River, 12/12/03 Cache County, UT BB, 12/23/03, 25%, 616 bugs MT, 01/05/04 5 C, 12 O, 20 T |
Use waterproof paper and either a waterproof pen or pencil. 
Put the label into one of the vials. If you do not know the county, search
for it at www.topozone.com.
10) Place a smaller label with the sample number
(#3, from the example above) in each vial and label the lids of the vials
with this same number using a dry erase marker. Label the "Big /Rare"
vial "B/R". Put a rubber band around the vials 
and put them on the appropriate shelf. 
11) The remaining portions of the sample,
the debris that has been sorted, and any unsorted portions of the sample should
be returned to the original sample jar and covered with preservative (70%
ethanol). Place an "X" on the jar lid to indicate that the sample has
been sorted and place it on the 
appropriate shelf. 
12) Record your sample sort time, the sample sort dates, and your initials on the sample tracking data sheet in the folder. Download this form.
Equipment
| preservative (70% ethanol)
500 & 250 micron meter sieves (of various diameters) waste container bucket enamel pans (of various sizes) separator bars (that fit each diameter of the sieves) coin plastic spoon and cups alcohol (ethanol) filled squeeze bottle distilled water filled squeeze bottle Parafilm dry erase marker |
petri dish (with permanent marker drawn lines)
dissecting microscope (7-20x magnification) fiber optic light source clicker/counter tweezers/foceps (both flat and pointed tipped) vial holder (vial sized holes drilled into a wooden block) scintillation vials desk lamp waterproof label paper waterproof pen or sharp tipped pencil rubber bands data recording sheet |
All the organisms removed during the sorting process are then identified by qualified taxonomists. We try to identify organisms to a consistent taxonomic level. Small (early instar) and poorly preserved specimens may be identified to a higher level than specified. Insects are normally identified to genus, some to species, and others to family. Non-insects are identified to the lowest taxonomic level feasible without slide-mounting. If you want your samples processed differently than described above, call us at (435) 797-3945.
See Results and Reports for our normal level of taxonomic resolution for all taxa. The identification keys we use most often can be found in Commonly used identification books.
