PEARL  Paleoecological Environmental Assessment and Research Laboratory

                                            Department of Biology, Queen's University, Kingston ON, Canada, K7L 3N6

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Invertebrate Preparation Methods                                                 

Preparation methods for the paleolimnological analysis of cladoceran remains

A Jeziorski (last updated October 5th, 2015)


Cladoceran analyses are an important paleolimnological tool and to obtain reproducible and reliable results it is essential that samples are processed correctly. At PEARL we follow the methods outlined in Frey (1986) and Korhola and Rautio (2001).


Queen's University requires that all students or staff using chemicals in a laboratory setting have WHMIS certification. Students are expected to use proper safety gear, including Latex gloves (in red box), protective eyewear, lab coat and appropriate footwear. Please ensure that the fumehood is cleaned both before and after use. The fumehood should be used by only one person at a time because of potentially incompatible chemicals.


Digesting: 250 mL beakers, KOH pellets, deionized water, glass stirring rods, weigh scale, scupula, and hot plate.

Sieving: 38-µm mesh, Nalgene® squirt bottle filled with deionized water, and 500 mL beaker to collect waste KOH.



·        Tare the scale. Weigh out ~1g of wet sediment into a 250mL beaker for each sample. The sediment volume may need to be adjusted depending on the concentration of cladoceran remains in your sediments.

·        Prepare adequate volume of 10% KOH solution (dissolve 10g of KOH pellets for every 100ml of deionized water).

·        Transfer ~150ml of the KOH solution to each sample beaker.

·        Heat samples to ~70-80°C for 30 min (or 1 hour for organic-rich sediment). Apply constant gentle stirring using a glass rod (a magnetic stirrer may cause unnecessary fragmentation of remains).

·        Pour the KOH/sediment mixture through the 37µm sieve (capture waste KOH in 500ml beaker for proper disposal, waste KOH can be added to the disposal container for bases located under the fumehood)

·        Proceed to wash the residue captured by the sieve using deionized water (using squirt bottle) until the water passing through the sieve is clear. Generally, if the bottom of the sieve feels 'slimy', or if the rinse water coming through the sieve is unclear, continue rinsing. It is important to fully rinse your sample to completely remove the KOH and smaller sediment particles.

·        Transfer the residue retained on the screen into a glass vial. It will require some practice to transfer all remains into the tube without using too much water. Add 2-3 drops of safranin-glycerin solution (the solution is made by adding a few drops of safranin dye to a small container of glycerin) to the vial to color the cladoceran remains and several drops of alcohol as a preservative.

Slide Preparation

·        Prepare glycerin jelly in a 250mL beaker by dissolving 15g of gelatin powder in 100mL of water using gentle heat (<75°C), then adding 100 mL of pure glycerin to the beaker. Add a few drops of formalin (to retard fungal growth) and a few drops of safranin dye. Stir thoroughly until mixed.

·        Pipette 50µl of the well-stirred sample onto a microscope slide; allow the sample to dry before mounting a cover slip.

·        Use hotplate to melt the glycerin jelly (mounting medium).

·        To transfer jelly from the beaker to the slide, break the tip off a glass pipette slightly below the widening point (use a folded paper towel to prevent injury).

·        Add two drops of jelly to the dried sample on the microscope slide, carefully place coverslip on top of sample, then allow the jelly to harden.


·        All slides should be counted in their entirety to avoid any bias from a non-random distribution of remains under the coverslip.

·        Dried, mounted samples can now be examined under a compound scope using brightfield illumination.

·        The number of slides required to identify at least 70 individuals (minimum count size; Kurek et al. 2010) will vary depending on the density of remains in the sample.

·        All cladoceran remains (e.g. carapaces, headshields, ephippia and postabdominal claws) should be tabulated separately, but only the most frequent body part for each taxon should be used to estimate species abundance. The clad.count function in the 'jezioro' R package can be used to simplify this step.

·        Badly fragmented remains should only be counted if they contain a clear diagnostic feature.


Frey DG (1986) Cladocera Analysis. In: Berglund BE (ed) Handbook of Holocene palaeoecology and palaeohydrology. John Wiley and Sons, New York, pp 667-692

Korhola A, Rautio M (2001) Cladocera and other branchiopod crustaceans. In: Smol JP, Birks HJB, Last WM (eds.) Tracking Environmental Change Using Lake Sediments. Volume 4: Zoological Indicators. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 4-41

Kurek J, Korosi JB, Jeziorski A, Smol JP (2010) Establishing reliable minimum count sizes for cladoceran subfossils sampled from lake sediments. Journal of Paleolimnology 44: 603-612

Taxonomic References:

Korosi JB, Smol JP (2012) An illustrated guide to the identification of cladoceran subfossils from lake sediments in northeastern North America: part 1—the Daphniidae, Leptodoridae, Bosminidae, Polyphemidae, Holopedidae, Sididae, and Macrothricidae. Journal of Paleolimnology 48: 571-586

Korosi JB, Smol JP (2012) An illustrated guide to the identification of cladoceran subfossils from lake sediments in northeastern North America: part 2—the Chydoridae. Journal of Paleolimnology 48: 587-622

Smirnov NN (1996) Cladocera: the Chydorinae and Sayciinae (Chydoridae) of the World. SPB Academic Publishing, Amsterdam.

Sweetman JN, Smol JP (2006) A guide to the identification of cladoceran remains (Crustacea, Branchipoda) in Alaskan lake sediments. Archiv für Hydrobiologie (Supplementbände) Monograph studies 151: 353-394

Szeroczyńska K, Sarmaja-Korjonen K (2007) Atlas of subfossil Cladocera from central and northern Europe. Friends of the Lower Vistula Society, Świecie.