A Quick Overview…
|Collection||Isolation of Tissue Pretreatment||Pretreatment||Drying||Grinding||Amount of Dry Tissue to Submit for SINLAB to weigh||Target Weight if you are weighing|
|Tissue is placed in labeled containers and preserved according to literature||Remove skin, scales and bones. Cut a 2x2cm section of muscle||Not typically required. SINLAB must be notified if any pretreatment was performed||60oC for 24-28 hrs or Freeze dry||Mortar & pestle or ball mill grinder to very fine powder||5 mg||1.000-1.200 mg|
|Tissue is placed in labeled containers and preserved according to literature||Remove dirt, debris, and non target plant material||Not typically required. SINLAB must be notified if any pretreatment was performed||60oC for 24-28 hrs or Freeze dry||Mortar & pestle or ball mill grinder to very fine powder||10 mg||3.000-3.200 mg|
|Place material in labeled containers||Wash feather with de-ionized (DI) H2O or 2:1 chloroform : methanol solution||Air dry 48 hrs||Not necessary||5 mg||1.000-1.200 mg|
|Blood is collected in vacutainers and kept frozen||Centrifuge if red blood cells and plasma are to be analyzed separately||60oC for 24-48 hrs or Freeze dry||Mortar & pestle or ball mill grinder to very find powder||5 mg||1.000-1.200 mg|
|Water samples are collected in labeled containers||Water is filtered using 4.7 cm diameter glass fiber filters**||60oC for 24-48 hrs||Not necessary||1 filter||Please send filter flat in sterile container (Petri dish or aluminum foil)|
|Samples are collected in labeled containers||Sticks, rocks or other debris should be removed prior to drying||Acid treatment if samples contain carbonate. *||60oC for 24-48 hrs||Mortar & pestle or ball mill grinder to very fine powder||5 g||Send to lab for processing|
*Please notify SINLAB if samples were acid treated
**Samples must be completely dried
***Subject to isotope concentration
****FILTERING NOTE: The method used for zooplankton collection is similar to the method used for the collection of particulate organic matter. The one difference is that the fresh or sea water is first filtered through a fine mesh to remove zooplankton and the remaining water is filtered on the glass fiber filters (GFFs). The GFFs can be 2.1or 4.7cm in diameter. The most important factor is to ensure the greatest amount of organic matter is collected on the filter as possible - an electronic filtering system works best for this task. Please send several blank filters (2 for every 30).
The Full Breakdown
How to collect, preserve, and store your samples before sending
Collection, Preservation and Storage of Samples
How much sample do I need?
The amount of material required for stable isotope analysis depends on the concentration of carbon, nitrogen, or hydrogen in the sample. Typically, C and N data can be obtained simultaneously for tissue with a C:N ratio of 3-6 (e.g. fish muscle, bird blood, invertebrates) and a C:N ratio of up to 15 for some plant material if weighed at our target weight.
See STEP 3 for weighing instructions and target weights.
It is the responsibility of the client to notify the SINLAB if samples have less than 0.5% N. Preliminary test runs can be done at the SINLAB to determine the amount of tissue required if the concentration is unknown. For samples with a very high C:N ratio (e.g. sediments, plants), separate analyses may be required to obtain data for both isotopes.
Ideally, 5-10 mg of dry tissue should be collected and submitted to the SINLAB for analysis. This allows for replicate samples as required. We recommend that 4% of samples be run in duplicate. If the researcher does not want replicate data it should be specified with the initial sample submission.
How should samples be handled and stored?
To prevent contamination of your sample, use aseptic techniques and clean storage containers. Clean scintillation vials, Eppendorf tubes, paper coin or glassine envelopes, vacutainers and whirl-pack bags are some examples of containers used for sample collection.
How do I preserve my samples until analysis?
The two main methods of preservation involve placing samples in a desiccators or freezer, however, there are a variety of sample storage and preservation techniques, each specific to sample type. A literature search in your research area should be conducted prior to sample collection to determine the most appropriate method of preservation. Please see the table below and/or contact the lab (firstname.lastname@example.org) for further information.
It should be noted that some preservation methods alter isotope ratios. For examples of papers investigating preservation techniques and pitfalls, please see ‘Literature on the preservation of samples' at the bottom of this page.
Samples must be dried using drying oven or a freeze-drier. Please see the following table for specific drying times. A mortar and pestle or ball-mill grinder can be used to grind the sample into a homogeneous fine powder (remember to always use aseptic techniques).
Samples must be dried using a freeze-drier or oven. Please see table for specific drying times. A mortar and pestle or ball-mill grinder can be used to grind the sample into a homogeneous fine powder (remember to always use aseptic techniques).
Sample preparation and target weights by tissue type
HYDROGEN ANALYSIS: Please Contact the SINLAB for Instructions
CARBON & NITROGEN ANALYSIS: Please refer to the table at the top of the page for specific guidelines.
ANIMAL Remove skin, scales and bone. Dry at 60° for 24-48 hours or freeze dry. Grind to a very fine powder. If you wish to submit samples for the SINLAB to weigh, please provide us with ~5mg. If you wish to submit preweighed samples, please aim for our target weight of 1.000 to 1.200mg.
PLANT Remove dirt, debris, and non-target plant material. Dry at 60° for 24-48 hours or freeze dry. Grind to a very fine powder. If you wish to submit samples for the SINLAB to weigh, please provide us with ~10mg. If you wish to submit preweighed samples, please aim for our target weight of 3.000 to 3.200mg.
FEATHER/CLAW/HAIR Wash with de-ionized water (DI) or 2:1 chloroform:methanol solution. Air dry for 48 hours. Do not powder/grind sample. If you wish to submit samples for the SINLAB to weigh, please provide us with ~5mg. If you wish to submit preweighed samples, please aim for our target weight of 1.000 to 1.200mg.
BLOOD Blood is collected in vacutainers and kept frozen. If plasma and red blood cells are to be analyzed separately, centrifuge samples. Dry at 60° for 24-48 hours or freeze dry. Grind to a very fine powder. If you wish to submit samples for the SINLAB to weigh, please provide us with ~5mg. If you wish to submit preweighed samples, please aim for our target weight of 1.000 to 1.200mg.
ZOOPLANKTON Filter water using 4.7cm diameter glass fibre filters. Dry at 60° for 24-48 hours. Please submit 1 whole filter in a sterile container (petri dish or aluminium foil).
SEDIMENT Prior to drying, remove sticks, rocks, or other debris. If samples contain carbonates, acid treatments will need to be done (the SINLAB must be notified if the samples submitted have either received acid treatments or will need the pre-treatment prior to weighing). Dry at 60° for 24-48 hours. Grind to a very fine powder. When sending samples to the SINLAB, please provide us with ~5g. As the C:N can vary in sediment samples, it is best to submit them to the SINLAB for preliminary tests to allow us to adjust our target weights and settings.
weighing and loading form instructions
You may opt to have the SINLAB staff prepare and weigh your samples (please see 'Services and Cost'). If this is the case, you may disregard this section and send your samples. If you are doing the weighing yourself please follow these instructions:
Please crush 3-4 blanks (blank=1 of your empty tin caps crushed) and place them in A1 of the loading tray.
The amount of material required for stable isotope analysis depends on the concentration of carbon, nitrogen, or hydrogen in the sample. C and N data can be obtained simultaneously if the samples are weighed at our "target" weights assuming C:N>3.
For tissue with a C:N ratio of 3-6 , 1.100 mg (± 0.100) of dry sample is analyzed (e.g. fish muscle, bird blood, invertebrates). If you do not have enough sample to meet the 1.100 mg target weight, we may still be able to analyze your samples for you. Please contact the lab prior to sending samples. Please download "LOADING FORM ANIMAL" and record your final weight. NOTE: FINAL WEIGHTS ARE RECORDED AFTER THE SAMPLE HAS BEEN FOLDED.
For a tissue with a C:N ratio of 15, 3.000 to 3.200 mg of dry sample is analyzed (e.g. some plant material) Please download "LOADING FORM PLANTS" and record your final weight. NOTE: FINAL WEIGHTS ARE RECORDED AFTER THE SAMPLE HAS BEEN FOLDED.
Weigh the samples into the smallest available tin cups (or, if analyzing for hydrogen, silver cups (Part # D2002)). At the SINLAB, we use 5 x 3.5 mm cups. For larger samples we use 8.5 x 4 mm cup (cups can be purchased from Elemental Microanalysis Ltd., distributed by Isomass, Inc. in Calgary, Canada, part # D1002 & D1091 respectively). Always clean your tools and work surface between samples (recommended: ethanol) to avoid sample cross-contamination.
Use a high precision microbalance (readability of 0.001mg or better) to ensure the reliable measurement of %C, %N or %H measurements (which are calculated based on the weights). Weights are then recorded into the appropriate loading form (see below).
In this photo, you can see an unfolded tin cup (top), an improperly folded tin cup (bottom left, tube shaped) and a properly folded tin cup (cube-shaped). After weighing the sample into the tin cup, fold it to remove atmosphere and shape it as close to a cube as possible. Avoid flattening the samples or leaving edges sticking out (they will get stuck in the auto-sampler).
Download the appropriate ‘Loading Form' below. Please fill out the ‘Sample Name' column with your identification codes and we will assign each one a SINLAB ID. Ignore the rows that already contain information, as these are for the standards that are to be run alongside your samples.
An excel spreadsheet with your sample identification codes must also accompany your sample shipment even if you are submitting a loading form.
Literature on the preservation of samples:
Arrington, D.A. and K.O. Winemiller. 2002. Preservation effects on stable isotope analysis of fish muscle. Transactions of the American Fisheries Society 131(2):337-342.
Bosley, K.L. and S.C. Wainright. 1999. Effects of preservation and acidification on the stable isotope ratios (15N:14N,13C:12C) of two species of marine animals. Canadian Journal of Fisheries and Aquatic Sciences 56:2181-2185.
Charette, M.R., Diamond, A.W., and T.D. Jardine. A note of caution when using different concentrations of blood and preservative in stable isotope analysis. Rapid Communications in Mass Spectrometry (unpub MS).
Edwards, M.S., T.F. Turner, and Z.D. Sharp. 2002. Short- and long-term effects of fixation and preservation on stable isotope values (delta C-13, delta N-15, delta S-34) of fluid-preserved museum specimens. Copeia 4:1106-1112.
Feuchtmayr, H. and J. Grey. Effect of preparation and preservation procedures on carbon and nitrogen stable isotope determinations from zooplankton. Rapid Communications in Mass Spectrometry 23(17):2605-2610.
Hobson, K.A., H.L. Gibbs, and M.L. Gloutney. 1997. Preservation of blood and tissue samples for stable-carbon and nitrogen isotope analysis. Canadian Journal of Zoology 75:1720-1723.
Jardine, T.D., S.A. McGeachy, C.M. Paton, M. Savoie, and R.A. Cunjak. 203. Stable Isotopes in Aquatic Systems, Sample Preparation, Analysis and Interpretation. Fisheries and Aquatic Sciences No. 2656.
Kaehler, S. and E.A. Pakhomov. 2001. Effects of storage and preservation on the delta C-13 and delta N-15 signatures of selected marine organisms. Marine Ecology-Progress Series 219:299-304.
Kelly, B., Dempson, J.B., and M. Power. 2006. The effects of preservation on fish tissue stable isotope signatures. Journal of Fish Biology 69(6):1595-1611.
Ponsard, S. and M. Amlou. 1999. Effects of several preservation methods on the isotopic content of Drosophila samples. Comptes Rendus De L'Academie Des Sciences Serie III-Sciences De La Vie 322(1):35-41.
Sarakinos, H.C., Johnson, M.L. and M.J. Vander Zanden. 2002. A synthesis of tissue-preservation effects on carbon nitrogen stable isotope signatures. Canadian Journal of Zoology 80(2):381-387.
Sweeting, C.J., Polunin, N.V.C., and S. Jennings. 2004. Tissue and fixative dependent shifts of delta C-13 and delta N-15 in preserved ecological material. Rapid Communications in Mass Spectrometry. 21(18):2587-2592.