The following are considerations and preparations for Stable Nitrogen Isotope Analysis, and are directly tailored to this method. Other types of water quality measurements and other sampling of limu (for example, FIB) may require different considerations and methods to have good quality data.
1. Shoreline Types
Depending on the habitat type or the shoreline, there may or may not be a safe way to enter the water, and there may not be algae growing to sample in certain area. If the shoreline and habitat is sandy with high wave activity, it is likely there will not be algae growth. If there is rocky shoreline, it is more likely that algae will grow there as they have a hard surface to attach to.
Shoreline Type |
Can we sample water? |
Can we sample limu? |
Sandy, high wave activity |
Can take water samples (but lots of mixing from high wave activity) |
Likely no limu growth, Cannot use limu analysis |
Rocky, some mixing with stream and submarine ground water |
Can take water samples easily |
Likely limu growth, Can use limu analysis |
2. Supplies Needed
Water sample requires sample bottles for collection which must be sterilized (usually by acid bath), filtration to remove sediment and particulates, collection bottles for analysis, freezer or fridge for analysis. In the field, it is standard to have an YSI or other probe to measure water quality characteristics (temperature, salinity, pH, DO%). It is also standard to have a cooler with ice.
Supplies |
Water |
Limu |
In Field |
- Coolers with Ice - Sample Bottles (x2, one for inorganic nutrients, one for stable isotope) - Gallon sized sample bags, labelled by site for organization - Filters (0.2 µm) and hand pump YSI Readings (for Salinity) |
- Sample Bags, Labelled by Site ID - Book/Camera (for in field ID) - Pan or container to rinse and remove epiphytes - Drying Supplies (Foil for Oven dry or wax paper, waterproof paper, newspaper, cardboard for herbarium, silica gel bags for). |
In Lab |
- Freezer - Inorganic Analysis (lab takes care) - Stable Isotope Analysis (see SOP Appendix B): Biosafety Level II room Bacterial culture (Pseudomonas aureofaciens) Flow through NO3 - Liquid N2 , etc. Personal Protective Equipment (PPE) |
- Oven (for drying) - Mortar and Pestle - Ethanol - Kim wipes - Spatula - Glass Vials - Tin Foil (3x5 mm) capsules - Analysis Tray - Microbalance |
3. Time & Effort and Costs
There is a tradeoff between time/effort and cost of having technician do the lab prep and analysis for you. It is considerably cheaper for both water analysis and limu analysis to do the lab prep and analysis yourself (you will need to have training! See the Training Section).
One consideration (even if you have the lab do the lab work for you) is the cost is only $18.50 per sample of limu compared to $65 per sample of water (a difference of $ 46.50, meaning that you could do 3.5 samples of limu for the price of one water sample. For good statistics and strong results, a minimum of 3 replicates are suggested. This would mean that you could have your three replicates of limu samples for the same price of a single sample of water.
One consideration (even if you have the lab do the lab work for you) is the cost is only $18.50 per sample of limu compared to $65 per sample of water (a difference of $ 46.50, meaning that you could do 3.5 samples of limu for the price of one water sample. For good statistics and strong results, a minimum of 3 replicates are suggested. This would mean that you could have your three replicates of limu samples for the same price of a single sample of water.
Time and Effort |
Water |
Limu |
In Field |
Two 50 mL samples bottle of water collected for analysis YSI Measurements Should be frozen ASAP (in cooler on ice) |
Limu needs to be collected and separate into different bags Dried to prepare analysis |
In Lab |
2 weeks worth of analysis for 30 samples 1st week growing bacteria 2nd week running samples |
After dried, ground into a powder (approximately 2 -5 minutes per sample) with mortar and pestle Encapsulate in tin foil small balls and microbalance weight |
Cost |
Water Stable Isotope Analysis |
Limu Stable Isotope Analysis |
Processed yourself (Requires Training) |
$20 Inorganic Nutrients (N+N)(required to accurately calculate final δ 15 N) + $25 Stable Isotope Analysis = $45 Total |
Drying, grinding, weighing, and tin foil ball yourself $11 |
Processed by Lab Technician |
Give dried sample to the lab $18.50 |
INORGANIC NUTRIENT ANALYSIS
UH Manoa website: http://www.soest.hawaii.edu/S-LAB/
UH Hilo also offers: https://hilo.hawaii.edu/analab/service.php
Other universities also provide these services
(but are not local and shipping costs and times can be long)
UH Hilo also offers: https://hilo.hawaii.edu/analab/service.php
Other universities also provide these services
(but are not local and shipping costs and times can be long)
Stable Isotope ANALYSIS
UH Manoa website: http://www.soest.hawaii.edu/GG/isotope_biogeochem/
UH Hilo also offers: https://hilo.hawaii.edu/analab/service.php
Other universities also provide these services
(but are not local and shipping costs and times can be long)
UH Hilo also offers: https://hilo.hawaii.edu/analab/service.php
Other universities also provide these services
(but are not local and shipping costs and times can be long)
4. Training Requirements
To be able to do the lab work by yourself there is are some required courses you must take depending on if you want to do the macroalgae samples by yourself, the water samples by yourself, or both. To just work in the lab in general, you need to take UH’s General Lab Safety Training. Then if you are grinding and weighing your macroalgae, the lab technician will show you how to use the microbalance, and fold the samples in the tiny 3 x 5 mm foil capsules. If you want to do the water stable isotopes yourself, you will have to take a Biosafety Lab Training, because you will be culturing bacteria, which will fractionate your stable isotopes of nitrogen.
Training Requirements |
For Water |
For Limu |
University of Hawai'i Training Requirements (must be done before even working in the Stable Isotope Laboratory) |
- General Lab Safety Training(http://www.hawaii.edu/ehso/lab-safety-training/) - Biosafety Lab Training (working with bacteria)(https://www.hawaii.edu/researchcompliance/biosafety-education) |
General Lab Safety Training(http://www.hawaii.edu/ehso/lab-safety-training/) |
Stable Isotope Laboratory Training Requirements |
Analysis Training by Stable Isotope Staff: - 1 week, growth of bacteria, and culture development - 2 week, samples with flow nitrogen through needles to samples and bacterial combinations in sealed containers - Use of mass spec, handling of liquid nitrogen Analysis of data read out from mass spec (how to read peaks etc.) |
Analysis Training by Stable Isotope Staff: - Cleaning equipment with ethanol - Use of Microbalance and Data Logging in Database of Weights and Species ID - Folding of 3 x 5 mm Tin Foil Capsules Storage in Desiccator before analysis |
5. storage and Shipping
For the island of Kaua‘i a constant challenge is having a location to stage and store the equipment (Table 18). My results of drying test showed that. Although different drying methods can be used for analysis, to standardize the method across the state and in the Pacific, it is best to have the samples dried in the sample oven. For the water collection, a freezer is needed. At a minimum, some electric input is needed for both the freezer and the oven in order to properly do this water sampling technique.
Storage and Shipment |
Water |
Limu |
Storage |
-Needs to be filtered before freezing (if using filters for DNA analysis) -Needs to be in freezer ASAP from field |
- Needs to be dried - Can be dried in variety of ways (as supported by mini study – oven, herbarium, silica gel, salt) - After dried, storage in desiccator |
Shipment |
- Needs to be air-carrier shipment or checked baggage - If flying over to lab to conduct lab work can do checked baggage - If not flying over for lab, air carrier Should be as frozen as possible before |
- Cannot be in water - Dried would be easier to ship over, again air-carrier or hand carry on flight would be easiest. - Dried before shipping preferable but can bring undried as long as no water in sample bags |