Jun 23, 2026
Zooplankton Collection and Processing for Elemental Composition: Methods for Abundance, Water Content, and Sample Preparation
- Ying-Yu Hu1,
- Kevan Merrow1,
- Aaraya Aad1,
- Zoe V. Finkel1
- 1Dalhousie University
- Marine Microbial Macroecology LabTech. support email: [email protected]
Protocol Citation: Ying-Yu Hu, Kevan Merrow, Aaraya Aad, Zoe V. Finkel 2026. Zooplankton Collection and Processing for Elemental Composition: Methods for Abundance, Water Content, and Sample Preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr3qq2vmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 07, 2023
Last Modified: June 23, 2026
Protocol Integer ID: 91958
Keywords: zooplankton, biomass, elements, abundance, sample preparation zooplankton sample, zooplankton collection, measurement of particulate carbon, particulate carbon, water content, water content of freeze, filtered seawater, particulate nitrogen, preparation of sample, processing for elemental composition, particulate phosphorus, elemental composition, homogeneous powder, methods for abundance, mesh screens by gentle filtration, gentle filtration, subsample of the powder, dried material, biogenic silica, powder
Funders Acknowledgements:
Simons Foundation
Grant ID: 549937
Simons Foundation
Grant ID: 723789
Abstract
Zooplankton samples are collected onto mesh screens by gentle filtration. To remove salts, the <500 µm size fraction is rinsed with filtered seawater, while the >500 µm fraction is rinsed with deionized (DI) water. Samples are flash frozen in the field and stored at –80 °C. In the laboratory, samples are freeze-dried until no further change in weight is observed, then ground to a fine, homogeneous powder using metal lysing tubes, or a mortar and pestle, or both. A subsample of the powder is further dried in an oven until a constant weight is achieved to determine the water content of freeze-dried material. This protocol also describes the preparation of samples for the measurement of particulate carbon (total and organic), particulate nitrogen, particulate phosphorus, biogenic silica, and chlorophyll a.
Safety warnings
Contact of skin with dry ice can cause severe frostbite, and can be an asphyxiation hazard if not contained properly.
Collect zooplankton samples
Filter microzooplankton and/or mesozooplankton onto 52 μm nitex mesh, using gentle vacuum pressure (130 mmHg).
Equipment
Nylon mesh filter screen
NAME
50 micrometer 102 cm wide nitex nylon mesh filter screen
TYPE
Dynamic Aqua Supply
BRAND
NTX50-102
SKU
Note
This single mesh size is used to collect all zooplankton samples size fractioned larger than 50 micrometer. A different mesh size can be used as long as it is smaller than the size of your collected samples.
Equipment
Filter forceps
NAME
blunt end, stainless steel
TYPE
Milipore
BRAND
XX6200006P
SKU
Rinse filter funnel with DI or filtered seawater to collect all sample onto the mesh and have salts removed. Use DI for any size fractioned samples >500
Note
Samples with organisms <500 μm may burst or lyse if rinsed with DI water due to osmosis and difference in salinity between seawater and DI water.
Place sample mesh in 5 mL Cryogenic vials or 100x15mm disposable petri-dish. The choice depends on the specific characteristics of the biomass.
Equipment
Cryogenic Vials with Closures
NAME
Polypropylene, 5 mL
TYPE
Corning
BRAND
66021-974
SKU
Equipment
disposable petri dishes
NAME
polystyrene 100 X 15 mm
TYPE
VWR
BRAND
25384-092
SKU
Flash freeze sample and store at -80 °C .
Weigh freeze-dried biomass
Log sheet for freeze-dried mass and water content.
assay_sheet_template_mass.pdf22.4KB Equipment
FreeZone Benchtop Freeze Dryer
NAME
2.5 L -50ºC Series
TYPE
FreeZone
BRAND
700202000
SKU
Every 24 hours:
Transfer samples to vacuum desiccator, allow samples to warm up to Room temperature , weigh each sample with its container.
If there is still change in weight, freeze samples and then freeze dry again.
Store freeze-dried samples at -80 °C .
Prepare metal lysing tubes.
Equipment
2 mL metal lysing matrix tube with grinding cylinder
NAME
metal lysing matrix tube
TYPE
MP Biomedicals
BRAND
116992006
SKU
Clean the lysing tubes, caps, and o-rings in phosphate free detergent.
Use a pipe cleaner to scrub the interior of the tubes and caps to remove any residue.
Rinse the tubes, caps and o-rings with DI water.
Place the metal lysing tubes and caps in a sanitation bath filled with DI water to further clean them.
Rinse each tube and cap with DI three times.
Rinse each tube and cap with 95% ethanol and air dry.
Wrap tubes in the tin foil pouch and autoclave tubes.
Keep tubes in the tin foil pouch, dry in the gravity oven.
Prepare agate mortar and pestle.
Equipment
Mortar and pestle deep form
NAME
100 mm diameter agate mortar and pestle
TYPE
Fisher Scientific
BRAND
FB970N
SKU
Use baking soda and DI water to clean mortar and pestle.
Rinse mortar and pestle with 95% ethanol and air dry.
Wrap mortar and pestle in tin foil pouch and autoclave mortar and pestle.
Keep mortar and pestle in the tin foil pouch, dry in the gravity oven.
Autoclave the following items:
(1) tin foil sheet (i.e. recommended size 3 " inch x 3 " inch)
(2) tweezers
(3) microscoopula
Equipment
Filter forceps
NAME
blunt end, stainless steel
TYPE
Millipore
BRAND
XX6200006P
SKU
Equipment
long straight precision tip forceps
NAME
precision tip forceps
TYPE
Fisher Scientific
BRAND
12-000-127
SKU
Prior to weighing, transfer the freeze-dried samples from the ULT freezer to a vacuum desiccator. It takes about 15 min for frozen samples to reach Room temperature .
Note
Loosen caps of 5 mL cryogenic vials before placing them into the vacuum desiccator.
Decontaminate biosafety cabinet while waiting for samples to return to Room temperature .
Place dry ice into a styrofoam box to prepare for sample storage.
Fill the thermoflask with liquid nitrogen (LN) if using a mortar and pestle to grind samples.
Ensure that the caps of the cryogenic vials are tightened. Remove samples from the vacuum desiccator.
Note
Ensure that the caps of the cryogenic vials are tightened.
Equipment
OHAUS PX85 pioneer Semi-Micro Analytical Balance
NAME
Semi-micro Analytical Balance
TYPE
OHAUS
BRAND
C210698907
SKU
Note
Adjust unit to "gram" (i.e. g).
Place the sample together with its container onto the balance and weigh it three times. Record all three measurements (W₁) in the ‘tube_freezedry_mg’ column of the data sheet.
Empty the cryogenic tube or petridish by transferring the sample into a grinding tube or mortar.
Grinding tube
19.1.1 Label the metal lysing tube.
19.1.2 Place a sterilized 3-inch × 3-inch tin foil sheet into a small autoclaved bowl or mortar of the same size. Fold the foil to form a funnel shape.
19.1.3 Using the folded foil as a funnel, transfer the sample from the original container into a metal lysing tube.
19.1.4 Keep metal lysing tube in dry ice.
19.1.5 Rinse tweezers and microscoopulas with 95% ethanol in between different samples.
Mortar
Equipment
mortar and pestle normal form
NAME
agate
TYPE
Fisherbrand
BRAND
FB970C
SKU
LINK
19.2.1 Fill the autoclaved mortar with liquid nitrogen (LN).
19.2.2 Transfer the sample from the original container into the mortar while submerged in LN.
19.2.3 Grind the sample in liquid nitrogen using the mortar and pestle, then transfer the powder into a new RNase-free cryogenic tube.
19.2.4 Immediately place the tube on dry ice.
19.2.5 Rinse tweezers and microscoopulas with 95% ethanol in between different samples.
Return the empty container with nylon mesh to the glass petri dish, weigh three times. Record the three weights (W2) in the "empty_tube_mg" column of the data sheet.
Calculate the mean value of freeze dry weight (FD) and the standard deviation (sd) in grams (g) of each sample weight using the following equation:
Prepare samples for water content estimation
6h
Combust acid-washed weighing bottles and tin foil sheets (cut to approximately 1.5 times the dimensions of the weighing bottle) at 500 °C for 06:00:00 .
Equipment
Weighing bottle with interchangeable ground glass stopper
NAME
5 mL
TYPE
Borosil
BRAND
76490-652
SKU
6h
Autoclave microscoopulas, tweezers and tin foil sheets.
Decontaminate biosafety cabinet.
Use a metal ice scooper, evenly fill the CoolPrep adapter with ~50 g dry ice to prepare for grinding using metal lysing tubes.
Equipment
FastPrep-24™ 5G bead beating grinder and lysis system
NAME
Sample homogenizer
TYPE
MP Biomedicals
BRAND
116005500
SKU
LINK
Equipment
CoolPrep™ adapter for 24 x 2 mL tube holder on FastPrep-24
NAME
Sample homogeniser adapter
TYPE
MP Biomedicals
BRAND
116002528
SKU
LINK
Load samples into the FastPrep attachment and log the orientation of each tube in case labels are removed during grinding.
Grind at 6.5 m/s speed for 00:00:05 .
5s
Restore the label if necessary. Keep ground samples in dry-ice box.
Log the three weights of the weighing bottle (W0) in the "empty_bottle_mg" column of the data sheet.
Wrap a sheet of tin foil around the bottom of the weighing bottle to create a stable base for labeling. Write the label directly on the foil, not on the bottle itself.
In the biosafety cabinet, transfer a portion of the samples from metal lysing tube into its corresponding weighing bottle. The table below lists estimated minimum biomass requirements for elemental and chlorophyll analysis across different size fractions.
| Size (um) | TPCN (mg) | POCN (mg) | TPP (mg) | bSi (mg) | Chla (mg) | Total (mg) | |
| 64~250 | 0.1 | 0.1 | 0.1 | 0.1 | 1 | 4.5 | |
| 200~500 | 0.3 | 0.3 | 0.1 | 0.6 | 0.3 | 4.5 | |
| 500~2000 | 0.3 | 0.3 | 0.2 | 2 | |||
| 1000~2000 | 0.3 | 0.3 | 0.2 | 2 |
Note
HINT: Remove the O-ring from the cap of the metal lysing tube and shake the tube with the cylinder inside to help transfer all remaining sample biomass to the weighing bottle and cryogenic vial. Scrap sample using microscoopula and tweezers.
Transfer a portion of the samples from the mortar into its corresponding weighing bottle, loosely wrap the opening with combusted foil.
Transfer the remaining sample into labelled RNase/DNase free cryogenic vial, store at -80 °C .
Estimate water content in freeze-dried biomass
2d
Weigh weighing bottle containing the sample three times. Record all three measurements (W1) in the "bottle_freezedry_mg" column of the data sheet.
Transfer weighing bottles containing samples into the forced-air oven at 60 °C for 48:00:00 .
Equipment
Forced air oven
NAME
oven
TYPE
VWR
BRAND
89511-410
SKU
Note
Place the weighing bottles into a vial rack and gently cover them with tin foil to prevent the vials from tipping over or their tin foil caps from falling off.
Equipment
Vial rack
NAME
Wheaton
BRAND
Z252425
SKU
2d
Transfer the weighing bottles to a vacuum desiccator. Loosen the caps and allow the samples to cool completely to Room temperature .
Weigh the weighing bottles with dried samples three times. Record all three measurements (W2) in the "bottle_ovendry_mg" column of the data sheet.
Water content is calculated from the mean pre- and post-oven-dry weights. Uncertainty is estimated by propagation of error using the standard errors of the mean (n = 3).
Prepare samples for TPC/PN analysis
Log sheet for CN samples
assay_sheet_template_cn.pdf21.8KB To avoid carbon contamination of tin-capsule, flattern a tin boat on 95% ethanol-rinsed and air-dried surface, and place it on the plate of the microbalance.
Move some weighing bottles from the vacuum desiccator into a box desiccator with desiccant.
Note
(1) Ensure the desiccant is active.
(2) Do not move too many samples out of the vacuum desiccator.
Use tweezers, gently place a D1103 tin capsule onto the microbalance plat lines with flatterned tin foil.
Equipment
Tin capsule
NAME
D1103
TYPE
Elemental Microanalysis
BRAND
D1103
SKU
LINK
Take one weighing bottle with sample from the box desiccator.
For the 250–2000 µm size fraction, weigh > 0.3 mg of sample.
For the 64–250 µm size fraction, weigh > 0.1 mg.
Record the weight in the "TPC_mg" column of the data sheet for the first replicate of the sample.
Encapsulate the sample:
Place the sample pellet into microplate. Record the well number in the "TPC_well" column of the data sheet.
Weigh another portion for the second replicate of the sample, encapsulate, record both the weight and the well number.
Weigh another portion for the third replicate of the sample, encapsulate, record both the weight and the well number.
Return the weighing bottle to the box desiccator.
Rinse tweezers and scoopulars between two different samples.
Blanks
Make three blank pellets to serve as triplicates.
Store the microplate containing the encapsulated samples in a vacuum desiccator until it is sent for elemental analysis (EA).
Prepare samples for POC/PN analysis
2d
Rinse tweezers with 95% ethanol, and air-dry.
Use tweezers to fold 25 mm GF/F filters in half, forming a semicircle.
Make a second fold perpendicular to the first, forming a quarter-circle.
Gently insert the folded filter into a vial (1DR 15X45MM 13-425) with the folded edge pointing down. Do not press down forcefully, just let it sit naturally and avoid deformation.
Place all vial+GFF sets into a foil pan. Cover the pan with foil, and combust at 450 °C for 04:00:00
Place an aluminum weighing boat on the plate of the microbalance, and tare the balance.
Move some weighing bottles from the vacuum desiccator into a box desiccator with desiccant.
Note
(1) Ensure the desiccant is active.
(2) Do not move too many samples out of the vacuum desiccator.
Take one weighing bottle with sample from the box desiccator.
For the 250–2000 µm size fraction, weigh > 0.3 mg of sample.
For the 64–250 µm size fraction, weigh > 0.1 mg.
Record the weight in the "POC_tin_sample_mg" column of the data sheet for the first replicate of the sample.
Label one pre-combusted vial+GFF set using the sample code followed by the replicate label "a", for example: 064a. Record the label number in the "POC_code" column of the data sheet.
Transfer sample from weighing boat into the folded GF/F filter sitting in the labelled vial.
Modify or select a vial rack that fits within the glass desiccator used for POC acid-fuming.
Rinse tin foil with 95% ethanol and allow it to air-dry. Place the vial+GFF set containing the sample into a rack and cover the rack with ethanol rinsed foil to prevent contamination.
Return weighing boat to the microbalance, record the weight in the "POC_tin_mg" column of the data sheet for the first replicate of the sample.
Weigh another portion for the second replicate of the sample, record "POC_tin_sample_mg" and "POC_tin_mg". Label the pre-combusted vial+GFF set using the sample code followed by the replicate label "b". Record the label number in the "POC_code" column of the data sheet.
Weigh another portion for the third replicate of the sample, record "POC_tin_sample_mg" and "POC_tin_mg". Label the pre-combusted vial+GFF set using the sample code followed by the replicate label "c". Record the label number in the "POC_code" column of the data sheet.
Return the weighing bottle to the box desiccator.
Rinse tweezers and scoopulars between two different samples.
For the next sample, use a clean, unused weighing boat.
Prepare three vial + GF/F filter sets without samples to serve as blanks. Label them as ‘blanka’, ‘blankb’, and ‘blankc’.
Rinse the inner surface of the desiccator lid with 95% ethanol, and allow it to air-dry.
In the fume hood, carefully place a porcelain rack into the glass desiccator.
Place the vial rack with the vial+GFF+sample sets on the porcelain rack.
Pour 500 mL concentrated HCl into a glass beaker.
Carefully pour concentrated HCl along the inner wall of the glass desiccator from the beaker, allowing the acid to flow directly to the bottom without splashing.
Do not apply vacuum grease to the rim of the desiccator lid
Place the lid onto the desiccator.
Leave the samples in the desiccator to be acid-fumed overnight.
Carefully remove the rack with the vial+GFF+sample sets from the desiccator. Place it in the fume hood.
Loosely cover the samples with a tent made of aluminum foil (95% ethanol rinsed and air-dried) to prevent contamination. Ensure the foil does not touch the samples, as acid released from them reacts with the foil.
In the glass desiccator, neutralize the acid using baking soda, then wash the desiccator and lid with DI, and allow them to air-dry.
Every hour, place a moistened pH strip above the samples. Once the strip indicates a neutral pH, transfer the rack containing the vial+GFF+sample sets to a forced-air oven.
Dry at 60 °C for 48:00:00
2d
Allow the rack containing the vial+GFF+sample sets to cool in the desiccator.
Encapsulate each GF/F filter with sample using D5055:
Equipment
Tin capsule
NAME
D5055
TYPE
Elemental Microanalysis
BRAND
D5055
SKU
LINK
Place the sample pellet into microplate. Record the well number in the "POC_well" column of the data sheet.
Store the microplate containing the encapsulated samples in a vacuum desiccator until it is sent for elemental analysis (EA).
Prepare samples for TPP analysis
Log sheet
assay_sheet_template_tpp_bsi.pdf Place an aluminum weighing boat on the plate of the microbalance, and tare the balance.
Move some weighing bottles from the vacuum desiccator into a box desiccator with desiccant.
Note
(1) Ensure the desiccant is active.
(2) Do not move too many samples out of the vacuum desiccator.
Take one weighing bottle with sample from the box desiccator.
For >250 µm size fraction, weigh > 0.2 mg of sample.
For <250 µm size fraction, weigh > 0.1 mg.
Record the weight in the "TPP_tin_sample_mg" column of the data sheet for the first replicate of the sample.
Assign an assay code for this sample and record this code in the "TPP_code" column of the data sheet.
Label a crucible with the same assay code using oil-based white sharpie.
Transfer sample from weighing boat into the labelled crucible.
Put crucible with the sample in a muffin tin to prevent the crucible falling over.
Return weighing boat to the microbalance, record the weight in the "TPP_tin_mg" column of the data sheet for the first replicate of the sample.
Log sheet
assay_sheet_template_tpp_bsi.pdf Weigh another portion for the second replicate of the sample, record "TPP_code", "TPP_tin_sample_mg" and "TPP_tin_mg". Label the crucible accordingly.
Weigh another portion for the third replicate of the sample, record "TPP_code", "TPP_tin_sample_mg" and "TPP_tin_mg". Label the crucible accordingly.
Return the weighing bottle to the box desiccator.
Rinse tweezers and scoopulars between two different samples.
For the next sample, use a clean, unused weighing boat.
Follow the TPP protocol:
Prepare samples for bSi analysis
Log sheet
assay_sheet_template_tpp_bsi.pdf Place an aluminum weighing boat on the plate of the microbalance, and tare the balance.
Move some weighing bottles from the vacuum desiccator into a box desiccator with desiccant.
Note
(1) Ensure the desiccant is active.
(2) Do not move too many samples out of the vacuum desiccator.
Take one weighing bottle with sample from the box desiccator.
For the 250–500 µm size fraction, weigh > 0.6 mg of sample.
For the 64–250 µm size fraction, weigh > 0.1 mg.
Record the weight in the "bSi_tin_sample_mg" column of the data sheet for the first replicate of the sample.
Assign an assay code for this sample and record this code in the "bSi_code" column of the data sheet.
Label a 50 mL falcon tube with the same assay code.
Transfer sample from weighing boat into the labelled 50 mL falcon tube.
Return weighing boat to the microbalance, record the weight in the "bSi_tin_mg" column of the data sheet for the first replicate of the sample.
Log sheet
assay_sheet_template_tpp_bsi.pdf Weigh another portion for the second replicate of the sample, record "bSi_code", "bSi_tin_sample_mg" and "bSi_tin_mg". Label the 50 mL falcon tube accordingly.
Weigh another portion for the third replicate of the sample, record "bSi_code", "bSi_tin_sample_mg" and "bSi_tin_mg". Label the 50 mL falcon tube accordingly.
Return the weighing bottle to the box desiccator.
Rinse tweezers and scoopulars between two different samples.
For the next sample, use a clean, unused weighing boat.
Follow the bSi protocol:
Prepare samples for chlorophyll analysis
Log sheet
assay_sheet_template_ch.pdf Place an aluminum weighing boat on the plate of the microbalance, and tare the balance.
Move some weighing bottles from the vacuum desiccator into a box desiccator with desiccant.
Note
(1) Ensure the desiccant is active.
(2) Do not move too many samples out of the vacuum desiccator.
Take one weighing bottle with sample from the box desiccator.
For the 250–2000 µm size fraction, weigh > 0.3 mg of sample.
For the 64–250 µm size fraction, weigh > 1 mg.
Record the weight in the "chla_tin_sample_mg" column of the data sheet for the first replicate of the sample.
Assign an assay code for this sample and record this code in the "assay code" column of the data sheet.
Label a 10 mL glass centrifuge tube with the same assay code.
Transfer sample from weighing boat into the labelled tube.
Return weighing boat to the microbalance, record the weight in the "chla_tin_mg" column of the data sheet for the first replicate of the sample.
Weigh another portion for the second replicate of the sample, record "assay code", "chla_tin_sample_mg" and "chla_tin_mg". Label the centrifuge tube accordingly.
Weigh another portion for the third replicate of the sample, record "assay code", "chla_tin_sample_mg" and "chla_tin_mg". Label the centrifuge tube accordingly.
Return the weighing bottle to the box desiccator.
Rinse tweezers and scoopulars between two different samples.
For the next sample, use a clean, unused weighing boat.
Follow the chlorophyll protocol: