TITLE: Extraction of meiofauna and meioflora from aquatic environmental samples
AUTHORS: Daisuke Shimada

[DESCRIPTION]
Meiofauna and microalgae are benthic communities of small animals and algae, respectively, that can pass through a 1-mm sieve but are retained in a 32-µm sieve. This simplified protocol collects meiofauna and microalgae living in or attached to aquatic substrates such as sediments, seagrasses, seaweeds, and sessile animals. Animals and algae are filtered, killed by heating, fixed with DESS solution, and sorted under a stereomicroscope. The advantage of this method is that both the morphological characteristics and DNA can be preserved for a long period (more than ten years).

[STEPS]
SECTION: Sampling
1. Collect substrates (sediments, seagrasses, seaweeds, sessile animals, etc.) by hand or by using sampling devices.
SECTION: Filtration
3. Transfer 100–500 ml of substrate into a bucket and wash the substrate well with approximately      10 times volume of tap water. If the substrate is mud, reduce the substrate volume to 50 ml or less. For fragile animals, such as turbellarians, use 3.5% MgCl2 instead of tap water and stir gently. Wait for 10 min or more and stir again gently.

References: Hulings & Gray (1971); Pfannkuche & Thirl (1988); Giere (2009)
 

 

 



SECTION: Sampling
2. Transport entire substrates to the laboratory at low temperature or immediately filter and fix the living organisms in the field by following the steps below. If performing this step is difficult, substrates should be frozen or stored in the DESS solution, despite the risk of injury to organisms.
SECTION: Filtration
4. Immediately pass the supernatants through a 10–100 μm (usually 32 or 63 μm) nylon mesh filter. Smaller mesh sizes can collect more organisms but are easily clogged with debris.
SECTION: Filtration
5. Repeat steps 3 and 4 once or twice.
SECTION: Filtration
6. Carefully remove the nylon mesh from the bottle, turn over, and wash in a Petri dish with 20 ml of filtered seawater. When handling a large substrate amounts or when the mesh appears clogged, the collected material can be transferred to a Petri dish with a dropper after each filtration.
SECTION: Filtration
7. Transfer the whole seawater with collected material to a 25–50 ml sampling bottle. If necessary, divide into several bottles.
SECTION: Fixation
9. Heat the sampling bottle in 80–90°C water for 2 min to kill the organisms and to inactivate the DNase. Several animals, especially nematodes, straighten when killed by heating. When looking for smaller animals, such as tardigrades, which are easier to find when they are alive, the order of heat killing,      fixation, and sorting can be switched.
SECTION: Fixation
10. Add 5 to 10 times the volume of DESS solution to the collected material and mix well. The sample can be stored in this state for a long period (more than 10 years).

Reference: Yoder et al. (2006)
 

SECTION: Fixation
10.1. How to make 1 liter of DESS solution
Dissolve 93.0 g of 2Na-EDTA in 400 ml of DW by stirring while gradually adding NaOH. Adjust pH to 7.5–8.0 and add DW until the total volume reaches 500 ml (= 0.5M EDTA).
*Buying a pre-conditioned EDTA solution is less time-consuming (but more expensive).
SECTION: Sorting
11. Transfer the sample to a Petri dish. Pick up organisms from the collected material using an Irwin loop under a stereomicroscope and store in 2.0-ml screw-cap micro tubes of DESS solution. To prevent rusting, rinse the loop with DW after each use.

References: Shirayama (2004); Schram & Davison (2012)
 

 

SECTION: Sorting
11.1. How to make an Irwin loop
Cut a stainless-steel wire (0.2–0.3 mm diameter) into 10 cm and 5 cm pieces.
SECTION: Fixation
10.2. Mix 500 ml of 0.5 M EDTA with 200 ml (or 200 g) of DMSO and add DW until a final volume of 1,000 ml.
SECTION: Fixation
10.3. Add a saturated amount of NaCl and stir well (precipitation of NaCl is not a problem).
SECTION: Filtration
4.1. How to make a filter
Remove the bottom from a 500 or 1,000 ml wide-mouth screw-cap plastic bottle and cut a piece of nylon mesh larger than the mouth of the bottle.

 

SECTION: Filtration
4.2. Place the mesh over the mouth of the bottle and fasten tightly with rubber bands.



 
 

SECTION: Sorting
11.2. Hook the pieces together and twist them using pliers, leaving small loops.


 

SECTION: Sorting
11.3. Remove the shorter piece and fix the longer piece to a suitable handle with water-resistant adhesive.
SECTION: Sorting
11.4. Bend the loop part to a manageable angle with pliers.



 
 

SECTION: Filtration
8. Wait 10 min for the organisms to settle at the bottom and remove as much of the supernatant as possible with a dropper.