Egg Counting, Storage, and Hatching

Counting Protocol

1. Wash freshly collected eggs free of debris, and deposit in a 50 ml Falcon tube, filled to the 30 ml waterline.
2. Vigorously agitate the tube and extract a 0.5 ml sample, dilute to 10 ml.
3. Extract and count a 0.5 ml sample of the diluted stock. This represents 1/1,200 of the total egg collection (1/60 x 1/20). The goal is to count 60-100 eggs per 0.5 ml of diluted sample.
4. If the sample is still too concentrated, remove 0.5 ml of the first dilution and bring to 10 ml.
5. Extract and count 0.5 ml of the second dilution. This represents 1/ 24,000 th of the total egg collection (1/60 x 1/20 x 1/20).
6. Count three replicates at a suitable dilution, average the three replicates, and compute the total egg collection for the 50 ml tube (use the correct dilution factor!).
7. Set out three counted replicates of 60-100 eggs each in 100 mm Petri dishes with covers for 48 h hatch-ratio determination. Fill 85% full with treated seawater, label, and cover
8. Label and store remaining eggs or set out to hatch. Label should include date, species, egg count, and tank number.

Storage Protocol

1. Storage of eggs is useful, both as a reservoir of nauplii to restart production tanks, and as a supply of nauplii for feeding fish or invertebrate larvae and adults.
2. Acartia non-diapause eggs can be stored for the two to six weeks. Shelf life may vary between Acartia from different locations.
3. To successfully store eggs, they must be fastidiously cleaned of all feces and detritus. Wash eggs through a 100 µm sieve into a beaker (to fracture the feces), pour that slurry through a 70 µm sieve, and wash carefully until little detritus remains.
4. Count eggs prior to storage and label the storage vessel to indicate species, collection date, egg count, and tank.
5. Store (up to) 5,000,000 eggs per 50 ml Falcon tube.
6. Storage is most successful when temperatures are stable around 1° C.
7. Egg viability from the Gulf of Mexico strain of A. tonsa, fed Cryptomonads exclusively, drops rapidly during storage. Presuming a 90% hatch ratio on the initial storage date, one week’s storage will reduce this to ~45% hatch, and two week’s storage will further reduce this to ~25%, presuming well cleaned eggs.

Hatching Protocol

1. Compute the number of nauplii required for hatch (nauplii required divided by the presumed hatch ratio plus a surplus for security). For example, to hatch 200,000 nauplii to start a 200 L tank:

Divide 200,000 by the 50% presumed hatch ratio for eggs stored a week.
200,000 eggs/ 0.50 hatch = 400,000 eggs
plus 25% more to avoid aggravation if the hatch ratio is lower
= 500,000 week-old eggs required


2. Count the eggs as previously described or read the label on the Falcon tube, compute the required portion of the 30 ml of stored eggs.
3. Rinse the eggs well on a 53 µm sieve.
4. Add approximately 50,000 eggs/L to treated, aerated seawater, cover the vessel, and use no aeration.
5. Incubate 48 h at 25° C.
6. Collect nauplii on a 53 µm sieve, dilute to 1000 ml, mix thoroughly, count nauplii in three 0.5 ml samples, average, and multiply by 2000 for total nauplii count. Record result.
7. For large naupliar counts serially dilute as for counting eggs. Keep accurate record of the dilutions used to facilitate population computation.