Materials Included In Kit

Additional Materials Required

Prelab Preparation

Cut the grid strips from the transparency sheet. Mix the artificial seawater (36 g of marine salts per liter of distilled water). The brine shrimp cysts can be subdivided into smaller batches so that more student groups can be retrieving cysts at once. Microcentrifuge tubes work well for this purpose.

Safety Precautions

Follow normal laboratory safety rules. Students should wash their hands thoroughly upon completion of the laboratory.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. Never release living specimens into the local ecosystem. Hatched brine shrimp larvae make excellent freshwater tropical fish food (after they are transferred briefly to spring water to diminish salt concentration). Use as fish food in a fish tank. If no fish tank is available, sterilize the culture of brine shrimp using Flinn Scientific Biological Waste Disposal Method I.

Teacher Tips

• Enough materials are provided for 30 students working in pairs or for 15 groups of students. The initial setup can be completed in a partial class period. Additional class time is required for the next four days for follow-up observation and record keeping.

• Students should be familiar with basic embryological concepts such as blastulation and gastrulation prior to doing this laboratory. A background reading is included for your use. It includes drawings of the various stages of the development of brine shrimp. You may choose to provide some of this information for your students during or after their lab work.

Further Extensions

1. Will hatching of cysts occur with lower or higher salinities? Suggestion: Compare hatching in normal seawater (= 100%), 10%, 1%, 0% and 200%. Relate the results to possible conditions in natural environments.
2. Will brine shrimp cysts, which fail to hatch after an initial hydration, successfully hatch after a second hydration? To answer this question, hydrate and hatch a batch of cysts that are attached to tape, as described above. Then, after allowing hatching for 4–5 days, pour off all excess water and let the tape, along with attached cysts, completely dry out for 1–2 days. Then rehydrate attached cysts in salt water. If some cysts do hatch after a second hydration, explain the possible biological/ecological significance of this result.
3. Will brine shrimp cysts hatch if they are confined and crowded into a small space? To address this question, use two separate transparency-grid strips, each with cysts attached to tape, prepared as described above. Use two additional pieces of tape to attach corners of the strips to the bottom of a dry Petri dish, one strip with cysts facing up and the other strip with cysts facing down. Be very careful not to apply direct pressure to cysts—they are fragile and may rupture. Add salt water. Compare the developmental progress of the two groups, especially noting when and how many cysts crack open as well as when and how many nauplius larvae emerge and swim away. Explain your results in relation to key environmental factors that could retard or enhance hatching and stimulate embryonic development.
4. Is the hatching success the same for cysts that are kept continually in the dark, as compared to cysts kept in continuous light?
5. Is the hatching success reduced or delayed if hydrated cysts are kept in the refrigerator at 5 °C, as compared to room temperature?

Teacher Handouts

10385_Teacher1.pdf

References

Special thanks to Dr. Charlie Drewes, Professor of Zoology and Genetics, Iowa State University, for providing this activity.

Introduction

Just add seawater to a small number of brine shrimp cysts and they seem to magically develop and hatch. Brine shrimp (Artemia) are amazing small organisms with the ability to survive severe environmental conditions. Now you can follow the developmental fate of individual cysts. Watch nauplii larvae hatch and swim!

Concepts

• Cryptobiosis

• Embryonic and larval development
• Environmental biology

Background

“Cryptobiosis” is a state of suspended animation induced by environmental adversity. An example of cryptobiosis is the arrested embryonic development that occurs in invertebrates, such as brine shrimp, fairy shrimp and tadpole shrimp. Prior to release by the female, the developing embryos of these organisms are encysted in a protective shell that enables them to survive desiccation and other environmental extremes. Embryos may remain viable in the encysted, cryptobiotic state for many years.

Within the brood sac of female brine shrimp, egg development proceeds rapidly through cleavage and blastula stages. Eggs are then deposited in the environment where they may remain encysted, with embryonic development arrested at the early gastrula stage. At this time, there are about 4,000 highly organized cells in the embryo, but no organs are discernible.

When encysted eggs are exposed to more favorable conditions (rehydration), the eggs swell and rapid development of the embryo resumes. Hatching typically occurs in 1–2 days, depending upon the temperature.

Brine shrimp cysts (in the gastrula stage) are abundant in environments such as the Great Salt Lake. The cysts are lightweight, small (about 0.25 mm diameter) and tend to float. This makes them difficult to handle, see, and study systematically. The techniques used in this lab help to anchor the cysts down in one place. This allows the tracking of individual cysts as they complete development and permits precise determination of hatching on a day-to-day basis.

Materials

Safety Precautions

Follow normal laboratory safety rules during this laboratory. Wash hands thoroughly upon completion of the lab work.

Procedure

1. Be sure your hands and the small brush are completely dry before working with the Artemia cysts.
2. Obtain a 2 x 5 cm transparency strip containing a 10 x 10 mm marked grid.
3. Cut a 2 cm long strip of double-stick tape making sure to handle the tape by the edge with a forceps. Do not get fingerprints on the tape.
4. Carefully place the strip of double-stick tape over the top of the grid pattern as shown in Figure 1.

5. Using the wooden tip of the brush handle, trace around the edge of the tape so that the tape is securely fastened to the transparency strip.
6. Using the brushy end of the brush, touch just the tip of the brush into the dried brine shrimp cysts. Numerous cysts should attach to the tips of the brush fibers. If too many cysts attach to the tip of the brush, lightly tap the tip of the brush on the rim of the container so that some cysts fall back into the container. Carefully and gently “paint” the cysts attached to the brush tip onto the sticky surface on top of the grid. Brush very gently back and forth with the tip of the brush to make sure the cysts are secured to the tape. Do not press the brush hard against the tape or the cysts will be damaged.
7. Repeat this procedure, if necessary, until a total of 50–100 cysts are stuck to the tape within the grid area (see Figure 2). Do not worry if a few cysts are attached outside the grid area.

8. Grasp the edge of the strip with your fingers and use a finger on your other hand to gently flick the edge of the transparency strip. This will dislodge any cysts which are not securely stuck to the tape. Do this gently since the cysts are fragile.
9. Attach a small piece of double-stick tape to the underside of the transparency as shown in Figure 2. Do not touch the cysts on the grid area.
10. Lay the transparency strip into the bottom of a dry Petri dish with the cysts and grid facing upwards. Press down on the strip where the small piece of tape was placed in step 9. This will fasten the transparency strip to the bottom of the dish and prevent it from floating once the seawater is added.
11. With the assistance of a stereo microscope, make a map showing the precise location of all cysts on the grid (see Figure 3, which shows 73 cysts attached). Count the original number of cysts that are anchored to the tape on your grid.

12. Flood the Petri dish about half-full with artificial seawater (about 20–25 mL) making sure the strip is fully immersed. Cover the Petri dish, label it, and place it in a room with continuous light at room temperature.
13. Inspect the cysts immediately after adding seawater and again after one day. Do cysts change in shape? Explain and make sketches of your observations and record them on the Artemia Worksheet.
14. Each day check for swimming nauplius larvae. Use a pipet to remove swimming larvae. (Note: It may be necessary to add some seawater to the dish if too much is removed when the nauplius larvae are transferred and counted.) Record the number of larvae removed each day on the data sheet. Plot the data as percent hatched against time. After four days, few if any brine shrimp should still be hatching.
15. Consult with your instructor for appropriate disposal procedures.

Student Worksheet PDF

10385_Student1.pdf