14 Echinodermata
159
- Purple sea urchins Strongylocentrotus purpuratus, or the red sea ur- Animals
chin Strongylocentrotus franciscanus. (These animals can be collected
on rocky beaches. Trauma and consequent bacterial contamination of
coelomic fluid are reduced by picking the urchins up quickly before
their tube feet can attach firmly to the substrate and, thus, be torn off.
Animals are placed in large plastic bags in buckets of seawater for
transport to the laboratory. Tube feet do not adhere firmly to the plastic bags, thus minimizing further tearing of tissues)
Procedure
1. Bleed the animals as soon as practicable after collection to reduce the Bleeding
time of exposure to stressful conditions that often lead to bacteremia.
2. Collect coelomic fluid through the peristomial membrane, using a different sterile siliconized syringe and 3.18 cm,22-G hypodermic needle
for each animal.
3. Draw the fluid slowly, remove the needle from the syringe, and dispense the fluid slowly down the side of a sterile siliconized IS-ml conical-bottom centrifuge tube. Between 1 and 15 ml fluid can be obtained
from a single animal. If repeating extraction of fluid from the same
animal, leave the needle in place in the peristomial membrane while
the fluid is being dispensed from the syringe into the centrifuge tube.
When the coelomic fluid can not be obtained sufficiently from a single
animal, the fluid from several animals may be pooled.
4. Keep the filled tubes at 4°C for 4-24 h to allow settling of the suspended cells.
1. Use sterile siliconized Pasteur pipettes, with tips drawn out further in Culture
the flame of a small alcohol lamp, to withdraw sedimented cells for set-up
hanging drop culture.
2. Prepare hanging drops with the cell suspension on cover glasses.
3. Turn the slide glass over a depression slide glass, and seal the rim of
the cover glass with melted bees wax.
4. Keep the preparations at 4°C.
Results
According to Johnson (1969), four types of cells are recognized in these
types of cultures. These cell types were phagocytic leukocytes, vibratile
cells, red spherule cells, and colorless spherule cells. Following contact
with a surface, the phagocytic leukocytes changed from bladder amoebocytes to flattened syncytial cells. Apparently only phagocytic leukocytes were involved in cellular clotting, and extracellular gelling of the
coelomic fluid was not observed. The cells survived for more than 1
month.
159
- Purple sea urchins Strongylocentrotus purpuratus, or the red sea ur- Animals
chin Strongylocentrotus franciscanus. (These animals can be collected
on rocky beaches. Trauma and consequent bacterial contamination of
coelomic fluid are reduced by picking the urchins up quickly before
their tube feet can attach firmly to the substrate and, thus, be torn off.
Animals are placed in large plastic bags in buckets of seawater for
transport to the laboratory. Tube feet do not adhere firmly to the plastic bags, thus minimizing further tearing of tissues)
Procedure
1. Bleed the animals as soon as practicable after collection to reduce the Bleeding
time of exposure to stressful conditions that often lead to bacteremia.
2. Collect coelomic fluid through the peristomial membrane, using a different sterile siliconized syringe and 3.18 cm,22-G hypodermic needle
for each animal.
3. Draw the fluid slowly, remove the needle from the syringe, and dispense the fluid slowly down the side of a sterile siliconized IS-ml conical-bottom centrifuge tube. Between 1 and 15 ml fluid can be obtained
from a single animal. If repeating extraction of fluid from the same
animal, leave the needle in place in the peristomial membrane while
the fluid is being dispensed from the syringe into the centrifuge tube.
When the coelomic fluid can not be obtained sufficiently from a single
animal, the fluid from several animals may be pooled.
4. Keep the filled tubes at 4°C for 4-24 h to allow settling of the suspended cells.
1. Use sterile siliconized Pasteur pipettes, with tips drawn out further in Culture
the flame of a small alcohol lamp, to withdraw sedimented cells for set-up
hanging drop culture.
2. Prepare hanging drops with the cell suspension on cover glasses.
3. Turn the slide glass over a depression slide glass, and seal the rim of
the cover glass with melted bees wax.
4. Keep the preparations at 4°C.
Results
According to Johnson (1969), four types of cells are recognized in these
types of cultures. These cell types were phagocytic leukocytes, vibratile
cells, red spherule cells, and colorless spherule cells. Following contact
with a surface, the phagocytic leukocytes changed from bladder amoebocytes to flattened syncytial cells. Apparently only phagocytic leukocytes were involved in cellular clotting, and extracellular gelling of the
coelomic fluid was not observed. The cells survived for more than 1
month.