12°_16°C. In order to verify the anatomical
integrity of nematocytes isolated with both
methods, the Trypan blue test (0.4% in ASW)
was applied, a dye that, owing to its molecular
weight (1200 dal), does not penetrate through
intact cell membrane.
The RVD test was carried out as follows: a
suspension placed on a slide was left for about an
hour in a damp room at 12°_16°C, in order to
render adhesion of nematocytes on the surface
possible; then, two strips of bi-adhesive tape
(Scotch 3 M) (Hidaka 1993) were placed at the
margins of the drop of suspension; these strips
were used to ftx a coverslip on the suspension to
allow the total replacement of all experimental
solutions. The test was carried out in three periods on a single nematocyte chosen for its strong
adhesion to the slide during the flow of ASW.
First period: isosmotic ASW for 5 min.; 2nd period: hyposmotic ASW (35%, 710 mOsm by a
reduction of concentration of NaCl) for 10 min.;
3rd period: isosmoticASW for 5 min. The osmotic pressure of all solutions was measured by an
osmometer (Fiske OS). About 20 images of
nematocytes, recorded at regular intervals, were
then elaborated, using a Macintosh Performa
6200 computer, equipped with the suitable programme, to measure the variation of the sagittal
area. The results were then expressed in area relative to the AI Ao control. Statistical analysis was
carried out using Student's t-Test (P
glutaraldehyde (10/0 in ASW) for 45 min, washed
several times with ASW, dehydrated in a series of
alcohols, coated with gold using an Edwards S150
metallizer, and observed with a DSM 950 Zeiss
microscope.
Results
Isolation
The incubation of tentacles of Aiptasia diaphana
in hypertonic ASW at 40°C for 10 min did not
allow us to obtain isolated cellular elements. By
extending treatment with hypertonic ASW to 20
min at 45°C, a few damaged cellular elements
were seen. In particular, nematocytes showed
disorganization of cytoplasm and the cell profile
did not appear well deftned, although control
conditions had gradually been restored replacing
the suspension with isotonic ASW even soon
Effects of Isolation Methods on Characteristics
299
after isolation. After treating the tentacle with
isotonic ASW at 40°C for 10 min with successive
mechanical shaking the tissue still appeared
intact and there were no isolated cellular elements whatsoever. At 45°C for 10 min a few isolated cellular elements were observed, but many
of these were still part of the tissue. After incubating the tentacles in isotonic ASW for 20 min at
45°C, many isolated cellular elements were
obtained, and even if there were a few still
attached to the tissue, they were detached by successive manual shaking. We observed nematocysts, nematocytes and other cells. Among the
nematocytes it was possible to identify microbasic-mastigophore nematocytes, with a length of
between 23-30 pm and basitric-isorhiza nematocytes, with a length of between 12-17 pm. For our
investigation in RVD, we studied, taking into consideration their size, microbasic-mastigophore
nematocytes, isolated by heat dissociation, at
45°C for 20 min.
As far as morphological features are concerned, microbasic-mastigophore nematocytes
showed their membrane to be intact at SEM
observation, and this was confrrmed by cytological test with Trypan blue. Cell volume is almost
completely occupied by the nematocysts, while
the cytoplasm seemed to be relegated into an
equatorial position. A peculiar feature of almost
all nematocytes was that of showing a distinct
cnidocil, (Fig. 1 a) sometimes surrounded by
stereocilia emerging from the same cell (Fig. 1 b).
In fact, immediately after isolation, nematocytes
showing the cnidocil made up 81.8%±5 of the
entire population of nematocytes. One hour after
isolation, keeping the cells at 12-16°C, the value
was 71 %± 15, which then further diminished,
after two hours, to 60%±9.9.
Due to the treatment of tissue with SCNonly microbasic-mastigophore nematocytes
were isolated. These appeared intact on SEM
observation, as also confrrmed by the Trypan
blue test, but they were lacking in the
mechanosensorial apparatus (Fig. 2a), which was
still present in those nematocytes which were
partially extruded in the tentacle (Fig.2b).
RVD
The exposure to a hypotonic stress (35%) of
nematocytes isolated by heat dissociation
induced the expected cell swelling, which after
integrity of nematocytes isolated with both
methods, the Trypan blue test (0.4% in ASW)
was applied, a dye that, owing to its molecular
weight (1200 dal), does not penetrate through
intact cell membrane.
The RVD test was carried out as follows: a
suspension placed on a slide was left for about an
hour in a damp room at 12°_16°C, in order to
render adhesion of nematocytes on the surface
possible; then, two strips of bi-adhesive tape
(Scotch 3 M) (Hidaka 1993) were placed at the
margins of the drop of suspension; these strips
were used to ftx a coverslip on the suspension to
allow the total replacement of all experimental
solutions. The test was carried out in three periods on a single nematocyte chosen for its strong
adhesion to the slide during the flow of ASW.
First period: isosmotic ASW for 5 min.; 2nd period: hyposmotic ASW (35%, 710 mOsm by a
reduction of concentration of NaCl) for 10 min.;
3rd period: isosmoticASW for 5 min. The osmotic pressure of all solutions was measured by an
osmometer (Fiske OS). About 20 images of
nematocytes, recorded at regular intervals, were
then elaborated, using a Macintosh Performa
6200 computer, equipped with the suitable programme, to measure the variation of the sagittal
area. The results were then expressed in area relative to the AI Ao control. Statistical analysis was
carried out using Student's t-Test (P
glutaraldehyde (10/0 in ASW) for 45 min, washed
several times with ASW, dehydrated in a series of
alcohols, coated with gold using an Edwards S150
metallizer, and observed with a DSM 950 Zeiss
microscope.
Results
Isolation
The incubation of tentacles of Aiptasia diaphana
in hypertonic ASW at 40°C for 10 min did not
allow us to obtain isolated cellular elements. By
extending treatment with hypertonic ASW to 20
min at 45°C, a few damaged cellular elements
were seen. In particular, nematocytes showed
disorganization of cytoplasm and the cell profile
did not appear well deftned, although control
conditions had gradually been restored replacing
the suspension with isotonic ASW even soon
Effects of Isolation Methods on Characteristics
299
after isolation. After treating the tentacle with
isotonic ASW at 40°C for 10 min with successive
mechanical shaking the tissue still appeared
intact and there were no isolated cellular elements whatsoever. At 45°C for 10 min a few isolated cellular elements were observed, but many
of these were still part of the tissue. After incubating the tentacles in isotonic ASW for 20 min at
45°C, many isolated cellular elements were
obtained, and even if there were a few still
attached to the tissue, they were detached by successive manual shaking. We observed nematocysts, nematocytes and other cells. Among the
nematocytes it was possible to identify microbasic-mastigophore nematocytes, with a length of
between 23-30 pm and basitric-isorhiza nematocytes, with a length of between 12-17 pm. For our
investigation in RVD, we studied, taking into consideration their size, microbasic-mastigophore
nematocytes, isolated by heat dissociation, at
45°C for 20 min.
As far as morphological features are concerned, microbasic-mastigophore nematocytes
showed their membrane to be intact at SEM
observation, and this was confrrmed by cytological test with Trypan blue. Cell volume is almost
completely occupied by the nematocysts, while
the cytoplasm seemed to be relegated into an
equatorial position. A peculiar feature of almost
all nematocytes was that of showing a distinct
cnidocil, (Fig. 1 a) sometimes surrounded by
stereocilia emerging from the same cell (Fig. 1 b).
In fact, immediately after isolation, nematocytes
showing the cnidocil made up 81.8%±5 of the
entire population of nematocytes. One hour after
isolation, keeping the cells at 12-16°C, the value
was 71 %± 15, which then further diminished,
after two hours, to 60%±9.9.
Due to the treatment of tissue with SCNonly microbasic-mastigophore nematocytes
were isolated. These appeared intact on SEM
observation, as also confrrmed by the Trypan
blue test, but they were lacking in the
mechanosensorial apparatus (Fig. 2a), which was
still present in those nematocytes which were
partially extruded in the tentacle (Fig.2b).
RVD
The exposure to a hypotonic stress (35%) of
nematocytes isolated by heat dissociation
induced the expected cell swelling, which after