16
carolinea, 68
(2010)
1978),
Eifel (F
riedrich
,
oral commun), Franken-
wald (S
teinberg
,
written commun), Sauer­land
­(
G
utowski
and F
oerster
,
oral commun). Never-
theless, a satisfying and clear-cut identification of
this organism has until now not been achieved.
In the present examination of materials from
known places it became clear that a definitive
description of this taxon can not be achieved
without a deeper critical taxonomic comparison
with a number of phormidian species which show
very similar cell dimensions, and without a de-
tailed knowledge of the boring behaviour and of
the resulting pattern of the endolithic layers. To
make progress in this field, special preparations
of the colonized carbonates were necessary, in-
cluding embedding methods with synthetic resin,
dissolving the rock matrix, and SEM treatments.
Based on the forthcoming results an assessment
of the taxon is intended.
Several investigations into limestone-boring were
done earlier (G
olubi
ć
et al.1970, G
olubi
ć
1973,
G
olubi
ć
et al. 1975, S
chneider
1977,
P
ente
-
cost
et R
iding
1986,
S
chneider
&
L
e
C
ampion
-
A
lsumard
1999,
A
rp
1999,
P
entecost
2003,
G
arcia
-
P
ichel
2006),
but there are lacking ex-
amples for carbonate boring Phormidians.
2
Materials and methods
The regional distribution of the cyanobacterium
in question is focused on the flowing waters of
the middle and southern Black Forest, were as
a consequence of changes in the geological
subsoil limestones increasingly appear. For the
present investigation settled limestones from the
river Breg near the town of Hüfingen and from
the river Elz above the town of Emmendingen
were applied.
2.1
Observations with light optical
microscope
Fresh bright blue-green colonized limestones
were transported cooled and wet into the labo-
ratory, where small notches were cut into them
using a scalpel blade. The excavations, as thin as
possible, were directly transferred to the micro-
scope and examined without further preparation.
The light optical microscope photographs were
taken with a NIKON-Coolpix 995 digital camera
(3,34
megapixel), connected with a C
arl
Z
eiss
Photomicroscope I. Sketching the drawings was
supported by the use of the “Great Z
eiss
drawing
apparatus”.
2.2
SEM examinations
In order to obtain information about the boring
procedure and the three-dimensional pattern of
the endolithic thallus, two different steps of inve-
stigation were needed: (1) The inspection of frac-
tured areas, (2) the release of resin-embedded
endolithic parts of thallus from the limestone ma-
trix.
2.2.1
Preparation of open fractures
Densely colonized limestones, just taken from
the river bed, were divided by hammer and chisel
into little blocks and fixed in 4 % formaldehyde
for 24 hours. They were then processed step
by step through increasing alcohol-water and
alcohol-acetone solutions until 100 % acetone
was ­reached. Afterwards the objects were dried
follow­ing the “critical point “-method in order to
avoid any shrinkage. Only just before the SEM-
procedure started, a little piece of dried limestone
was broken up at a prepared position (sawing),
treated according to the usual SEM-preparation
methods, and examined immediately so as to
prevent abrasions of the area in the interim.
2.2.2
Embedding – casting technique
After fixing in 4 % formaldehyde, pieces of re-
cently sampled limestones were treated following
the method given by G
olubi
ć
et al. (1975): Putting
the fixed objects into NaOCl (Clorox, M
erck
)
for
24-48
hours, rinsing with tap water and gradually
increasing the acetone content to 100 %. During
this process, negative pressure was temporarily
applied (3 hours in an exsicator) in order to re-
move possible small air bubbles out of the pores.
From acetone the objects were transferred into a
covered vessel containing a 1:1 mixture of ace-
tone : Araldite (compl.). Used Araldite: C
arl
R
oth
,
Karlsruhe, type 11 (CY212), 1:1 composed with
hardening agent HY 964, incl. accelerator 964
(
DY 064). The time in the acetone-resin solution
lasted 6 hours in total. After 3 hours, the lid was
taken away in order to start the evaporation proc-
ess of the acetone. The following transfer into a
pure complete Araldite-mixture was performed in
little troughs moulded out of copper foil. In order
to achieve the hardening of the resin, the troughs
remained uncovered over night at room tempera-
ture and were then heated at 60 °C for 48 hours.
From the hardened blocks the overlapping resin
was sanded down and the stone broken up to
provide open areas for acid treatment. The car-
bonate substrate was etched by 2 % hydrochlo-
ric acid. The remaining Araldite-framework of