The use of
confocal laser scanning microscopy (CLSM) for the study of inclusions in amber
is a recent development. Böker & Brocksch (2002) produced a series of images of
insects in Baltic amber using CLSM and identified the potential for 3D imaging
of minute detail of taxonomically important morphological structures such as the
mandibles and genital organs. Since then, however, very little has been
published on CLSM analysis of amber inclusions despite the apparent benefits.
for microvertebrate recovery have rarely (Mateus et al. 1997) been applied to
the Lourinhã Formation. The Lourinhã Formation is comprised of fossiliferous
fluviodeltaic deposits that outcrop extensively in the Lusitanian Basin, Western
Portugal. This formation is mainly composed of intercalated sandstone channels
with extensive alluvial mudstone layers (Hill 1989). Within the extensive
mudstone layers most fossils including many large vertebrates (Antunes and
Mateus, 2003) and microfossils are found (e.g. Ramalho 1967). Microvertebrate
fossil faunas have been collected for many years in the Lourinhã Formation,
mainly by surface collecting. However, in 2008 the Museum of Lourinhã started a
systematic sieving campaign to better understand the overall fauna of the
Some of the
first microvertebrate finds in Portugal were discovered in the Guimarota mine in
1960 by palaeontologists from the Freilicht Universitat, Berlin (Krebs, 2000).
The first account of sieving methods being used in Portugal were published by
Kühne (1968), using a constant flow of water and sieves incorportated on a metal
barrel. Sieving was applied at the Paimogo theropod embryo nest site (Mateus et
al. 1997) from 1994 to 1996, in the search for embryo bones and eggshells (Mateus,
1998) and was also occasionally applied to the Porto das Barcas fossil site, but
without much success.
geographical information of sieving sites in the Lourinhã Formation was acquired
using GPS coordinates, stratigraphic information and a measured section was
acquired by plotting the site photograph with detailed geologic annotation.
Samples were taken using a pick axe at 30 - 40cm stratigraphic intervals and
within 1 - 2m horizontal spacing. This systematic sieving campaign, applied to
the Lourinhã Formation for the very first time, is being used to investigate and
assess the composition and diversity of the microfossil fauna.
The first use
of sieving in the search of microvertebrate remains was performed ca. 1847 by
Plieninger, in Germany (McKenna et al. 1994). Early methods of sieving were also
used by Moore in England (1867) and later by Wortman and Brown in the United
States (1891). Sieving became well implemented in the palaeontological
community after Hibbard (1949) reported using the technique to collect Cenozoic
mammal fossils from an unconsolidated sandstone matrix (McKenna et al., 1994).
Hibbard introduced the use of screen boxes (wooden boxes with a brass mesh).
However, this method requires having water near the work site, is laborious, and
requires a large staff (Ward 1984). McKenna (1962; 1965) provided further
insight into the screen box technique and proposed a standard model using
manufactured rectangular wooden boxes and a regular size steel mesh. Both
McKenna’s and Hibbard’s techniques are field-oriented and require the presence
of nearby water. McKenna simply optimized Hibbard’s technique by processing a
larger quantity of matrix using more screen boxes (almost 300, compared to
Hibbard’s dozen), and adapting the method to the constraints of particular
sites. Grady (1979) described a new method using mosquito nets instead of the
classical material of screen boxes with brass mesh, providing a more
field-oriented method with easily transported equipment (Ward, 1984).
the method reported in this paper is similar to other laboratory-oriented
techniques. Described in further detail by Kühne (1971) and Krebs (2000), the
“Henkel technique” (Henkel, 1966) is a laboratory-oriented technique that was
used for more than a decade during the time that microvertebrates were being
collected from the Guimarota Mine. This technique is a static sieve method
making use of a jet of water that passes through a barrel with a 500µm -mesh on
the side. Freudenthal (1976) developed a table technique. This “table” stood 1m
high and used a 500µm mesh as a replacement for the table top. Solid side bars
were attached in order to avoid sediment loss. A jet of water was used to wash
the sediment through the table top.
above described provide excellent guidance for new field researchers, but due to
peculiarities of each site/investigation, modifications to the methods may be
required. Aspects such as the location of the fossiliferous horizon (i.e.
remoteness), budget and laboratory conditions (i.e. pre-existing
infrastructures) can hinder recovery of specimens in an identifiable condition.
The methodology here described does not aim to process vast amounts of sediment,
as others can. Instead, it does allow individual horizons to be processed
without the potential of mixing sedimentary beds. When a sedimentary bed is of
limited vertical extent the field-orientated techniques require extensive
excavation of the target horizon, losing possible valuable horizons, or mixing
multiple sedimentary layers. This can hinder the investigation of fine scale
ecological, climatological and evolutionary patterns.
The list below
is the equipment used for this technique during the 2008 Museu da Lourinhã field
season with approximate prices (see fig. 1):
· Glass bottles (donated free by local cafés)
· Fine paint brushes (diameters 0.25 to 0.5cm)
· Sieves of different mesh sizes (15000µm, 750µm, 500µm. Sizes
estimated using a grain size comparator chart) - four of each
bowls (four sets of circular: 40cm, 30cm, and squared: 30x30cm.
Different coloured sets provides an easy way to avoid mixing up samples
· Plastic Trays (20, for drying, 20cm in diameter)
· Latex gloves (one pair per worker per day, 200 pairs)
· Hard - water softeners like polycarboxylates (e.g. Calgon®) (10 -
· Packet of Self - adhesive labels
· Funnels (two)
· Metal Ashtrays (Two, 10x10cm)
addition, supplementary laboratory supplies and equipment are required:
· Hand lens (~10€), Respirators (~30€), Washbasin (preferably with
shower), Lab coat(s), hydrogen peroxide (1l, 5%v/v), Optical microscope
(1000 - 4000€, Magnifications range 0,63 to 8,65X).
Materials used for sieving: A-- transparent bowls used to dissolve the boulders
collected in the field; B- 15000µm sieve; C-garden shovel used to move sediment
between bowls; D- 750µm sieve; E- various small-sized bowls (30x30cm); F-
large-sized bowl (30cm diameter); G-trays to dry sediment; H- painted ashtray
for picking, and various brushes; I- labeling material; J- small transparent
boxes used to store sorted specimens and glass juice bottle used to store
unpicked sediment; K- binocular microscope.
45 rock samples were collected, each sample weighing approximately 5 kg. We took
into consideration precise geographical and stratigraphic location, broad
objectives for scientific outputs, and sediment trauma. The rock samples were
stored in sealable polythene bags (Ziplock®) to avoid any contamination and were
labeled using a water resistant black marker pen.
start the sieving process, a 30cm diameter plastic bowl (Fig. 2A) was filled
with hot water, at a temperature below boiling (60 to 70ᵒC) in order to avoid
weaken the structural integrity of the plastic.
5 to 20 ml of hydrogen peroxide was added to the hot water, different quantities
can be added depending on how compact the sediment is. The use of hydrogen
peroxide has been shown to be an effective way of liberating clay minerals from
microvertebrate specimens without the damaging effects of other commonly used
chemicals, such as acids (Wilborn, 2009).
15000µm sieve was placed into the bowl of hot water and as much sample as
possible was put into the now submerged sieve, typically around 500 grams (Fig.
sediment disaggregated without disturbance (Fig. 2C). This process normally took
about 30 minutes.
the sediment was still compact after 30 minutes. The water was changed for fresh
hot water, except for a 1cm layer above any disaggregated sample (to avoid any
loss), and 5 - 20 ml of hydrogen peroxide was added.
there was only
a little sediment (and perhaps some fossils) left in the 15000µm sieve, the
sediment in the sieve was emptied onto a tray. Each tray used was labeled with
the appropriate sample number and left to dry in the sun. Spotlights or a
radiator within the laboratory can achieve the same result by increasing the
40cm diameter plastic bowl was filled with cold water. A small garden trowel was
used to transfer the sediment to the 750µm sieve. Using a trowel or any similar
tool instead of hands is an effective way to transport sediment without loss.
The sieve was filled to the top as it proved quicker to sieve larger portions of
the sample than smaller ones (Fig. D, E, F). Agitation was performed with the
sediment continually immersed in water as the sieve was shaken by hand in
circular movements. As the water became cloudy it was necessary to replace it,
taking care not to lose any of the sieved material. When refilling the bowl
caution should be taken in directing the flow of water against the side of the
bowl, thus causing minimal agitation to the sieved material. Once the sample
within the sieve was considered clean (no visible clay
streak appeared in the water when shaking) it was transferred to a labeled
plastic tray. Any objects trapped within the sieve mesh were gently freed by
gently tapping the sieve (Fig. 2 G, H).
the sample was sieved through the 750µm sieve, the remaining proportion of the
sample (the <750µm portion of the sample) is left in the bowl and the fraction
from the sieve was transferred to a plastic tray. The tray was labeled and left
to dry (Fig. 2I).
<750µm portion was then sieved again with a 500µm sieve. After this step, there
remained a portion of the sample with a grain size under 500µm.
<500µm portion was washed using a shower nozzle with low pressure. The operator
used his hand to create turbulence within the plastic bowl to carefully put the
sediment into suspension, helping to liberate the clay fraction. The <500µm
fraction was allowed to dry.
all the sieved samples
were dried they were transferred to appropriate containers
for study under the microscope. The containers used for this field season were
glass juice bottles that were washed, cleaned with water, and dried prior to
this procedure. The containers were
labeled using adhesive labels for outside the bottle and a
small slip of paper within the bottle (Fig. 2K).
the case a sample needs to be left part-way through processing, it is advised
that the water used during sieving be drained and replaced with fresh cold
water. This way the clay does not dry overnight which can cause clumping of the
Some aspects of the sieving methodology: A-- Lab during the sieving
season (note the different colored bowls), B-- the clay boulders are left to
disaggregation in the 15000µm sieves, C-- a dose of hydrogen peroxide can be
added if the boulders are hard to dissolve, D-- with a garden shovel the
sediment is transferred to the 750µm sieve, E-- the bowl to which the sieved
sediment will go to can be filled with cold water, F-- sediment passes through
the sieve with circular movements in the water, G-- in order to minimize the
amount of time sieving, small parcels of sediment should be done at a time, H--
sediment passes through the 500µm sieve, I-- the sediment is left to dry at
atmospheric conditions, J-- if the weather does not permit the sieved sediment
can be left to dry under strong light, K-- store the dried sediment in juice
glass bottles, L-- by the end of the day clean the pipes using deflocculating
agents and hot water.
The major risk
associated with this technique is when handling the hydrogen peroxide. Gloves,
respirator, lab coat and boots should be used by the operator. The process is
best done in pairs to allow one person to poor the hydrogen peroxide and another
to ensure there is no spillage.
To avoid sample
cross contamination sieves should be cleaned after they are used. Washing the
sieve in water and using a stiff brush is typically enough to remove most
particles. Whilst a toothpick can be used to free the most stubborn quartz
grains from a sieve mesh.
amounts of sediment were dumped into the laboratory sink, hard - water softeners
(e.g. Calgon®) and hot water were poured down the drain at the end of each
working day. To further prevent blockages from occurring, the trap below the
sink was emptied and manually cleaned regularly (Fig. 2L). An alternative to
dumping residue down the drain is to let it settle in the basin overnight, and
decant the water off the top down the drain. Let the residue dry and throw it
hydrogen peroxide should be handled with acid gloves, acid-proof glasses, and an
acid-resistant rubber coat.