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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.


Sieving methods 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 Lourinhã Formation. 

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.

Precise 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).

In contrast, 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. 

The methods 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


·    Plastic bowls (four sets of circular: 40cm, 30cm, and squared: 30x30cm. Different coloured sets provides  an easy way to avoid mixing up samples during processing)


·    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 - 30g/day)


·    Packet of Self - adhesive labels


·    Funnels (two)


·    Metal Ashtrays (Two, 10x10cm)


·    One garden trowel


·    TOTAL



In 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).


Figure 1. 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.



1.    Approximately 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.

2.   To 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.

3.   3.Next, 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).

4.    A 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. 2B).

5.    The sediment disaggregated without disturbance (Fig. 2C). This process normally took about 30 minutes.

6.    If 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.

7.    Once 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 temperature.

8.    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 coloured 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).

9.    Once 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).

10.The <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.

11.The <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.

12.Once 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).

13. In 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 sediment.

Figure 2. 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.

Because large 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 away.

To handle hydrogen peroxide should be handled with acid gloves, acid-proof glasses, and an acid-resistant rubber coat.


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