The ideal calcareous nannofossil slide contains particles with sizes ranging from 0 to 20 μm, exhibiting an even spatial distribution across the slide. The gap between individual particles should be sufficient to allow calcareous nannofossils to be easily located and identified.
The particle size variation must remain consistent among nannologists but can be adjusted based on the age of the sediments. This size variation can be controlled by the time allowed for particles to settle from suspension before pipetting the sample. The spatial distribution of particles may vary between nannologists and can be influenced by the amount of distilled water used during the sample preparation process.
During sample preparation, it is essential to exercise utmost care to avoid contamination. To minimise risks, use inexpensive disposable equipment whenever possible. For reusable equipment, such as glassware and brushes, ensure proper cleaning by soaking them in a 10% hydrochloric acid (HCl) solution to remove any calcareous residues. Afterwards, rinse the equipment thoroughly with distilled water to ensure it is contamination-free before reuse.
Standard smear and spray slide methods are valuable for quantitative studies, particularly for determining absolute abundances. However, counting accuracy can be enhanced using a consistent sediment weight for each sample (Henriksson, 1993).
Okada (1992) enhanced counting accuracy by incorporating microbeads of equal weight into sediments of the same weight. The mixture of sediments and microbeads was then smeared or sprayed onto the slide using an exact measured weight.
Beauford (1991) developed a random-settling technique to ensure a more uniform particle distribution on the slide, further improving the reliability of counting.
The short centrifuge technique, introduced by Edwards (1963) and modified by Pienaar (1966), effectively concentrates particles within the size range of calcareous nannofossils, resulting in clean sediment preparations. This technique is especially beneficial for SEM examination and capturing high-quality SEM and optical microscopy images.
The thin-section preparation technique is seldom used and is typically reserved for cases where carbonate-rich sediments cannot be disaggregated. Standard thin sections are approximately 30 μm thick, but are manually thinned further to about 10 μm for specific applications. This technique is particularly useful for studying larger specimens, such as Nannoconus.
The mobile mount technique facilitates the observation and documentation of morphologies and optical properties across different profiles of the same specimen. It ensures the reliable identification of species that settle randomly on either side or in a plan view, owing to their consistent height-to-width ratio. This approach is especially valuable for examining species that may appear to exhibit superficially similar taxonomic characteristics in one profile but display significant morphological variations in another. Understanding the profile of the specimen is crucial when performing extinction angle measurements and determining the direction of extinction. The mobile mount technique plays a vital role in facilitating this process.
Standard Smear Slide
• Clearly label the microscope slide.
• Place a small sample piece representing sedimentary rock directly onto the glass slide. Add one or two drops of distilled water.
• Crush the sample with a glass rod to create a thick suspension (clean the rod with diluted hydrochloric acid after each sample preparation).
• Using a paintbrush (clean the brush with diluted hydrochloric acid after each sample) or a toothpick (use a new toothpick for each sample), smear the suspension evenly and thinly across the slide surface.
• Dry the slide on a hot plate.
• Affix the coverslip using an optical mounting medium (Norland Optical Adhesive 61).
Spray Slide
• Wrap the small, representative sample in a paper towel.
• Gently crush it with a small hammer, then place the crushed sediment into a beaker and add distilled water.
• Soak the mixture for 1-12 hours, depending on preference. To minimise the risk of etching, dissolution, or overgrowth of nannofossils, consider reducing the soaking time or skipping this step entirely.
• Stir the mixture and let it disintegrate as needed.
• Thinly spray the suspension onto the labelled glass slide using a disposable pipette.
• Dry the slide on a hot plate.
• Affix the coverslip using an optical mounting medium (Norland Optical Adhesive 61).
Short Centrifuging
• Wrap the small, representative sample in a paper towel and gently crush it with a small hammer.
• Place the crushed sediment into a beaker and add distilled water. Soak for 12 hours.
• Decant the suspension into a labelled test tube and place it into a centrifuge.
• Spin at 350 rpm for 30 seconds, then stop the centrifuge to discard particles greater than 30 microns.
• Decant the supernatant into another labelled test tube and add distilled water.
• Spin the decanted supernatant at 850 rpm for 1 minute, discarding the supernatant containing particles less than 2 microns.
• Re-suspend the settled sediments in distilled water and repeat the process until the supernatant is clear.
• Dilute the settled sediments and spray them onto the slide.
• Dry the slide on a hot plate.
• Affix the coverslip using an optical mounting medium (Norland Optical Adhesive 61).
Gravity Settling
• Wrap the small, representative sample in a paper towel and gently crush it with a small hammer.
• Place the crushed sediment into a beaker and add distilled water.
• Stir the suspension and wait 1-2 minutes for the sediment to settle, then discard the settled fraction.
• Transfer the supernatant to another beaker.
• Allow the suspended sediment to settle for 10-15 minutes, then discard the supernatant.
• Use the settled fraction for further preparation.
• Dilute the settled sediments and spray them onto the slide.
• Dry the slide on a hot plate.
• Affix the coverslip using an optical mounting medium (Norland Optical Adhesive 61).
SEM Preparation
Prepare and dilute the suspension using one of the above techniques (preferably the short centrifuge method), and spread it evenly onto 10 mm or 24 mm round coverslips. Allow the suspension to dry on a hot plate set to low heat. Once dried, mount the coverslips onto 10 mm or 24 mm aluminium SEM stubs using colloidal silver or carbon adhesive dots. Alternatively, the diluted suspension can be directly applied onto the carbon adhesive dots and left to dry. Finally, coat the dried suspension on the coverslips or carbon dots with a gold or palladium layer approximately 500 Å thick.
Round 10 mm or 24 mm coverslips with dried suspension can be utilised to analyse the exact specimen in both LM and SEM, ensuring the identification of the specimen with certainty under different illuminations. Techniques involving LM examination followed by SEM analysis were described by Moshkovitz (1974), Jafar (1977), Greig (1983), Gallagher (1988), and Varol (1989). For LM analysis, it is recommended to use a dry 63× objective lens with high NA to avoid the need for cleaning immersion oil. Conversely, techniques that begin with SEM examination, followed by LM analysis of the same specimen, were detailed by Thierstein et al. (1971), Smith (1975), and Moshkovitz (1989).
Mobile Mount
• Place a small piece of the sample onto a glass slide. Add one or two drops of distilled water to the sample to create a mixture.
• Smear the mixture evenly and thinly across the surface of the glass slide.
• Dry the slide on a hot plate.
• Scrape the dried mixture onto a separate glass slide.
• Stir the scraped, dried mixture with immersion oil (viscosity: 21,000 cSt).
• Place a coverslip on top of the mixture.
• Wait approximately 3-4 hours (or until the mixture settles between the glass slide and the coverslip) before examining the slide.
• To examine the slide, gently disturb the mixture by poking the coverslip