CHAPTER NINE

OCCULAR MICROMETRY

9.1Preparation of Diatoms for Microscopic examination

From the diatom samples, the living motile diatoms can be separated in several ways:

Supernatant Removal: In a petri dish, spread the sample, mix with water and allow to settle overnight. The following day, all the supernatant is drained from the petri dish. Thereafter place coverslips on the damp sediments for 4 hours, exposing the sediment to light. Remove the coverslips and rinse gently to remove sand particles. The cover slips can then be placed on a clean slide for microscopic examination.

Submersed Coverslips: Place moist sediment on a tray or petri dish and stick coverslips on the sediment surface. Live diatoms adhere to the surface of the coverslip. Remove sand grains by carefully rinsing with water.

Tissue Paper: This is used if the sediment sample contains large sand grains. The tissue paper is paced between the coverslip and the sediment, thus allowing the passage of motile diatoms onto the coverslip while preventing unwanted sediment grains.

Glass Slides: Glass slides can be stuck onto soil, mud or sediment up to three-quarter of the length of the slide. Diatoms adhere to the slide and can be viewed under the microscope.

9.2Importance of Micrometry in Diatoms

Measurement of diatoms species is important for following reasons:

Variation in size measurement of a given species is important in a given diatom community, as co-occurring small and large diatom species respond differently to environmental constraints (Snoeijs et al (2002).

Bio-volume determination and consequently biomass can reveal the variation in size and shape. Variation of size and shape of diatoms is of ecological significance because it affects surface to volume ratio and hence the area of the cell exposed to the environment. Bio-volume determination is done by comparing the diatom cell to a simple geometric solid fore example, a sphere, cylinder, etc. The corresponding formula (volume) for such solid is then applied, after taking the dimension (diameter, height, length, etc.) of the diatom.

Size analysis of diatom populations is a potentially powerful tool for understanding diatom life histories, population dynamics, and phylogenetic relationships (Spaulding et al, 2012).

Water quality estimation using "Omnidia” software for taxonomy, (Lecointe et al., 1993).

Diatom size is determined by morphometric measurement of such diatoms. Dimensions such as length, width and diameter are usually made. Measurement of cell dimensions of diatoms is done using micrometry with a micrometer eye piece graticule.

9.3Microscopic Examination of Diatoms

Diatoms make for very interesting specimen under the microscope. They show complex patterns with very fine punctures on their surface. With some of the species, fine pores in the frustules are used for testing the resolving power of the lens of a microscope.

Diatoms are prepared for viewing under the microscope using wet (temporary) mounts. Here, the sample is simply smeared on the slide using such liquids as water (sample smeared on the slide using such liquids as water) or permanent (sample mounted in a mountant, for example, Naphrax) mount. In some cases, hydrogen peroxide (or other oxidising agents, may be used to remove the organic matter of the frustules for better viewing. Here, a small amount of hydrochloric acid (HCL) may be used for the purposes of removing calcium carbonate followed by rinsing in distilled water to remove the acid. The sample can then be dried and placed and placed on a slide for viewing.

Diatoms are examined using microscopes of different types. Basically, light microscope is the first choice. Other microscopes such as Electron microscopes (Scanning Electron microscope (SEM), Transmission electron microscope (TEM)) are used for further detailed examination.

9.3.1Light Microscopy

The light microscope is an instrument for visualizing fine detail of an object. It does this by creating a magnified image using a series of glass lenses, which first focus a beam of light onto or through an object, and convex objective lenses to enlarge the image formed. To increase contrast, a mounting medium of higher refractive index (naphrax) can be used. Brightfield and phase contrast microscopy can be used for observing diatoms. Measurements in the light microscope are usually made in a permanent or temporary mounting medium of high refractive index (1.65 for Naphrax) using micrometer eyepiece. The eyepiece graticule or other measuring equipment is calibrated against a stage micrometer prior to the analysis to allow for measurement of dimensions and taxonomic features.

9.3.2Electron Microscopy

Electron microscopy (EM) is a technique for obtaining high resolution images of biological and non-biological specimens. The high resolution of EM images results from the use of electrons (which have very short wavelengths) as the source of illuminating radiation. There are two main types of electron microscopy—the transmission electron microscope (TEM) and the scanning electron microscope (SEM). The transmission electron microscope is used to view thin specimens (tissue sections, molecules, etc.) through which electrons can pass generating a projection image.

Figure 13: Electron microscopy collection depicting different diatom species

Scanning electron microscopy depends on the emission of secondary electrons from the surface of a specimen. It provides detailed images of the surfaces of the cells and whole organisms that are not possible by TEM.

9.4Digital flow Cytometry on a FlowCAM® (Fluid Imaging Technologies)

This captures and records several (hundreds, or even thousands), of images of a chosen taxon from a single sample within minutes. Morphological measures can be quantified through post- processing of the high-resolution images. Measurements in the FlowCAM are made on moving particles in an aqueous medium of lower refractive index (1.33 for H2O). For asymmetric cells, ferret or caliper or geodesic diameter are taken. The advantage of FlowCAM is increased speed of data acquisition.