CHAPTER TEN
ILLUSTRATIONS AND IMAGING OF DIATOMS
10.1Illustration of Diatoms
Illustrations of diatoms are made as follows:
Drawing of Diatoms: Drawings of diatoms are done by freehand or with drawing aid or attachment. The drawing aid is attached to the microscope. Example of such drawing aids is camera Lucida. This fits around the tube of the microscope, enabling the simultaneous viewing and drawing of the specimen. It consists of a prism (placed over the eyepiece) with a side arm bearing a mirror, which reflects the specimen’s image onto a paper placed on the bench for tracing the image and filters which balance illumination. The illumination of the microscope and the room lighting are adjusted to enable accurate tracing around the virtual image of the specimen on a paper.
Photomicrography of Diatoms: Digital photography is used in contemporary times for diatom imaging. These include use of phase-contrast microscopy, fluorescence microscopy, light microscopy or Scanning/Transmission Electron microscopy. Photomicrography is cheaper and less time consuming.
Video images: can also be made of diatoms and used to illustrate diatom movement, stock footage videos for movies and advertisements.
10.2Enumeration of Diatoms
Different methods are applied in the enumeration/counting of diatoms, using a compound microscope. Different types of counters include Sedgewick counting chamber and Haemocytometer.
10.2.1Sedgewick Counter is also known as Sedgewick Rafter Counting Chamber.
It is a premier counting cell designed specifically for the quantitative measurement of the exact number of particles in a precise volume of a fluid. It is available in glass and plastic versions.
For Sedgewick counter, place 1 ml in Sedgewick-Rafter counting chamber. Count the diatoms in as many cells to obtain a count of 400 organisms. The density of diatoms is obtained from the following formula:
Calculations:
Diatoms/mL = Diatom Count x 1000
Gird Cells Counted
Diatom cell density(diatoms/cm2) = Diatom Per mL x Storage Volume
197.6 (Surface Area of 5 Slides)
10.2.2Haemocytometer
The haemocytometer is a counting chamber device originally designed for counting blood cells. It consists of a thick glass microscope slide with a rectangular indentation that creates a precision volume chamber. The device is carefully crafted so that the area bounded by the lines is known, and the depth of the chamber is also known. By observing a defined area of the grid, it is possible to count the number of cells or particles in a specific volume of fluid, and thereby calculate the concentration of cells in the fluid overall. A well-used type of haemocytometer is the Neubauer counting chamber.
To calculate the number of viable cells/mL:
1. Take the average cell count from each of the sets of 16 corner squares. 2. Multiply by 10,000 (104). Or:
Cell Density = \\frac{AverageCellperSmallSquareXDilutionFactor}{VolumeofaSmallSquare(mL)}
Volume of a small square = width x height x depth
= 1mm x 0.1mm x 0.1mm
= 0.1mm3
= 0.0001mL
If sample was diluted, then appropriate dilution factor is applied.
Total cells per mL = Total cells counted x \\frac{\\mathrm{d}\\mathrm{i}\\mathrm{l}\\mathrm{u}\\mathrm{t}\\mathrm{i}\\mathrm{o}\\mathrm{n}\\mathrm{f}\\mathrm{a}\\mathrm{c}\\mathrm{t}\\mathrm{o}\\mathrm{r}}{\\mathrm{N}\\mathrm{o}.\\mathrm{o}\\mathrm{f}\\mathrm{S}\\mathrm{q}\\mathrm{u}\\mathrm{a}\\mathrm{r}\\mathrm{e}\\mathrm{s}} x 10,000 cells per mL
10.2.3Automated Cell Counters
Consisting of advanced autofocusing and counting algorithms which allow quick, convenient and accurate counting of cells, while avoiding user variation associated with counting. The sample is loaded into an automated cell counter and is forced through a small tube while the automated cell counter uses optical or electrical sensors or electronic detectors.