Preparation of diatom slides

Most of the ultra-structural details of diatoms lie at the limit of resolution of light. In addition, all mounting media generally used in cytology have a refractive index similar to that of diatom valves, with the result that slides with diatoms mounted in these media are too low in contrast for satisfactory investigation. Hence, diatoms are enclosed in a medium of higher refractive index than that of the diatom valves (Krammer and Lange-Bertalot, 2000). Three types of mounting media are generally used: ‘Hyrax’ r.i. (refractive index) 1.71 (Hanna, 1930); ‘Naphrax’ r.i. 1.69 (Flemming, 1954) and ‘Pleurax’, r.i. 1.73 (Hanna, (1949); refractive indices after Meller, (1985)). ‘Naphrax’ is available from Brunel Microscopes Ltd, Chippenham, SN14 6QA, England (http://www.brunelmicroscopes.co.uk/acatalog/Diatom_Mountants.html); while ‘Pleurax’ may be obtained from Dr Jonathan Taylor, North-West University, South Africa (Jonathan.Taylor@nwu.ac.za).

Slides should be free of contamination by other diatomaceous material and should display an assemblage of diatoms that is as close as possible, in terms of composition, to that of the original sample. For this reason, strewn slides are used almost exclusively (Lohman, 1982), and can be prepared following the methods of Welsh (1964), described below:

(Note: It is always necessary to keep the sample well mixed or shaken, as the larger diatom cells will tend to settle out of solution quicker than the smaller cells and thus the community counts will be skewed and unreliable).

1. Slides and cover slips should be thoroughly cleaned with detergent soap and stored in ethanol until used.
2. Using a pipette, a portion is drawn from a well-shaken numbered vial of cleaned material. The cleaned diatom suspension is diluted until the sample appears slightly cloudy (to a naked eye).
3. A single drop of ammonium chloride (NH₄Cl; 10% solution) is added for every 10ml of diluted diatom suspension to neutralise electrostatic charges on the suspended particles and reduce aggregation¹⁰⁴.
4. Using a pipette or straw ~0.5-1ml (depending on the size of the cover-slip) of this suspension is placed on a clean, dry cover-slip.
5. The diatom suspension placed on the cover slip is allowed to dry in a dust free environment at room temperature. Care should be taken not to disturb until dry, as vibration causes clumping of the diatom valves.
6. The drying of cover slips on a hot plate is not recommended because the resultant convection currents form more or less concentric rings of diatoms. If more rapid drying is required the samples may be dried under a 60W light globe.
7. After the water has evaporated, diatom-coated cover slips are placed on a hot plate at ~350°C for 1 minute to drive off the excess moisture and to sublimate the residual ammonium chloride.
8. The cooled cover slip can be examined under 400 x magnification to determine if the concentration of diatoms in the solution was correct. At least 10, but not more than 40, valves should be visible per field. When the sample is finally viewed at 1000 x magnification there should ideally be between 5 and 15, but not more than 20, valves visible in each field. If the concentration is too high or low, steps 1–7 need to be followed again, using a more, or less, dilute suspension, before proceeding further.
9. After the diatom-coated cover slips have been allowed to cool, one or two drops of mountant are placed onto each by means of a glass rod or pipette.
10. Heat the mountant on the cover-slips gently for 30 seconds to 1 min.
11. A previously cleaned glass slide is then lowered onto the cover slip, inverted, and then heated at 90–120°C on a  hot plate until the mounting medium ‘boils’ and all the solvent evaporates.
12. The solvent of the mounting medium should be evaporated quickly. If this is not done, a ring of exuded medium will harden around the edge of the cover slip, while the mounting medium under the cover slip remains more or less viscous.
13. Under no circumstances should the mounting medium be heated for too long, because it will then turn dark in colour.
14. Depending on temperature and the quality of the mounting medium, it is necessary to heat the slide on the hot plate for two to five minutes.
15. After the mounting medium is boiled for sufficient length of time and while it is still viscous, the hot slide is quickly removed from the hot plate, and laid on the work bench.
16. The cover slip is then adjusted into position. If this operation is not successful for the first time, the slide need only be re-heated for few minutes and positioning is repeated.
17. When the slide is thoroughly cooled, all the mounting medium should be hard and brittle and capable of being easily chipped off with the point of a scalpel.
18. Surplus medium, which has been exuded and has set round the edge of the cover slip, may be carefully removed with the point of a scalpel, after which the slide is then wiped clean with a soft rag soaked in the particular mounting medium’s solvent (iso-propyl alcohol for ‘Pleurax’) and toluene (which is carcinogenic for ‘Hyrax’).
19. The slide should be carefully labeled with sample details - date of collection, site location and co-ordinates, habitat, collector and type of mounting medium. The slide should also be labeled with the date of preparation and the name of the technician.
20. The slide is ready for microscopic examination and archiving.