A Methods Manual for the Collection, Preparation and Analysis of ...

Section SummaryPreparation:1. Pre-preparation examination for live cells.2. Cleaning of cells:a. In laboratory equipped with a fume cabinet: KMnO 4 + hot HClmethod, hot H 2 SO 4 + HNO 3 (2:1) method, hot H 2 O 2 method.b. In WELL VENTILATED laboratory without fume cabinet: ColdH 2 O 2 .c. Rinsing: Centrifuge available: centrifuge with distilled water untilsample is circumneutral (4-5 runs for 10 min. at 2500 rpm)d. No centrifuge available: decant supernatant using an aspirator.Resuspend sample using distilled water and allow to settle for 8 hours(repeat 4-5 times)3. Slide preparation:a. Concentrated diatom solution diluted with distilled water until onlyslightly cloudy,b. Add 1-2 drops of 10% NH 4 Cl to dilute solution to prevent clumpingof cells,c. 1.5 – 2 ml of dilute solution is placed on cover slip, depending on sizeof cover slip,d. Sample allowed to air-dry ( Takes approximately 24 hrs),e. Cover slip heated to drive off excess moisture and sublimate NH 4 Cl,f. Sample mounted with high-resolution mountant.Archiving:1. Cleaned samples should be stored in ethanol, at a concentration high enoughto prevent the growth of bacteria and fungi and to prevent the dissolution ofsilica.2. Slides should be stored flat until mountant is dry.3. All relevant information on the location, date of collection, substratum andcollector should be stored both with the sample and the slide, not simply areference number.28