Methods
METHODS
Samples of diatomite were initially broken up by soaking them in water and then alternately freezing and thawing. The number of cycles needed to break them down to a smooth, creamy suspension varied from about ten, to over one hundred for the hardest and most compressed samples.
I used an acid technique (Hartley 1990) for cleaning and dissolving all organic material, followed by bleaching. This left individual diatoms as clear, transparent frustules. The cleaning process, although violent, did not break up the material to any degree.
After cleaning, most samples contained fine particles of broken diatoms and the remains of Foramanifera and other plankton that settled alongside them. These were separated with fine sieves and by swirling the samples in a petri dish to separate different components, being careful not to throw out the baby with the bath water!
Cleaned material was then suspended in de-ionised water as a dilute mixture. Several drops of the mixture were pipetted onto a clean microscope slide so that it covered the surface evenly with a single layer of diatoms. The slide was dried, which resulted in the material loosely adhering to the glass; generally referred to as a “dry spread”.
Several hundreds of these spreads were prepared and each observed under a x20 objective and a x8 eye-piece. Using a mechanical stage the slide was passed backwards and forwards underneath the objective in a series of overlapping transects, until all the dry spread had been observed.
When I found a diatom that I wished to preserve in a selective mount, I marked its position on the slide with a fine ink dot. These “dotted” slides were then passed on to Klaus Kemp who, with his amazing skill, picked-up the selected diatoms and mounted them on slides in naphrax as permanent mounts. In fact, five diatoms were mounted in a line on each slide to save material and time. Each diatom was numbered in ascending order in which they were found and these references I used throughout the study.
All records were logged and each diatom was photographed. For this I used an Olympus AH3 photomicroscope fitted with Olympus Plan Apo objectives. The images were acquired digitally on a Nikon D1X DSLR camera and transmitted directly to my PC. I decided not to use any optical contrast enhancing methods, but rather obtain a “straight” record which I subsequently enhanced using Adobe Photoshop. To record all the characters of each diatom at different depths within the field of view (and therefore not all focussed within a single picture) I used an image-stacking software programme in my PC. I took between 3 and 30 pictures of each diatom, at different depths, to include all its structure – referred to as “montage” in the captions on the photographs. The programme then combined all the sharp elements of each image to produce a single “in focus” picture. I used a very early computer programme dating from about 1994, “Auto Montage” by Synoptics, for my study. By now this software has been largely replaced by the, very much cheaper, Helicon Focus software which is widely used by many microscopists. Either way, I believe this method greatly enhances the information available from each photograph
Samples of diatomite were initially broken up by soaking them in water and then alternately freezing and thawing. The number of cycles needed to break them down to a smooth, creamy suspension varied from about ten, to over one hundred for the hardest and most compressed samples.
I used an acid technique (Hartley 1990) for cleaning and dissolving all organic material, followed by bleaching. This left individual diatoms as clear, transparent frustules. The cleaning process, although violent, did not break up the material to any degree.
After cleaning, most samples contained fine particles of broken diatoms and the remains of Foramanifera and other plankton that settled alongside them. These were separated with fine sieves and by swirling the samples in a petri dish to separate different components, being careful not to throw out the baby with the bath water!
Cleaned material was then suspended in de-ionised water as a dilute mixture. Several drops of the mixture were pipetted onto a clean microscope slide so that it covered the surface evenly with a single layer of diatoms. The slide was dried, which resulted in the material loosely adhering to the glass; generally referred to as a “dry spread”.
Several hundreds of these spreads were prepared and each observed under a x20 objective and a x8 eye-piece. Using a mechanical stage the slide was passed backwards and forwards underneath the objective in a series of overlapping transects, until all the dry spread had been observed.
When I found a diatom that I wished to preserve in a selective mount, I marked its position on the slide with a fine ink dot. These “dotted” slides were then passed on to Klaus Kemp who, with his amazing skill, picked-up the selected diatoms and mounted them on slides in naphrax as permanent mounts. In fact, five diatoms were mounted in a line on each slide to save material and time. Each diatom was numbered in ascending order in which they were found and these references I used throughout the study.
All records were logged and each diatom was photographed. For this I used an Olympus AH3 photomicroscope fitted with Olympus Plan Apo objectives. The images were acquired digitally on a Nikon D1X DSLR camera and transmitted directly to my PC. I decided not to use any optical contrast enhancing methods, but rather obtain a “straight” record which I subsequently enhanced using Adobe Photoshop. To record all the characters of each diatom at different depths within the field of view (and therefore not all focussed within a single picture) I used an image-stacking software programme in my PC. I took between 3 and 30 pictures of each diatom, at different depths, to include all its structure – referred to as “montage” in the captions on the photographs. The programme then combined all the sharp elements of each image to produce a single “in focus” picture. I used a very early computer programme dating from about 1994, “Auto Montage” by Synoptics, for my study. By now this software has been largely replaced by the, very much cheaper, Helicon Focus software which is widely used by many microscopists. Either way, I believe this method greatly enhances the information available from each photograph