Preparing the sample. Your sample tubes should have a random number label if they come from an experimental treatment group and they should be processed for counting at random!!! The counter should have no idea what treatment the sample comes from to avoid bias. Do not loose your key! • Optional: Add a dropor more of Lugol’s reagent (An aqueous solution of iodine and potassium iodide in water) to the homogenate before spinning down pellet. Note: if the algae you are counting are motile (e.g. live cultured algae) you must immobilize them first, Lugol’s does this as well. Lugol stains starch granules and makes cells darker and also heavier. They are therefore easier to visualize and sink faster ontoyour counting chamber surface and plane of focus. Make sure your pellets are dry, then you can freeze these. Add an exact (known) but minimum volume of filtered sea water (0.45um filter; FSW) to the pellet and resuspend well by vigorous shaking or vortexing until no visible clumps remain. Take an aliquot of the resuspension to dilute if necessary. I recommend using a dilution that will give aconcentration of 25 cells/square. I try to count the 4 corner squares, so that in total I count about 100 cells per loading of the chamber. If the sample is very low in cells, count the whole chamber (9 squares). If you count less than 4 squares, always count the same ones, e.g. the top left and bottom right. If you count only one always count the center square. Keep track of your dilution factor(DF=final vol/initial vol).
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Filling the chamber:
NOTE: CONSISTENCY IN FILLING TECHNIQUE IS PARAMOUNT TO SUCCESSFUL COUNTS!!!!! (Practice first with mock samples and not with your precious samples!) • This is so critical that it is recommended that one person do the counts, or at least does the counts of one type of samples. For example one person counts the expelled algae and theother counts the expelled algae for the measurement of bleaching.
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Clean chamber and coverslip with lens paper with a little ethanol to remove any grease. Always use the correct coverslip (rectangular glass provided with chamber), a regular coverslip will not give you accurate values. Make sure you have extra coverslips! Use a glass Pasteur pipette to mix yoursample well. I recommend that you use the same pipette to load all samples, just have a little beaker with water to rinse the pipette between samples. Do not use plastic pipettes since they can bind significant amounts of debris and algae. The surface of a fresh glass pipette will also bind some algae but it quickly coats and equilibrates. The initial mixing will help achieve this. With the coverslipon the chamber quickly touch the chamber inlet groove with the tip of the pipette making sure there is liquid at the tip. Hold the pipette with one hand and use the upper surface of the index finger of your other hand to guide the tip; the pipette should be held at 45° angle to the chamber. The chamber should fill very quickly (almost instantaneously) with liquid by capillary action. Not muchpipette bulb pressure is required. If the is any slow filling or if the liquid front is not even (e.g. when it forms a bubble) start over. If it takes too long to fill (I count three or five seconds) while you are filling, redo. Each haemocytometer has two chambers. Remix your sample and draw a new sample for every chamber. i.e. do not fill both chambers with liquid from the same draw. The part ofthe haemocytometer that is filled is just the plateau, polished glass surfaces that has the grid etched onto it, not the whole slide (i.e. not the deep grooves around the plateaus. Quickly draw any excess liquid from the filling port. Mount slide onto microscope stage, being careful to have secure mounting. Allow time (about 0.5 -1.0 minute) for the algae to settle. Tip: Have two (or three)...