First trial of centrifuge on pond water 14/12/2017

“First Light” for my centrifuge – the second hand Eppendorf 5415c. I followed a procedure detailed on the Micscape website for using the centrifuge to concentrate the organisms in a pond water sample from Stowe Pool in Lichfield collected today.

The centrifuge worked well and did indeed concentrate the organisms (see photos below for procedure I followed) although the variety was limited in the sample.

Andy

1-The-Eppendorf-5145c-centrifuge

2-Pond-water-sample-including-piece-of-plant-root

3-Stirring-sample-to-release-organisms-into-water

4-Filtering-out-large-particles using tea strainer

5-Filling-Ependorf-1-5ml-tubes-with-pond-water – each one needs to have same amount fluid in order for centrifuge to balance:

6-Placing-Eppendorf-tubes-in-centrifuge- note that they are evenly spread around centrifuge to obtain balance at high G-forces – you should always use pairs and never one on its own:

7-Screwing-on-airtight-lid-onto-rotor:

8-Centrifuged-samples-on-homemade-stand:

9-Pipetting-off-excess-fluid-after-pellet-the idea is to carefully remove the fluid above the pellet of centrifuged solids at bottom of tube without disturbing the pellet. Ideally, the pellet and single drop fluid left after pipetting.

10-View-of-pellet-at-bottom-of-Eppendorf-tube. after pipetting – it is the brown spot at bottom of tube in picture. I used pipette to transfer the pellets and small amounts of fluid from 6 tubes to 2 tubes and centrifuged again to further concentrate the contents into two pellets:

11-Slide-after-pellet-and-drop-water-pipetted-onto-it-showing-concentrated-protozoa & debris. I pipetted the final 2 pellets onto a slide and the picture below shows many dark areas in the water drop – these are from the pellets:

12 Coverslip & nail varnish to seal it before viewing under microscope:

Photos of organism from sample today seen under Zeiss IM microscope x32 magnification. The first photo shows well one particular feature evident in many of the photos – which is that the centrifuge has caused chloroplasts to be concentrated on one side of the cell rather than spread throughout. Other photos show large vacuoles which I think are from damage to the cell. Most of the cells were inactive – I presume killed by the process, although the banana-shaped ciliated organism in the third photo below was alive and moving.

 

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