Effective sand removal protocol

[zzffnn]

I was asked about the specifics of my sand removal method, so here it goes.

I tried pushing aside sand particles using a glass slide or covet slip, that did remove sand but remove most of my protists as well. So I tried other methods and found that the following worked best.

The key items include a 200 microliter micropipette and a small 1.5 ml clear wall microcentrigue tube, which allow you to see and remove different layers of precipitation (of different weight/density). Then we can do rounds of filtration, precipitation and concentration, as needed.

I usually take layers of pond water (for example, bottom, middle layer vegetation and top layer - what layer epends on where your target protists lives), put them into a small container and give them a good hard shake. This shaking step separates protists from sand and vegetation, if they attach to those.

Then I filter the shake through the finest kitchen strainer that I have found - this step removes big sand particles and vegetation, but won't remove small particles.

Then I put the flow-through into a 1.5 ml clear wall microcrntrifuge tube. Let things precipitate for at least 10 minutes. Then I remove and toss away, as much as possible, the clear top layer of water (using a 200 microlittle micropipette for precision). This step concentrates the organic matter with protists.

Then I shake the precipitates inside the tube. Next, I hold the tube close to my eyes, wait for around 10 seconds (for heavier sand particles to fall down) and use micropipette to precisely remove a water layer close to the top. If one wants cleaner sample at the cost of getting less varieties of protists, then wait longer then 10 seconds and get the very top layer. If one wants more protist varieties at the cost of having a little more sand particles, then wait less than 10 seconds and take a lower layer.

Now sample is ready to be loaded under cover slip for microscopy observation. This protocol removed most of sand in my samples and I was able to focus high NA (>=0.9) objectives sharply and precisely. An example, using a 70x NA 1.23 objective looking at a tiny Arcella, will be posted here once I get home.

One can choose to repeat one of the above steps several times for cleaner sample with less sand, at the risk of losing more protists.

If you have a micro centrifuge that can spin at around 200 rounds per minute, then you don't need to wait for precipitation. Note spinning speed higher than 800 rpm may kill protists by centrifugal force. Used micro centrifuge and micropipette can be bought used cheaply off eBay, or try your local university surplus auctions.

[mrsonchus]

Nice technique and walk-through, very interesting. I use a hand-centrifuge (bought new online very cheaply) for certain stages of pollen preparation in a similar fashion. Thanks for the useful information.

[zzffnn]

Nice technique and walk-through, very interesting. I use a hand-centrifuge (bought new online very cheaply) for certain stages of pollen preparation in a similar fashion. Thanks for the useful information.

I think I have seen similar kinds of hand centrifuge at eBay (http://m.ebay.com/itm/Hand-held-centrif ... 1660997638). It looked too large for microscpoy sample prep though (centrifuge itself is not small and container also seems slightly too large). Does yours work well?

[mrsonchus]

It does indeed, I use the micro-pots with the snap on lids, I simply plug the full-size (test-tube diameter-ish) holders with cotton wool then push the microtubes into them snugly, works a treat. It takes a bit of practice to gain good control over the separation process and achieving what is usually termed a 'plug' is not easy, but also not necessary for the 'lightweight' application of pollen processing.

Here's a quick video I just made to show the centrifuge in action - here I use tissue to hold the micro-tubes in the larger pots. Each handle-turn spins the pots 16 times and 60 handle-turns per minute is an easy pace, as seen in the video, giving nearly 1000 rpm for the pots, a nice and relatively-gentle speed. A cracking piece of kit that as I remember was very cheap to buy.



A couple of pictures,

ws_700x525_hand_centrifuge-3.jpg (119.4 KiB) Viewed 4107 times

and with micro-tubes in tissue-beds,

ws_700x525_hand_centrifuge-6.jpg (87.9 KiB) Viewed 4107 times

Hope this helps a bit.

[zzffnn]

Thank you John B. No worry about counting the rpm, with protists we don't need high rpm anyway. Approximately how big is your hand centrifuge though?

As I promised in my first post, the following is the Arcella video shot with 70x NA 1.23 objective. Previously I had a much bigger Arcella with more sand and the same objective, but I did not get image quality as clear as this one: https://youtu.be/IL3rk8MWE9g

[mrsonchus]

Video just posted!

[zzffnn]

Oops, I did not see your post above mine just now. I found it very helpful. Thank you very much John B.

That hand centrifuge does not look too huge and seems pretty sturdy. Where did you buy it and how much did it cost, if you don't mind?

[mrsonchus]

Here's a picture from the site just now, the 'better one' is a little dearer but I paid just under £10 a year ago for the grey 'basic' one and it works perfectly. The company is called 'SciChem'..

scichem_hand_centrifuge.JPG (34.71 KiB) Viewed 4097 times

Here's the web-page link, just search for 'hand centrifuge'..
http://laboratory.scichem.com/Catalogue ... 0000000000

Good luck.