Currently I am working on a drug sensitivity test on P. falciparum. In the mean time I just wanted to preserve the parasites in a deep freeze.
Transfer culture to a 15ml centrifuge tube. Centrifuge culture at 2000RPM for 10mins, and then discard supernatant. Add the same amount of glycerolyte to pellet (Ex., 0.5ml pellet add .5 ml glycerolyte) , and transfer it to cryovial and freeze at -80 freezer or to cryobank. (make sure culture is in ring stage mostly for freezing)
http://ki.se/content/1/c6/06/57/28/Methods%20in%20malaria%20research.pdf
Go to the above link. Download and/or open the pdf file. You can get Acrobat reader free from adobe if you don't already have a program to view pdf's. Go to page 14 (Freezing of patient isolates and strains with glycerolyte
by Johan Normark) and follow the protocol. Follow the protocol on page 15 for thawing of parasites. I have used both of these protocols for many years with complete success. Make certain that you synchronize the parasites before freezing so that parasitemia is high and that they are in early ring stage before freezing. Good Luck!
Go to the above link and you will find all protocols !
http://www.mr4.org/Publications/MethodsinMalariaResearch.aspx
Good luck !
Transfer 15ml of the red blood cell centrifuge and discard supernatant and transfer the deposit into cryovial and add liquid nitrogen and store at -70 and is ready to be used
thank you all for ur information. I highly appreciate ur efforts!!
In our lab we kept alive trpansoma parasit in liqiuid nitrogen container, so I think you can do the same with plasmodium.
Simon correa MRC unit The Gambia
Choose cultures of approximately 5% parasitaemia with a high proportion of high ring forms
centrifuge culture @2000rpm for 5 minutes.remove the supernatant
ADD EQUAL VOLUME OF DEEP FREEZE solution
28%glycerol,3%sorbitol,0.65%Nacl
Solution made by adding 28ml glycerol to 72ml of4.2%sorbitol or mannitol in0.9%Nacl.Sterilise by filtration.
Add this solution slowly to packed blood cells,drop by drop,at room temperature to allow glycerol to penetrate cells
Place no more than 0.5ml into each ampoule.
freeze rapidly by plunging into liquid nitrogen or -70c deep freeze.
Thanks
Good luck
The method above that suggests using glutaraldehyde is surely a mistake. This is a fixative that will preserve the parasite for EM or other studies, but these will not be viable when thawed. Perhaps you menat to say glycerol or glycerolite.
I recommend you follow Michel's link to the MR4 protocols. Also note the equally important steps of thawing the parasites following cryopreservation.
Dear Yared,
I am not sure if the you have the asexual stages in culture or blood obtained from patients. It you have whole blood from patients you need to wash with alsevers solution to remove the complement proteins. For asexual stages in culture use protocols provided by Samsom.
The following reference will assist you.
Trager W, Jensen JB. 1976. Human malaria parasites in continuous culture. Science
193(4254):673-675.
Regards,
I use the Stockholm Sorbitol Method included in the Methods in Malaria Research
http://ki.se/sites/default/files/methods_in_malaria_research.pdf
http://www.mr4.org/Portals/3/Methods_In_Malaria_Research-5theditionv5-2.pdf
It has worked perfectly for me.
BTW, there is a brand new edition of the protocols available for download at the EVIMalaR site
http://www.evimalar.org/resources/
Hello Yared,
Best is follow the procedure given in "METHODS IN MALARIA RESEARCH" - Sixth Edition - edited by - Kirsten Moll, Akira Kaneko, Arthur Scherf and Mats Wahlgren (MR4/ATCC, Manassas, VA, USA, 2013 ).
Hope this will help you.
All the Best..
Dear Yared Debebe,
This link is the one I suppose can help you Article Malaria Methods and Protocols
particularly its part entitled " Maintenance of the Plasmodium berghei Life Cycle" which can be cited as Sinden R.E., Butcher G.A., Beetsma A.L. (2002) Maintenance of the Plasmodium berghei Life Cycle. In: Doolan D.L. (eds) Malaria Methods and Protocols. Methods in Molecular Medicine™, vol 72. Humana Press.Article Maintenance of the Plasmodium berghei Life Cycle
.
This is another paper that can be helpful I hope. Article Freeze-thaw lysates of Plasmodium falciparum-infected red bl...
Dear Yared Debebe
1. This is helpful book https://www.beiresources.org/portals/2/MR4/Methods_In_Malaria_Research-6th_edition.pdf
2. Here is the article from Experimental Parasitology published in 1987 entitled Article Plasmodium falciparum: Freeze-fracture of the gametocyte pel...
3. Where as the one I considered the latest that can help you is the method of Freeze-Thawing of P. falciparum Blood-Stage Parasites https://www.burnet.edu.au/system/publication/file/5544/Yap_et_al_2019.pdf
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