Seeking a diluting fluid that will lyse the erythrocytes and the leukocytes. Will appreciate help on diluting fluid formulation or possibly a branded one.
this will depend on how you are estimating the numbers. FACS or under the microscope?
The following ILRI reference might answer your question:
http://www.ilri.cgiar.org/InfoServ/Webpub/fulldocs/LivProd/chapter2.htm
...particularly, the section on blood smears and use of a haemocytometer - though I've never bothered staining with Giemsa (as recommmended in the above); these are the only approaches I've ever used, and I've not worried too much about the presence/absence of blood cells (though in recent years I've only ever worked with cultured T. brucei!). Hope this helps.
I am so grateful for all your contributions. Sarah, it's under the microscope.
Alternatively, you can try using stained-thin blood slides. Counting the number of the tryps and divide with the number of the erythrocytes as seen in one field. The approximate percentage can be estimated from here. Use many fields (min. 5 fields) to get the means.
you can use any counting slide like haemocytometer or malassez slide
It calculates tryps per slide or chamber/chambers then you can measure the parasitemia per ml
I will suggest you use a haemocytometer. You can count the number of parasite in the selected haemocytometer chamber, relate that to the volume of the chamber and then to the entire volume of the sample. It is relatively straight forward and I remember using this method to count both malaria-infected and uninfected red cells in the past.
Thank you Akinola Adisa. We have been using that method but I want to be sure we count tryps without erythrocytes and leucocytes.
Sam Alsford, I really appreciate your contribution, but I really care about the presence of erythrocytes and leucocytes, this is the reason behind my experiment.
Working with malaria parasite, we developed a method using SLO or Equinatoxin II to differentially lyse uninfected and infected RBC (uRBC and iRBC). Using the right concentrations (you would need to titrate and work out the right concentration), SLO lyses uRBC while EqII lyses both iRBC an uRBC. The PV (parasitophorous vacuole) membrane was preserved in both cases. This enabled us to clearly identify iRBC under the light microscope with relative ease, although we backed the data up with a lot of biochemical analyses. We did not look at leucocytes in the paper because we grew or plasmodium in washed rbc in the lab.
Here is a copy:
Selective permeabilization of the host cell membrane of Plasmodium falciparum-infected red blood cells with streptolysin O and equinatoxin II. Biochem J. 2007 April 1; 403(Pt 1): 167–175.
SLO can be purchased from sigma but equinatoxin was a reagent generated in the lab. Remember that SLO may required activation before use, so read the manual that comes with the product. I hope this helps.
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