TITLE:  Plasmodium berghei   ookinete culture
AUTHORS: Benito Recio Totoro

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-style:italic;">Plasmodium</span><span style = "font-style:italic;font-style:italic;"> </span><span style = "font-style:italic;">berghei</span><span> is one of the main non-human malaria models used in research. It readily infects laboratory strains of mice and rats as well as mosquitoes. Because of this, it is safe to manipulate and it does not require human fluids/tissues. This document provides the Operational Procedure for producing and culturing ookinetes of </span><span style = "font-style:italic;">P. berghei in vitro</span><span>. The method has been optimized for the </span><span style = "font-style:italic;">P. berghei</span><span> ANKA train clone 2.34 and the derived GFP-expressing clone, but should work for other strains.</span></div><div class = "text-block">This method is adapted and modified from:</div></div>

[STEPS]
?. [Mice infections for passages.]
Mice are inoculated via intraperitoneally (IP) with no more than  either from a cryopreserved stock or by passage from other mice.From cryopreserved stock: allow the blood-cryopreservation solution to thaw slowly and inoculate  IP.Passage form another mouse: The number of parasites per ml of blood has to be determined to always inoculate a constant amount.
200 µl
200 µl
?. [Mice infections for passages.]
To determine the number of parasites per ml of blood, collect a drop of tail blood from the infected mouse and make a thin smear. Let it dry and fix with methanol.
?. [Mice infections for passages.]
Prepare  of 30% Giemsa per slide and stain for .
1 mL
?. [Mice infections for passages.]
Rinse slide with distilled water and dry by gently applying pressure with an absorbent paper towel. Do not rub the paper towel on the slide.
?. [Mice infections for passages.]
Determine the parasitemia by counting at least 500 red blood cells (RBC) at 1000x magnifications and determine the percentage of infected RBC.parasitemia=(iRBC/RBC)(100)where iRBC = infected RBC counted, RBC = total number of RBC counted.
Infected RBC with more than one parasite count as one.
?. [Mice infections for passages.]
Collect an additional  of tail blood and dilute it to  with PBS with  of heparin.
1 µl
1 mL
1 µl
?. [Mice infections for passages.]
Mix well and determine the number of RBC per ml of blood with the Neubauer chamber.
?. [Mice infections for passages.]
Determine the number of infected RBC per ml of blood using the parasitemia obtained previously.iRBC/ml=(parasitemia/100)(RBC/ml)
?. [Mice infections for passages.]
For maintenance passages, dilute the blood with PBS to obtain 4 x 105 infected RBC per  and inoculate IP the mice.
200 µl
Make no more than 8 passages as gametocytes lose the ability to mature into gametes. Also, keep infected mice for no more than a week as they will suffer unnecessarily. With this inoculum, the parasitemia should reach between 15 to 25% in one week.
?. [Mice infections for passages.]
Euthanize the mouse in the CO2 chamber and collect the blood via cardiac puncture with a 1 ml syringe preloaded with  of heparin. Before puncture, clean the puncture site with a cotton ball soaked in 70% ethanol.
10 µl
?. [Mice infections for ookinete culture]
For ookinete culture, inoculate mice with  of phenylhydrazine IP three days before the inoculation of the parasites to induce reticulocytosis.
200 µl
?. [Mice infections for passages.]
Fill the two slots of the Neubauer chamber with ∼ of blood solution (which is diluted 1/1000). Do not overflow the chambers.
10 µl
?. [Mice infections for passages.]
View under the microscope first with the 10x objective to locate the grid, and then with a 40x objective to zoom into the centre grid (the one with the smallest squares). The centre square is a 5 by 5 grid, each of these smaller squares are a 4 by 4 grid. Count the number of RBC in 5 squares of the 5 by 5 grid (the 4 squares in the corners and the centre one). Cells touching the top and right margins are counted, while cells touching the bottom and left margins are not counted.
?. [Mice infections for passages.]
RBC/ml=(xRBC/20\times10^6)(1\times10^{12})(1000)
?. [Mice infections for ookinete culture]
Determine the parasitemia and infected RBC concentration of the donor mouse as explained in steps 2 to 8.
?. [Mice infections for ookinete culture]
Collect the infected blood as explained in step 9.
?. [Mice infections for ookinete culture]
Dilute the infected blood with PBS to obtain 4 x 107 infected RBC per  and inoculate the donor mouse IP.
200 µl
With this inoculum, the parasitemia must reach between 15 to 25% in three days.
?. [Ookinete culture]
Three days after the inoculum of 4 x 107 infected RBC verify the parasitemia as explained in steps 2 to 5, and verify the viability of the gametocytes by performing an exflagellation test.
?. [Ookinete culture]
For the exflagellation test, collect an additional  of tail blood and place in a  0.6 ml microcentrifuge tube with 7 µl of ookinete medium and  of heparin. Mix very gently.Take the resulting  and place them on a microscope slide, cover with a coverslip and incubate for  at .
2 µl
1 µl
10 µl
20 °C
?. [Ookinete culture]
Determine the number of exflagellation centres in 10 random fields of view.
We only use mice with 15 to 25% parasitemia and >7 exflagellation centres per field of view.We find it not useful to determine the gametocytaemia because the correlation of parasitemia vs gametocytaemia, and the correlation between gametocytaemia vs exflagellation is low (Fig  2).
?. [Ookinete culture]
Prepare a T25 culture flask with 4 ml of ookinete medium. Sacrifice the mice and collect the blood as explained in step 9.
About 1 ml of blood is obtained.It is critical to collect the blood in a gentle manner but fast. Do not pull the syringe plunger too much as to generate vacuum. Mix the blood with the heparin very well. Try to succeed in the cardiac puncture in the first attempt.Rough manipulation of the blood at this step decreases considerably the ookinete culture yield.
?. [Ookinete culture]
Add the blood to the culture flask. Again treat the blood gently, do not push the syringe plunger too hard.Mix slightly and incubate at  for .
20 °C
?. [Ookinete culture]
After incubation, determine the number of ookinetes in the culture by taking a sample of . Dilute in  of PBS and count in the Neubauer chamber.
50 µl
950 µl
?. [Ookinete culture]
Add ∼ of the ookinete culture dilution on each chamber and count the number of ookinetes in the four big squares in the corners. For reference, these big squares have a 4 by 4 grid.Ookinetes/ml=(xOokinetes/4squares)(20)(10000)This is the number of ookinetes over the number of squares counted, times the dilution factor, times a conversion factor.
10 µl
Usually, between 25 and 35 million ookinetes are obtained in total per culture (from one mouse).