I am George Dimopoulos at the Malaria Research Institute at the Johns Hopkins School of Public Health, and now we will show you a procedure on how to infect an awfully Gambia mosquitoes with plasmodium faci and parasites, and how to determine the infection phenotype on the mosquito Midgut. For this Procedure, the basic equipment needed are we need a CO2 incubator for the parasite culture. Alternatively, if you don't have a CO2 incubator, you can use a candle jar.
We also need an insectary, of course, standard molecular biology and cell biology equipment such as dissection microscopes, light microscopes. We need water baths. We need a mosquito membrane feeder system and centrifuge's.
Hi, my name is ISH and I'm in the lab of Dr.George Depos. I'm a postdoc. My name Is Lindsay Garver and I'm a PhD student.
The first step of the plasm Infection is, is to have the plasm culture ready. If you don't have a carbon dioxide incubator, then you can have a candle candle jar where you can put the six well plate, having the gato set culture, then light the candle, and then seal the jar. The first step is to have the human, fresh, human blood and serum and put them at 37 in a water bath first to thaw and also to have them like warm because you need really warm blood and warm serum.
To prepare the blood you need to allocate one animal of the fresh human blood in an eend off. Yeah, the fresh blood has to be spun at 2000 RPM for five minutes to peel it down the erythrocytes. After the spin is over, the clear soup has to be discarded and the red pellets, which is the RBC, has needs to be washed three times in human serum.
We add equal volume of human serum to the pellet, mix them and spin again at 2000 RPM for five minutes. This step is repeated three times. Finally, to have a red blood cell pellets ready to be added to the ganote culture.
Okay, the plasmodium culture Culture, which is in the CO2 incubator, it has to be maintained in RPMA medium for 16 days, and on day 16, they're ready for the infection. Since we're working with the plasmodium, culture, care must be taken at every steps. The first step is that the parasites should be grown in a BC two lab and it should be performed in a hood.
All the pipet tips and all of the append offs, everything has to be discarded in a biohazard box and any other solutions has to be like our pipe pitman. They have to be washed in 10%bleach and you have to wear gloves. Lab coats from the day 16 culture need to take a small drop of blood on the glass slide, make a smear and and do against a stain, and another, the light microscope.
We can determine the GAMMAT percentage from the 16 day old gametocyte culture. Remove most of the media taking care not to disturb or suck up any parasites, which is easily at the bottom of the, well pipe it the rest of the media and parasites. Mixing the parasites in the red blood cells into an pen off the parasites, which is in the append of is then put in a centrifuge to spin at 2000 RPM for five minutes.
After the spin, the parasites will pellet with the red blood cells. The supine is the RPMI media, and at the bottom the pellet has the parasites. With the RBC, remove the soup, which is the RPMI.
Media care should be taken not to disturb the parasite and the red blood cells. The GATOT percentage, which is determined by the GIMs 10, is usually in the range of two to 4%Then when the red blood cells and the parasites are ready after the spin, they're mixed together and the volume of blood that needs to be added is determined by the final gatot percentage. It needs to be acute, and it's usually in the range of 0.3 to 1%and this is what is what will be used for the feeding of the phys mosquitoes.
For the Presidium S Infection, this is a glass feeder, Which we use for the membrane feeding for the mosquitoes. We put the blood inside through the small hole that you see, and it is contained in that. At the bottom on the side are two nozzles, which are connected to our water birth, which is mentioned at 37 degree.
To prepare the feeder for the feeding, we take a small piece of affil. We stretch from the two ends on from all the four sides, and then put that on the open side of the glass feeder so as to make a thin sheet of membrane just as to mimic the skin. This is what is called the membrane feeding.
This is a circulating water bath, which maintains the water at 37 and also circulates the water through the glass feeder so that the blood is maintain warm at 37. So we connect the glass feeder through the small capillaries and connect that to the circulating water bath and turn the water bath on so that the water circulates and the water and blood they never mix as they're in two separate chambers. This is a small cup containing the phis Gambi mosquitoes.
To begin with, the glass feeder is put on the cage and a weight is put on the glass feeder so that the membrane, which is the phim, is in flush with the mesh net. Around 200 microliter of the blood, which contains the parasites. With the required gambotto percentage are added to the feeder, making sure that the blood goes all the way down and the mosquitoes can get access to the blood.
The fitting is continued for around 30 minutes. Once the fitting is complete, the glass feeder is washed thoroughly in 10, in 10%bleach, and the rest of the membranes and tape every append off, they're discarded in biohazard box. After the blood fitting, the unfit mosquitoes have to be removed from the Fed ones.
To do this, we knocked on the mosquitoes by putting the small cup cups containing the blood fed mosquitoes in cold room for five to 10 minutes. As we can see, the mosquitoes are already down in the cold. We open the lid and transfer the mosquitoes in a small petro dish, which is kept on a ice bucket so that the mosquitoes are kept in cold all through and they cannot escape.
Remember, these are plasmodium infected mosquitoes, so all care must be taken not to let them go loose. Once the mosquitoes are down in the cold room, we transfer the mosquitoes to the normal insectory, which is maintain at the room temperature, but we put the mosquitoes always in the petrol dish, which is kept in the ice bucket, so they're there so that they're always down. You can see how the Fed mosquitoes can be removed from the UNFI ones.
The Fed ones have the blood in them. They're fully engorged. Some them have fed on blood partially so they're not fully engorged.
They have a small blood in their abdomen, but others have a lot of blood in their abdomen. The UNFI ones do not have any blood in their abdomen, and so you can easily, easily distinguish between the Fed and the unfit ones.Shown. Here are the adult female mosquitoes which are fed on the blood, so we'll transfer these mosquitoes from the petro dish to the wax line cardboard cups.
After putting the blood fed mosquitoes in the cups, we put a small piece of cotton soaked in 10%sucrose so that they can feed on the 10%sucrose for the next seven days that you're going to incubate and also put a small folded we pepper towel, so to ment the moisture within the cup and covered with a petrol dish. The plasmon infected mosquitoes are put in another small cage, so it has to have a double protection and will put the cage in a bill three lab, which is maintain at 27 degrees Rogate, and 80%humidity seven days after the blood Feeding. The mosquitoes, which were kept in the insectory, in the double caged box, they're taken out from the insectory and taken to the cold room to knock the mosquitoes down.
After the mosquitoes are down, they're transferred to a petro dish, which is kept on an ice bucket. Now we are getting ready to dissect the mosquitoes. We'll dissect the mid gut and look for the cyst.
The first step is to add a hundred microliter of PBS on a glass slide mounted on a dissection microscope. The next step is using a plaintiff forceps. We transfer a single mosquito from the petro dish to the PBS, which is on the glass slide mounted on the dissection microscope.
The others are still left on the chilled petro dish. To dissect the mosquitoes, hold the mosquitoes at the thorax with one of the fps and the posterior abdomen with another fp. Then pull the body part slowly until the mid gut is exposed.
It looks like a white sack, like body use forceps to remove the excess tissue and the other body parts. Yeah, this is the mid gut, the which looks like a white sac like body, which has the os. We'll transfer this midgut now to a 12 Well glass light and immerse in a five to 10 microliter of 0.2%micro pro, which will stand the midgut recover the slide.
With the cover slip, we do this step for all the infected mosquitoes. The gut looks like a bright pink, and we'll transfer this slide to a light microscope To count the OS load. We are now looking at the Midgut dissected from the infected mosquitoes.
The midgut look like a slike body and with the rom stain, they have a very slight pinkish color, and the O SST should have a bright pink against the faint pink gut tissue. These midgut though, they're from the infected mosquitoes, but they do not show any sst, so they may be not infected. But if a midgut would have been infected, the S would've shown as a small, bright pink circle, and there can be numerous lag between 22 500 sometimes within one single Mid gut.
This Procedure involved infecting human blood with plasmodium rum parasites, feeding that infected blood to ly Gambian mosquitoes, dissecting the midgut out of those mosquitoes, and determining the OSIS counts within those infected midgut. This technique is important because it is the closest approximation of human malaria transmission that we can replicate in the laboratory.
