Toxoplasma Goia Osis are typically obtained from infected cat feces and isolated using a flotation procedure resulting in a partially purified suspension, molecular and tissue culture. Experiments on this parasite, however, require highly purified osis to achieve this. Osis are further purified using a sucrose flotation step where osis are resuspended and sucrose overlaid with water and centrifuge to separate the osis away from most of the fecal matter.
The osis layer is then transferred into a new tube underlaid with cesium, chloride and centrifuged. The resulting distinct OSIS band is then collected and washed, ready to use on experiments requiring highly purified teon io assist. Hi, I'm Dr.Sarah Staggs, an employee of Dynam, a contractor for the National Exposure Research Laboratory of the US Environmental Protection Agency.
Today we will show you a procedure for purifying Toxoplasma Gandhi io Oasis from the feces of laboratory infected cats using a discontinuous cesium chloride gradient. This procedure is routinely used in our laboratory to isolate OSI, assist for use of molecular biology and tissue culture experiments. So let's get started.
Before beginning any work with TG O Osis, important safety considerations should be taken into account. People who are immunocompromised, pregnant, or actively trying to get pregnant should not handle the pathogen due to the risk of acquiring toxoplasmosis, which can cause illness and severe birth defects. X.To reduce the risk of infection, set aside a designated space for this procedure and post signs to warn others before they enter the room.
Remember to wear a lab coat, disposable gown, disposable gloves, and proper eye protection or a face shield when handling T-One IO cysts. Be sure to change your gloves often and avoid touching any equipment with contaminated gloves. It's also important to work on metal autoclavable trays lined with disposable absorbent liners.
And along those lines, take a minute to make sure all of the racks tubes and other equipment you plan to use are either disposable or autoclavable. If you're using non-disposable items, be sure to autoclave them after use for two one hour cycles. You'll also want to connect all vacuum lines to a vacuum shield filter to avoid contaminating the vacuum pump.
When you're finished isolating cysts, you will need to apply a fresh 10%hypochlorite solution to your work surface. Allow it to dry and then rinse the area several times with water. Once the proper safety precautions have been put into place, you can proceed with your experiments.
Prior to T Guni OYS collection, one must first prepare all of the necessary solutions for the sucrose, float and cesium chloride gradient. Centrifugation steps. The solutions that you will need include a 2.2 molar solution of sucrose sterilized by a 20 minute cycle in the autoclave, one liter of TE buffer pH 7.2 A one normal solution of sodium hydroxide, a 2%solution of sulfuric acid and a stock solution of caesium chloride with a specific gravity of one point 15.
The one point 15 caesium chloride stock solution will be used to make solutions A, B, and C, which will be used to prepare the cesium chloride gradient. Solution A is made by mixing 30 milliliters of TE with 20 milliliters of one point 15 cesium chloride stock solution solution B is made by mixing 20 milliliters of TE with 30 milliliters of one point 15 cesium chloride stock solution, and then adding 12.5 microliters of pheno red solution. Finally, solution C is made by mixing 10 milliliters of TE with 40 milliliters of one point 15 cesium chloride solution.
We'll begin our preparation of the sucrose float by taking a 10 milliliter fecal suspension of T osis in 2%sulfuric acid and transferring it to a 50 milliliter conical centrifuge tube. The source of the fecal matter comes from feces of infected cats that has already been processed through a sucrose flotation procedure previously described in a textbook written by Dr.JP Duby, entitled Toxoplasmosis of Animals and Humans. This additional step further reduces the amount of fecal debris in the cesium chloride purification process and helps achieve the highest purity of the T goi cysts.
We then neutralize the sulfuric acid by adding three fifths volume of one normal sodium hydroxide solution to the fecal suspension. Mix the suspension well by vortexing. Next, add an equal volume of sucrose and mix the suspension well by vortexing.
Using a 10 milliliter per pet, carefully overlay the sucrose fecal suspension with 10 milliliters of double distilled water. Once you are done carefully, place the tubes in the centrifuge and spin the at 1200 times G for 20 minutes at room temperature with no break after the spin. Being careful not to swell your perpe gently perpe from the air water interface to collect the top water and interface layers.
It's very important to try to minimize the amount of the sucrose layer that is carried over while you're collecting the interface layer. Transfer these layers to a new 50 milliliter conical centrifuge tube. Vortex the tube to mix the remaining sucrose fecal pellet solution.
Repeat the previous water overlay in centrifugation steps once more. After the spin, collect the top water and interface layers and transfer them to a new tube. Use double distilled water to bring the volume of the two oh oh cyst interface solutions to 50 milliliters.
Then centrifuge the tubes at 2000 times G for 10 minutes at room temperature. Finally aspirate the S supernatant from each tube. Re suspend the pellets in five milliliters of tea buffer and pull the osis suspensions.
The osis are now ready for the cesium chloride gradient step. We'll start by preparing a discontinuous cesium chloride gradient in a 50 milliliter polycarbonate Oak ridge tube to the polycarbonate tube. Add 10 milliliters of the suspension containing TE and osis using a 50 milliliter syringe with an 18 gauge blunt ended autoclavable steel needle.
And a two-way stop cock slowly add the following solutions directly to the bottom of the tube. Eight milliliters of solution a eight milliliters of solution B at eight milliliters of solution C.Make sure the flow rate doesn't exceed N 0.5 milliliters per second. It's also important to note that the phenol red in solution B helps distinguish the gradient layers.
Now you are ready to centrifuge the Oak ridge tube at 12, 000 times G for 60 minutes at four degrees Celsius with no break using a fixed angle, high speed rotor. Now that the centrifugation is finished, we collect the opaque white band between solutions A and B.This band contains the ooc assists. Using a 10 milliliter per pet, go directly to the ooc assists band without disturbing the gradient and collect the ooc.
Try to minimize the amount of caesium chloride solution that you aspirate while you're collecting the cyst, because excess caesium chloride might make it difficult to pellet the cysts during the wash. Step later on, transfer the OSIS to a new 50 milliliter conical centrifuge tube. Now wash the O cysts with 30 to 40 milliliters of double distilled water and then centrifuge the tube at 2000 times G at room temperature for 10 minutes with no break, carefully aspirate the SUP natant being careful not to disturb the O cyst pellet.
Repeat the wash two more times. Once the final washers have finished, carefully aspirate the sup natant and resuspend the pellet in 10 milliliters of 2%sulfuric acid. Check the OSIS under the microscope to ensure that all fecal debris has been successfully removed from the sample.
The osis are now ready for further manipulation, or you can store them at four degrees Celsius until you're ready to use them. Here is a look at what un purified osis look like using a differential interference contrast microscope at 400 x magnification. Notice the presence of large amounts of fecal contaminants in the field making it very difficult to identify the OSI assists.
Here is what the OISS look like under 400 x after the cesium chloride gradient purification protocol. Notice the absence of fecal debris. We've just shown you how to purify Toxoplasma Gandhi Iasis from the feces of laboratory infected cats using a discontinuous cesium chloride gradient.
When doing this procedure, it's important to remember to follow all safety guidelines and to always be careful when underlaying the cesium chloride gradient by adding each layer very slowly and trying not to disturb or mix the gradient. So that's it. Thanks for watching and good luck with your experiments.
