Maintaining Wolbachia in Cell-free Medium

My name is Courtney Ston. I work at the Johns Hopkins Bloomberg School of Public Health in the laboratory of Dr.Jason Rascon. Today we'll be doing bacterial purification from cultured insect cells.

It's actually a very straightforward procedure. We start by harvesting our cells and then physically disrupting the eukaryotic host cell membranes using glass bead lysis. And then we use differential centrifugation to separate the bacteria from the host cell debris.

And then we use a series of filtration steps to further remove host cell particles so that at the end we are left with just the purified bacterial pellet. This is actually one of the major requirements. It's a cell culture hood and it's used to keep everything sterile because cell culture is really easy to contaminate.

We also need cell culture media, the cells themselves and we grow them in plastic flasks. We also need three millimeter blast beads, 50 ml conical tubes, and we need a centrifuge that is capable of a high speed centrifugation, approximately 18, 000 G to pellet our bacteria, but also capable of doing about a 3000 G or 2, 500 G to pellet our host cells. So the first thing we'll do is we'll start with Our cultured cells.

They're grown right? These are T 75 flasks. They have 15 mls of culture media as well as ourselves.

So the first thing we'll do is start by disrupting the cell layer and collecting them in a 50 ml centrifuge tube. And you just more or less have to squeegee up and down on the cell layer and it displaces all the cells into the media. And then once our cells are in the media, we just pipette them into put the ML conical.

Now to the two cell suspensions, we are gonna add a three millimeter glass beads. And these have been autoclaved and they can actually be reused. They can be washed and soaked in bleach and then autoclave before they're used again.

You just pour being sure not to touch the mouth of the glass B jar to the mouth of the centrifuge tube. And we put approximately five up to the five milliliter volume mark worth of glass beads into each centrifuge tube. So the next thing we do is we wanna physically disrupt the the eary cells to release our bacteria into the suspension.

So we use a vortex, we put it on its highest vortex feet, and then we vortex for five minutes. So now we Vortex the cells with the three Millimeter glass beets for five minutes and we will pipette off the supernat with a clean pipette into a a clean and sterile 50 ML centrifuge tube. We will cast our newly created suspension and this suspension consists of the release bacteria and solution as well as the whole cell debris.

So the next step we're gonna do is a low speed centrifugation step so that we can pellet out the wholesale material and leave the bacteria in suspension. So we're gonna use the centrifuge, we're gonna use it at 2, 500 times G for 10 minutes. We're also gonna centrifuge at four degrees Celsius to help collect the pellet at the bottom.

And so we just place it in here, make sure it's balanced, close it, and centrifuge for 10 minutes. I've now centri fused our supernatant containing host cell debris as well as the purified bacteria. I did it at 2, 500 G for 10 minutes.

And as you can see, I have a pellet at the bottom of my tube that consists of theary cell debris. My newly freed bacteria are still in the supernat because the centrifugation was not high enough to pellet them as well. So the next thing that I will be doing is taking off the supernat and filtering it through a five micron filter.

So this is a five micron filter and it fits onto actually any sort of syringe. I'm about to use a 30 ml syringe, but you just pull this, the the plunger up, and then without touching the actual filter, especially the part down here, it needs to remain sterile. We attach the syringe to the filter just by screwing it in, and then we gently depress the plunger to pass the sup natan through and into our tube.

You don't wanna press too hard cause then you can actually break the filter and it will no longer work. If it gets to the point where you have to apply a lot of pressure, then you need to take your filter off and replace it cause it means that it's clogged with a lot of cell debris. And at this point, what we normally do, depending on our volume as another added method of obtaining a very pure bacterial culture, we add 250 millimolar sucrose solution that has been filter sterilized to our high speed centrifuge tube.

To fill the bottle to about at least 80%full. We usually fill it into the neck. These tubes need to be that full so that they don't implode inside of our high speed centrifuge.

Okay, so now we are going to spin our supernate and contending our purified bacteria at 18, 000 times G in this high speed centrifuge. So we make sure to place our tubes in balanced of course, and then we close it. We're gonna set it for 10 minutes.

And we like to do this at 10 degrees Celsius or lower, which our temperature is set over here. And then our angular velocity for this specific rotor in centrifuge is 12.5 revolutions per minute. We have a conversion chart, so it will spin at approximately 18, 670 times G.So now we've performed our high speed centrifuge spin to pellet our purified bacteria.

Now as you can see, we, we have a nice bacterial pellet at the bottom of our high speed tube. So what we'll do now is remove all of the SUP natin into a liquid waste container. So what we need to do at this point is resuspend our bacteria.

I'm going to resuspend it in two milliliters of our culture medium, which is Schneider's insect medium, supplemented with 10%heat inactivated fetal bovine serum. And I'll just resuspend the pellet by pipetting up and down until all of the bacteria are back into solution. So now that our bacteria back in suspension, we are going to do one more purification step just to get rid of any other possible host cell debris.

So for this, we are going to use a 2.7 micron filter. What I like to do is to use the plastic casing from our five micron filter as a holder hold. So we can take up our solution, screw your syringe onto your 2.7 micron filter, and now we will filter into a long term storage tube.

So like the other filter, you just press the plunger gently. And so now we Have our solution of purified bacteria. So the next thing that I'm gonna show you Is how we assay for viability.

We use the backlight live dead bacterial viability kit by molecular probes. This kit includes two stains, propidium, iodide, and cyto nine propidium. Iodide is red, fluoresce is red, and it stains only bacteria who have a bacterial membrane that's not completely intact and cyto nine stains all bacteria.

So when you look at these bacterial bacteria under the microscope fitted with fluorescent optics, all the bacteria will fluoresce green and then a subset, those that are dead will fluoresce red. We take a small aliquot from our purified sample and in this case I'm gonna take just 10 microliters, just enough to make one slide. And I'm gonna dispense it into another two.

And then I'm gonna add our other two stains, just a microliter of the mixture of the propidium iodide and the cyto nine to our bacterial suspension. And then you can let this incubate in the dark for 15 minutes. We find however that it actually you can go ahead and mount it and look at it nearly immediately and see your results.

Okay, so now I am going to mountain view my slide containing the bacteria stain with the backlight life dead stain. Let's dispense it onto my slide, a cover slip on and then view. I am gonna use our red green mixed filter so that I can assess the viability of my bacteria.

So when I open the shutter, I see my bacteria, they're just pinpricks more or less. But I see here approximately 90 to 95%of the bacteria are green, while only approximately I'd say approximately 5%are red. So then We have a very high viability rate, right?

So I've just walked you through the Purification of viable Akia from cultured insect cells. This protocol included the physical disruption of eukaryotic host cells and then differential fication to isolate the purified bacteria as well as filtration a series of filtration steps to ensure the quality of your purified bacteria to completely purify it from all host cell components. And then we walked through the backlight live dead assay, which uses two stains to assess the viability of your bacteria.

And you can use, now use your purified bacteria in a variety of applications, including infection of other cultured cell systems to increase infection of a specific system. You can also do metabolic studies or numerous other studies using Your live purified bacteria.